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Key Points: A growing body of evidence suggests that epithelial Na + channels (ENaCs) in the brain play a significant role in the regulation of blood pressure; however, the brain structures that mediate the effect are not well understood. Because vasopressin (VP) neurons play a pivotal role in coordinating neuroendocrine and autonomic responses to maintain cardiovascular homeostasis, a basic understanding of the regulation and activity of ENaC in VP neurons is of great interest. We show that high dietary salt intake caused an increase in the expression and activity of ENaC which resulted in the steady state depolarization of VP neurons. The results help us understand one of the mechanisms underlying how dietary salt intake affects the activity of VP neurons via ENaC activity., Abstract: All three epithelial Na + channel (ENaC) subunits (α, β and γ) are located in vasopressin (VP) magnocellular neurons in the hypothalamic supraoptic (SON) and paraventricular nuclei. Our previous study demonstrated that ENaC mediates a Na + leak current that affects the steady state membrane potential in VP neurons. In the present study, we evaluated the effect of dietary salt intake on ENaC regulation and activity in VP neurons. High dietary salt intake for 7 days caused an increase in expression of β- and γENaC subunits in the SON and the translocation of αENaC immunoreactivity towards the plasma membrane. Patch clamp experiments on hypothalamic slices showed that the mean amplitude of the putative ENaC currents was significantly greater in VP neurons from animals that were fed a high salt diet compared with controls. The enhanced ENaC current contributed to the more depolarized basal membrane potential observed in VP neurons in the high salt diet group. These findings indicate that high dietary NaCl intake enhances the expression and activity of ENaCs, which augments synaptic drive by depolarizing the basal membrane potential close to the action potential threshold during hormonal demand. However, ENaCs appear to have only a minor role in the regulation of the firing activity of VP neurons in the absence of synaptic inputs as neither the mean intraburst frequency, burst duration, nor interspike interval variability of phasic bursting activity was affected. Moreover, ENaC activity did not affect the initiation, sustention, or termination of the phasic bursting generated in an intrinsic manner without synaptic inputs., (© 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Key Points: The nucleus accumbens (nAc) is involved in addiction-related behaviour caused by several drugs of abuse, including alcohol. Glycine receptors (GlyRs) are potentiated by ethanol and they have been implicated in the regulation of accumbal dopamine levels. We investigated the presence of GlyR subunits in nAc and their modulation by ethanol in medium spiny neurons (MSNs) of the mouse nAc. We found that the GlyR α1 subunit is preferentially expressed in nAc and is potentiated by ethanol. Our study shows that GlyR α1 in nAc is a new target for development of novel pharmacological tools for behavioural intervention in drug abuse., Abstract: Alcohol abuse causes major social, economic and health-related problems worldwide. Alcohol, like other drugs of abuse, increases levels of dopamine in the nucleus accumbens (nAc), facilitating behavioural reinforcement and substance abuse. Previous studies suggested that glycine receptors (GlyRs) are involved in the regulation of accumbal dopamine levels. Here, we investigated the presence of GlyRs in accumbal dopamine receptor medium spiny neurons (MSNs) of C57BL/6J mice, analysing mRNA expression levels and immunoreactivity of GlyR subunits, as well as ethanol sensitivity. We found that GlyR α1 subunits are expressed at higher levels than α2, α3 and β in the mouse nAc and were located preferentially in dopamine receptor 1 (DRD1)-positive MSNs. Interestingly, the glycine-evoked currents in dissociated DRD1-positive MSNs were potentiated by ethanol. Also, the potentiation of the GlyR-mediated tonic current by ethanol suggests that they modulate the excitability of DRD1-positive MSNs in nAc. This study should contribute to understanding the role of GlyR α1 in the reward system and might help to develop novel pharmacological therapies to treat alcoholism and other addiction-related and compulsive behaviours., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Key Points: β-Adrenergic stimulation enhances Ca 2+ entry via L-type Ca V 1.2 channels, causing stronger contraction of cardiac muscle cells. The signalling pathway involves activation of protein kinase A (PKA), but the molecular details of PKA regulation of Ca V 1.2 remain controversial despite extensive research. We show that PKA regulation of Ca V 1.2 can be reconstituted in Xenopus oocytes when the distal C-terminus (dCT) of the main subunit, α 1C , is truncated. The PKA upregulation of Ca V 1.2 does not require key factors previously implicated in this mechanism: the clipped dCT, the A kinase-anchoring protein 15 (AKAP15), the phosphorylation sites S1700, T1704 and S1928, or the β subunit of Ca V 1.2. The gating element within the initial segment of the N-terminus of the cardiac isoform of α 1C is essential for the PKA effect. We propose that the regulation described here is one of two or several mechanisms that jointly mediate the PKA regulation of Ca V 1.2 in the heart., Abstract: β-Adrenergic stimulation enhances Ca 2+ currents via L-type, voltage-gated Ca V 1.2 channels, strengthening cardiac contraction. The signalling via β-adrenergic receptors (β-ARs) involves elevation of cyclic AMP (cAMP) levels and activation of protein kinase A (PKA). However, how PKA affects the channel remains controversial. Recent studies in heterologous systems and genetically engineered mice stress the importance of the post-translational proteolytic truncation of the distal C-terminus (dCT) of the main (α 1C ) subunit. Here, we successfully reconstituted the cAMP/PKA regulation of the dCT-truncated Ca V 1.2 in Xenopus oocytes, which previously failed with the non-truncated α 1C . cAMP and the purified catalytic subunit of PKA, PKA-CS, injected into intact oocytes, enhanced Ca V 1.2 currents by ∼40% (rabbit α 1C ) to ∼130% (mouse α 1C ). PKA blockers were used to confirm specificity and the need for dissociation of the PKA holoenzyme. The regulation persisted in the absence of the clipped dCT (as a separate protein), the A kinase-anchoring protein AKAP15, and the phosphorylation sites S1700 and T1704, previously proposed as essential for the PKA effect. The Ca V β 2b subunit was not involved, as suggested by extensive mutagenesis. Using deletion/chimeric mutagenesis, we have identified the initial segment of the cardiac long-N-terminal isoform of α 1C as a previously unrecognized essential element involved in PKA regulation. We propose that the observed regulation, that exclusively involves the α 1C subunit, is one of several mechanisms underlying the overall PKA action on Ca V 1.2 in the heart. We hypothesize that PKA is acting on Ca V 1.2, in part, by affecting a structural 'scaffold' comprising the interacting cytosolic N- and C-termini of α 1C ., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Key Points: The functional importance of residues in loop G of the GABA A receptor has not been investigated. D43 and T47 in the α1 subunit are of particular significance as their structural modification inhibits activation by GABA. While the T47C substitution had no significant effect, non-conservative substitution of either residue (D43C or T47R) reduced the apparent potency of GABA. Propofol potentiated maximal GABA-evoked currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Non-stationary variance analysis revealed a reduction in maximal GABA-evoked P open , suggesting impaired agonist efficacy. Further analysis of α1(T47R)β2γ2 receptors revealed that the efficacy of the partial agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol) relative to GABA was impaired. GABA-, THIP- and propofol-evoked currents mediated by α1(T47R)β2γ2 receptors deactivated faster than those mediated by α1β2γ2 receptors, indicating that the mutation impairs agonist-evoked gating. Spontaneous gating caused by the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to α1β2(L285R)γ2 receptors, confirming that α1(T47R) impairs gating independently of agonist activation., Abstract: The modification of cysteine residues (substituted for D43 and T47) by 2-aminoethyl methanethiosulfonate in the GABA A α1 subunit loop G is known to impair activation of α1β2γ2 receptors by GABA and propofol. While the T47C substitution had no significant effect, non-conservative substitution of either residue (D43C or T47R) reduced the apparent potency of GABA. Propofol (1 μm), which potentiates sub-maximal but not maximal GABA-evoked currents mediated by α1β2γ2 receptors, also potentiated maximal currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors. Furthermore, the peak open probabilities of α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were reduced. The kinetics of macroscopic currents mediated by α1(D43C)β2γ2 and α1(T47R)β2γ2 receptors were characterised by slower desensitisation and faster deactivation. Similar changes in macroscopic current kinetics, together with a slower activation rate, were observed with the loop D α1(F64C) substitution, known to impair both efficacy and agonist binding, and when the partial agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol) was used to activate WT or α1(T47R)β2γ2 receptors. Propofol-evoked currents mediated by α1(T47R)β2γ2 and α1(F64C)β2γ2 receptors also exhibited faster deactivation than their WT counterparts, revealing that these substitutions impair gating through a mechanism independent of orthosteric binding. Spontaneous gating caused by the introduction of the β2(L285R) mutation was also reduced in α1(T47R)β2(L285R)γ2 compared to α1β2(L285R)γ2 receptors, confirming that α1(T47R) impairs gating independently of activation by any agonist. These findings implicate movement of the GABA A receptor α1 subunit's β1 strand during agonist-dependent and spontaneous gating. Immobilisation of the β1 strand may provide a mechanism for the inhibition of gating by inverse agonists such as bicuculline., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Voltage-gated calcium channels are essential players in many physiological processes in excitable cells. There are three main subdivisions of calcium channel, defined by the pore-forming α1 subunit, the CaV 1, CaV 2 and CaV 3 channels. For all the subtypes of voltage-gated calcium channel, their gating properties are key for the precise control of neurotransmitter release, muscle contraction and cell excitability, among many other processes. For the CaV 1 and CaV 2 channels, their ability to reach their required destinations in the cell membrane, their activation and the fine tuning of their biophysical properties are all dramatically influenced by the auxiliary subunits that associate with them. Furthermore, there are many diseases, both genetic and acquired, involving voltage-gated calcium channels. This review will provide a general introduction and then concentrate particularly on the role of auxiliary α2 δ subunits in both physiological and pathological processes involving calcium channels, and as a therapeutic target., (© 2016 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Key Points: The role of the β1 strand in GABAA receptor function is unclear. It lies anti-parallel to the β2 strand, which is known to participate in receptor activation. Molecular dynamics simulation revealed solvent accessible residues within the β1 strand of the GABAA β3 homopentamer that might be amenable to analysis using the substituted Cys accessibility method. Cys substitutions from Asp43 to Thr47 in the GABAA α1 subunit showed that D43C and T47C reduced the apparent potency of GABA. F45C caused a biphasic GABA concentration-response relationship and increased spontaneous gating. Cys43 and Cys47 were accessible to 2-aminoethyl methanethiosulphonate (MTSEA) modification, whereas Cys45 was not. Both GABA and the allosteric agonist propofol reduced MTSEA modification of Cys43 and Cys47. By contrast, modification of Cys64 in the β2 strand loop D was impeded by GABA but unaffected by propofol. These data reveal movement of β1 strand loop G residues during agonist activation of the GABAA receptor., Abstract: The GABAA receptor α subunit β1 strand runs anti-parallel to the β2 strand, which contains loop D, known to participate in receptor activation and agonist binding. However, a role for the β1 strand has yet to be established. We used molecular dynamics simulation to quantify the solvent accessible surface area (SASA) of β1 strand residues in the GABAA β3 homopentamer structure. Residues in the complementary interface equivalent to those between Asp43 and Thr47 in the α1 subunit have an alternating pattern of high and low SASA consistent with a β strand structure. We investigated the functional role of these β1 strand residues in the α1 subunit by individually replacing them with Cys residues. D43C and T47C substitutions reduced the apparent potency of GABA at α1β2γ2 receptors by 50-fold and eight-fold, respectively, whereas the F45C substitution caused a biphasic GABA concentration-response relationship and increased spontaneous gating. Receptors with D43C or T47C substitutions were sensitive to 2-aminoethyl methanethiosulphonate (MTSEA) modification. However, GABA-evoked currents mediated by α1(F45C)β2γ2 receptors were unaffected by MTSEA, suggesting that this residue is inaccessible. Both GABA and the allosteric agonist propofol reduced MTSEA modification of α1(D43C)β2γ2 and α1(T47C)β2γ2 receptors, indicating movement of the β1 strand even during allosteric activation. This is in contrast to α1(F64C)β2γ2 receptors, where only GABA, but not propofol, reduced MTSEA modification. These findings provide the first functional evidence for movement of the β1 strand during gating of the receptor and identify residues that are critical for maintaining GABAA receptor function., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Key Points: Lateral superior olive (LSO) principal neurons receive AMPA receptor (AMPAR) - and NMDA receptor (NMDAR)-mediated EPSCs and glycinergic IPSCs. Both EPSCs and IPSCs have slow kinetics in prehearing animals, which during developmental maturation accelerate to sub-millisecond decay time-constants. This correlates with a change in glutamate and glycine receptor subunit composition quantified via mRNA levels. The NMDAR-EPSCs accelerate over development to achieve decay time-constants of 2.5 ms. This is the fastest NMDAR-mediated EPSC reported. Acoustic trauma (AT, loud sounds) slow AMPAR-EPSC decay times, increasing GluA1 and decreasing GluA4 mRNA. Modelling of interaural intensity difference suggests that the increased EPSC duration after AT shifts interaural level difference to the right and compensates for hearing loss. Two months after AT the EPSC decay times recovered to control values. Synaptic transmission in the LSO matures by postnatal day 20, with EPSCs and IPSCs having fast kinetics. AT changes the AMPAR subunits expressed and slows the EPSC time-course at synapses in the central auditory system., Abstract: Damaging levels of sound (acoustic trauma, AT) diminish peripheral synapses, but what is the impact on the central auditory pathway? Developmental maturation of synaptic function and hearing were characterized in the mouse lateral superior olive (LSO) from postnatal day 7 (P7) to P96 using voltage-clamp and auditory brainstem responses. IPSCs and EPSCs show rapid acceleration during development, so that decay kinetics converge to similar sub-millisecond time-constants (τ, 0.87 ± 0.11 and 0.77 ± 0.08 ms, respectively) in adult mice. This correlated with LSO mRNA levels for glycinergic and glutamatergic ionotropic receptor subunits, confirming a switch from Glyα2 to Glyα1 for IPSCs and increased expression of GluA3 and GluA4 subunits for EPSCs. The NMDA receptor (NMDAR)-EPSC decay τ accelerated from >40 ms in prehearing animals to 2.6 ± 0.4 ms in adults, as GluN2C expression increased. In vivo induction of AT at around P20 disrupted IPSC and EPSC integration in the LSO, so that 1 week later the AMPA receptor (AMPAR)-EPSC decay was slowed and mRNA for GluA1 increased while GluA4 decreased. In contrast, GlyR IPSC and NMDAR-EPSC decay times were unchanged. Computational modelling confirmed that matched IPSC and EPSC kinetics are required to generate mature interaural level difference functions, and that longer-lasting EPSCs compensate to maintain binaural function with raised auditory thresholds after AT. We conclude that LSO excitatory and inhibitory synaptic drive matures to identical time-courses, that AT changes synaptic AMPARs by expression of subunits with slow kinetics (which recover over 2 months) and that loud sounds reversibly modify excitatory synapses in the brain, changing synaptic function for several weeks after exposure., (© 2016 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Potassium channels participate in many biological functions, from ion homeostasis to generation and modulation of the electrical membrane potential. They are involved in a large variety of diseases. In the human genome, 15 genes code for K(+) channels with two pore domains (K2P ). These channels form dimers of pore-forming subunits that produce background conductances finely regulated by a range of natural and chemical effectors, including signalling lipids, temperature, pressure, pH, antidepressants and volatile anaesthetics. Since the cloning of TWIK1, the prototypical member of this family, a lot of work has been carried out on their structure and biology. These studies are still in progress, but data gathered so far show that K2P channels are central players in many processes, including ion homeostasis, hormone secretion, cell development and excitability. A growing number of studies underline their implication in physiopathological mechanisms, such as vascular and pulmonary hypertension, cardiac arrhythmias, nociception, neuroprotection and depression. This review gives a synthetic view of the most noticeable features of these channels., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

The past fifteen years has seen a revolution in our understanding of ionotropic glutamate receptor (iGluR) structure, starting with the first view of the ligand binding domain (LBD) published in 1998, and in many ways culminating in the publication of the full-length structure of GluA2 in 2009. These reports have revealed not only the central role played by subunit interfaces in iGluR function, but also myriad binding sites within interfaces for endogenous and exogenous factors. Changes in the conformation of inter-subunit interfaces are central to transmission of ligand gating into pore opening (itself a rearrangement of interfaces), and subsequent closure through desensitization. With the exception of the agonist binding site, which is located entirely within individual subunits, almost all modulatory factors affecting iGluRs appear to bind to sites in subunit interfaces. This review seeks to summarize what we currently understand about the diverse roles interfaces play in iGluR function, and to highlight questions for future research., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

During the past decade, considerable evidence has accumulated that non-NMDA glutamate receptors (both AMPA and kainate subtypes) are modulated by the association of the core tetrameric receptor with auxiliary proteins that are integral components of native receptor assemblies. This short review focuses on the effect of two types of auxiliary subunits on the biophysical properties and kinetic behaviour of AMPA and kainate receptors at the level of single receptor molecules. Type I transmembrane AMPA receptor proteins increase the number of AMPA receptor openings that result from a single receptor activation as well as the proportion of openings to conductance levels above 30 pS, resulting in larger peak ensemble currents that decay more slowly and bi-exponentially. Co-expression of Neto1 and 2 with pore-forming kainate receptor subunits also increases the duration of bursts and destabilizes desensitized states, resulting in a rapid component of recovery and clusters of bursts that produce a slow component in desensitization decays. The distinct gating seen in the presence of auxiliary subunits reflects slow switching between gating modes with different single-channel kinetics and open probability. At any given time, the relative proportions of receptors in each gating mode determine both the shape and the amplitude of synaptic currents., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

NMDA receptors (NMDARs) are a class of ionotropic glutamate receptors (iGluRs) that are essential for neuronal development, synaptic plasticity, learning and cell survival. Several features distinguish NMDARs from other iGluRs and underlie the crucial roles NMDARs play in nervous system physiology. NMDARs display slow deactivation kinetics, are highly Ca(2+) permeable, and require depolarization to relieve channel block by external Mg(2+) ions, thereby making them effective coincidence detectors. These properties and others differ among NMDAR subtypes, which are defined by the subunits that compose the receptor. NMDARs, which are heterotetrameric, commonly are composed of two GluN1 subunits and two GluN2 subunits, of which there are four types, GluN2A-D. 'Diheteromeric' NMDARs contain two identical GluN2 subunits. Gating and ligand-binding properties (e.g. deactivation kinetics) and channel properties (e.g. channel block by Mg(2+)) depend strongly on the GluN2 subunit contained in diheteromeric NMDARs. Recent work shows that two distinct regions of GluN2 subunits control most diheteromeric NMDAR subtype-dependent properties: the N-terminal domain is responsible for most subtype dependence of gating and ligand-binding properties; a single residue difference between GluN2 subunits at a site termed the GluN2 S/L site is responsible for most subtype dependence of channel properties. Thus, two structurally and functionally distinct regions underlie the majority of subtype dependence of NMDAR properties. This topical review highlights recent studies of recombinant diheteromeric NMDARs that uncovered the involvement of the N-terminal domain and of the GluN2 S/L site in the subtype dependence of NMDAR properties., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

Previous attempts to measure the functional properties of recombinant nicotinic acetylcholine receptors (nAChRs) composed of known receptor subunits have yielded conflicting results. The use of knockout mice that lack α5, β2, α5β2 or α5β2α7 nAChR subunits enabled us to measure the single-channel properties of distinct α3β4, α3β4α5 and α3β4β2 receptors in superior cervical ganglion (SCG) neurons. Using this approach, we found that α3β4 receptors had a principal conductance level of 32.6 ± 0.8 pS (mean ± SEM) and both higher and lower secondary conductance levels. α3β4α5 receptors had the same conductance as α3β4 receptors, but differed from α3β4 receptors by having an increased channel open time and increased burst duration. By contrast, α3β4β2 receptors differed from α3β4 and α3β4α5 receptors by having a significantly smaller conductance level (13.6 ± 0.5 pS). After dissecting the single-channel properties of these receptors using our knockout models, we then identified these properties - and hence the receptors themselves - in wild-type SCG neurons. This study is the first to identify the single-channel properties of distinct neuronal nicotinic receptors in their native environment.

Dystonia is characterized by excessive involuntary and prolonged simultaneous contractions of both agonist and antagonist muscles. Although the basal ganglia have long been proposed as the primary region, recent studies indicated that the cerebellum also plays a key role in the expression of dystonia. One hereditary form of dystonia, rapid-onset dystonia with parkinsonism (RDP), is caused by loss of function mutations of the gene for the Na pump α3 subunit (ATP1A3). Little information is available on the affected brain regions and mechanism for dystonia by the mutations in RDP. The Na pump is composed of α and β subunits and maintains ionic gradients of Na(+) and K(+) across the cell membrane. The gradients are utilized for neurotransmitter reuptake and their alteration modulates neural excitability. To provide insight into the molecular aetiology of RDP, we generated and analysed knockout heterozygous mice (Atp1a3(+/-)). Atp1a3(+/-) showed increased symptoms of dystonia that is induced by kainate injection into the cerebellar vermis. Atp1a3 mRNA was highly expressed in Purkinje cells and molecular-layer interneurons, and its product was concentrated at Purkinje cell soma, the site of abundant vesicular γ-aminobutyric acid transporter (VGAT) signal, suggesting the presynaptic localization of the α3 subunit in the inhibitory synapse. Electrophysiological studies showed that the inhibitory neurotransmission at molecular-layer interneuron-Purkinje cell synapses was enhanced in Atp1a3(+/-) cerebellar cortex, and that the enhancement originated via a presynaptic mechanism. Our results shed light on the role of Atp1a3 in the inhibitory synapse, and potential involvement of inhibitory synaptic dysfunction for the pathophysiology of dystonia.

The T1R1 receptor subunit acts as an umami taste receptor in combination with its partner, T1R3. In addition, metabotropic glutamate receptors (brain and taste variants of mGluR1 and mGluR4) are thought to function as umami taste receptors. To elucidate the function of T1R1 and the contribution of mGluRs to umami taste detection in vivo, we used newly developed knock-out (T1R1(-/-)) mice, which lack the entire coding region of the Tas1r1 gene and express mCherry in T1R1-expressing cells. Gustatory nerve recordings demonstrated that T1R1(-/-) mice exhibited a serious deficit in inosine monophosphate-elicited synergy but substantial residual responses to glutamate alone in both chorda tympani and glossopharyngeal nerves. Interestingly, chorda tympani nerve responses to sweeteners were smaller in T1R1(-/-) mice. Taste cell recordings demonstrated that many mCherry-expressing taste cells in T1R1(+/-) mice responded to sweet and umami compounds, whereas those in T1R1(-/-) mice responded to sweet stimuli. The proportion of sweet-responsive cells was smaller in T1R1(-/-) than in T1R1(+/-) mice. Single-cell RT-PCR demonstrated that some single mCherry-expressing cells expressed all three T1R subunits. Chorda tympani and glossopharyngeal nerve responses to glutamate were significantly inhibited by addition of mGluR antagonists in both T1R1(-/-) and T1R1(+/-) mice. Conditioned taste aversion tests demonstrated that both T1R1(-/-) and T1R1(+/-) mice were equally capable of discriminating glutamate from other basic taste stimuli. Avoidance conditioned to glutamate was significantly reduced by addition of mGluR antagonists. These results suggest that T1R1-expressing cells mainly contribute to umami taste synergism and partly to sweet sensitivity and that mGluRs are involved in the detection of umami compounds.

Linkage of certain neurological diseases to Na(+) pump mutations and some mood disorders to altered Na(+) pump function has renewed interest in brain Na(+) pumps. We tested nanomolar ouabain on Ca(2+) signalling (fura-2) in rat hippocampal neurone-astrocyte co-cultures. The neurones and astrocytes express Na(+) pumps with a high-ouabain-affinity catalytic subunit (α3 and α2, respectively); both also express pumps with a ouabain-resistant α1 subunit. Neurones and astrocytes were identified by immunocytochemistry and by stimulation; 3-4 μM L-glutamate (Glu) and 3 μM carbachol (CCh) evoked rapid Ca(2+) transients only in neurones, and small, delayed transients in some astrocytes, whereas 0.5-1 μM ATP evoked Ca(2+) transients only in astrocytes. Both cell types responded to 5-10 μM Glu or ATP. The signals evoked by 3-4 μM Glu in neurones were markedly inhibited by 3-10 μm MPEP (blocks metabotropic glutamate receptor mGluR5) and 10 μm LY341495 (non-selective mGluR blocker), but not by 80 μm AP5 (NMDA receptor blocker) or by selective block of mGluR1 or mGluR2. Pre-incubation (0.5-10 min) with 1-10 nm ouabain (EC50 < 1 nm) augmented Glu- and CCh-evoked signals in neurones. This augmentation was abolished by a blocker of the Na(+)-Ca(2+) exchanger, SEA0400 (300 nm). Ouabain (3 nm) pre-incubation also augmented 10 μM cyclopiazonic acid plus 10 mm caffeine-evoked release of Ca(2+) from the neuronal endoplasmic reticulum (ER). The implication is that nanomolar ouabain inhibits α3 Na(+) pumps, increases (local) intracellular Na(+), and promotes Na(+)-Ca(2+) exchanger-mediated Ca(2+) gain and increased storage in the adjacent ER. Ouabain (3 nm) also increased ER Ca(2+) release and enhanced 0.5 μM ATP-evoked transients in astrocytes; these effects were mediated by α2 Na(+) pumps. Thus, nanomolar ouabain may strongly influence synaptic transmission in the brain as a result of its actions on the high-ouabain-affinity Na(+) pumps in both neurones and astrocytes. The significance of these effects is heightened by the evidence that ouabain is endogenous in mammals.

Activation of N-methyl-D-aspartate (NMDA) receptors (NMDARs) is a crucial mechanism underlying the development and maintenance of pain. Traditionally, the role of NMDARs in the pathogenesis of pain is ascribed to their activation and signalling cascades in postsynaptic neurons. In this study, we determined if presynaptic NMDARs in the primary afferent central terminals play a role in synaptic plasticity of the spinal first sensory synapse in a rat model of neuropathic pain induced by spinal nerve ligation. Excitatory postsynaptic currents (EPSCs) were recorded from superficial dorsal horn neurons of spinal slices taken from young adult rats. We showed that increased glutamate release from the primary afferents contributed to the enhanced amplitudes of EPSCs evoked by input from the primary afferents in neuropathic rats. Endogenous activation of presynaptic NMDARs increased glutamate release from the primary afferents in neuropathic rats. Presynaptic NMDARs in neuropathic rats were mainly composed of NR2B receptors. The action of presynaptic NMDARs in neuropathic rats was enhanced by exogenous D-serine and/or NMDA and dependent on activation of protein kinase C. In contrast, glutamate release from the primary afferents in sham-operated rats was not regulated by presynaptic NMDARs. We demonstrated that the lack of NMDAR-mediated regulation of glutamate release in sham-operated rats was not attributable to low extracellular levels of the NMDAR agonist and/or coagonist (D-serine), but rather was due to the insufficient function and/or number of presynaptic NMDARs. This was supported by an increase of NR2B receptor protein expression in both the dorsal root ganglion and spinal dorsal horn ipsilateral to the injury site in neuropathic rats. Hence, suppression of the presynaptic NMDAR activity in the primary sensory afferents is an effective approach to attenuate the enhanced glutamatergic response in the spinal first sensory synapse induced by peripheral nerve injury, and presynaptic NMDARs might be a novel target for the development of analgesics.

The output of the cerebellum to the motor axis of the central nervous system is orchestrated mainly by synaptic inputs and intrinsic pacemaker activity of deep cerebellar nuclear (DCN) projection neurons. Herein, we demonstrate that the soma of these cells is enriched with K(V)1 channels produced by mandatory multi-merization of K(V)1.1, 1.2 α and KV β2 subunits. Being constitutively active, the K(+) current (IK(V)1) mediated by these channels stabilizes the rate and regulates the temporal precision of self-sustained firing of these neurons. Placed strategically, IK(V)1 provides a powerful counter-balance to prolonged depolarizing inputs, attenuates the rebound excitation, and dampens the membrane potential bi-stability. Somatic location with low activation threshold render IK(V)1 instrumental in voltage-dependent de-coupling of the axon initial segment from the cell body of projection neurons, impeding invasion of back-propagating action potentials into the somato-dendritic compartment. The latter is also demonstrated to secure the dominance of clock-like somatic pacemaking in driving the regenerative firing activity of these neurons, to encode time variant inputs with high fidelity. Through the use of multi-compartmental modelling and retro-axonal labelling, the physiological significance of the described functions for processing and communication of information from the lateral DCN to thalamic relay nuclei is established.

Voltage-dependent calcium channels (VDCCs) serve a wide range of physiological functions and their activity is modulated by different neurotransmitter systems. GABAergic inhibition of VDCCs in neurons has an important impact in controlling transmitter release, neuronal plasticity, gene expression and neuronal excitability. We investigated the molecular signalling mechanisms by which GABA(B) receptors inhibit calcium-mediated electrogenesis (Ca(2+) spikes) in the distal apical dendrite of cortical layer 5 pyramidal neurons. Ca(2+) spikes are the basis of coincidence detection and signal amplification of distal tuft synaptic inputs characteristic for the computational function of cortical pyramidal neurons. By combining dendritic whole-cell recordings with two-photon fluorescence Ca(2+) imaging we found that all subtypes of VDCCs were present in the Ca(2+) spike initiation zone, but that they contribute differently to the initiation and sustaining of dendritic Ca(2+) spikes. Particularly, Ca(v)1 VDCCs are the most abundant VDCC present in this dendritic compartment and they generated the sustained plateau potential characteristic for the Ca(2+) spike. Activation of GABA(B) receptors specifically inhibited Ca(v)1 channels. This inhibition of L-type Ca(2+) currents was transiently relieved by strong depolarization but did not depend on protein kinase activity. Therefore, our findings suggest a novel membrane-delimited interaction of the G(i/o)-βγ-subunit with Ca(v)1 channels identifying this mechanism as the general pathway of GABA(B) receptor-mediated inhibition of VDCCs. Furthermore, the characterization of the contribution of the different VDCCs to the generation of the Ca(2+) spike provides new insights into the molecular mechanism of dendritic computation.

The increased vascular tone that defines essential hypertension is associated with depolarization of vascular smooth muscle cells (VSMCs) and involves a change in the expression profile of ion channels promoting arterial contraction. As a major regulator of VSMC resting membrane potential (V(M)), K(+) channel activity is an important determinant of vascular tone and vessel diameter. However, hypertension-associated changes in the expression and/or modulation of K(+) channels are poorly defined, due to their large molecular diversity and their bed-specific pattern of expression. Moreover, the impact of these changes on the integrated vessel function and their contribution to the development of altered vascular tone under physiological conditions need to be confirmed. Hypertensive (BPH) and normotensive (BPN) mice strains obtained by phenotypic selection were used to explore whether changes in the functional expression of VSMC inward rectifier K(+) channels contribute to the more depolarized resting V(M) and the increased vascular reactivity of hypertensive arteries. We determined the expression levels of inward rectifier K(+) channel mRNA in several vascular beds from BPN and BPH animals, and their functional contribution to VSMC excitability and vascular tone in mesenteric arteries. We found a decrease in the expression of Kir2.1, Kir4.1, Kir6.x and SUR2 mRNA in BPH VSMCs, and a decreased functional contribution of both K(IR) and K(ATP) channels in isolated BPH VSMCs. However, only the effect of K(ATP) channel modulators was impaired when exploring vascular tone, suggesting that decreased functional expression of K(ATP) channels may be an important element in the remodelling of VSMCs in essential hypertension.

Endogenous neurosteroids are among the most potent and efficacious potentiators of activation of GABA(A) receptors. It has been proposed that a conserved glutamine residue in the first membrane-spanning region (TM1 region) of the α subunits is required for binding of potentiating neurosteroids. Mutations of this residue can reduce or remove the ability of steroids to potentiate function. However, it is not known whether potentiation requires that a steroid interact with the α subunit, or not. To examine this question we mutated the homologous residue in the β2 and γ2L subunits to glutamine, and found that these mutations could not confer potentiation by allopregnanolone (3α5αP) when expressed in receptors containing ineffective α1 subunits. However, potentiation is restored when the entire TM1 region from the α1 subunit is transferred to the β2 or γ2L subunit. Mutations in the TM1 region that affect potentiation when made in the α1 subunit have similar effects when made in transferred TM1 region. Further, the effects of 3α5αP on single-channel kinetics are similar for wild-type receptors and receptors with moved TM1 regions. These results support the idea that steroids bind in the transmembrane regions of the receptor. The observations are consistent with previous work indicating that neurosteroid potentiation is mediated by an action that affects the receptor as a whole, rather than an individual subunit or pair of subunits, and in addition demonstrate that the mechanism is independent of the nature of the subunit that interacts with steroid.

Kainate receptors (KARs) are a subfamily of ionotropic glutamate receptors (iGluRs) that mediate excitatory synaptic transmission, regulate neurotransmitter release, and show a remarkably selective distribution in the brain. Compared to other iGluRs, the precise contribution of KARs to brain function is less understood. Unlike recombinant KARs, native KARs exhibit characteristically slow channel kinetics. The underlying explanation for this dissimilar kinetics has remained elusive until recently. New research has identified Neto1 and Neto2 as KAR auxiliary subunits that determine unique properties of synaptic KARs, including their slow kinetics and high affinity for agonist. Whether these auxiliary subunits regulate KAR trafficking and targeting at the synapse is less clear. By regulating channel gating, Neto1 and Neto2 can increase the diversity of KAR functional properties. These auxiliary subunits may represent a starting point for a better understanding of the role played by neuronal KARs under normal and pathological conditions, but also, they may provide an alternative target for the development of new drugs regulating KARs and brain function.

Mutations in Kv3.3 cause spinocerebellar ataxia type 13 (SCA13). Depending on the causative mutation, SCA13 is either a neurodevelopmental disorder that is evident in infancy or a progressive neurodegenerative disease that emerges during adulthood. Previous studies did not clarify the relationship between these distinct clinical phenotypes and the effects of SCA13 mutations on Kv3.3 function. The F448L mutation alters channel gating and causes early-onset SCA13. R420H and R423H suppress Kv3 current amplitude by a dominant negative mechanism. However, R420H results in the adult form of the disease whereas R423H produces the early-onset, neurodevelopmental form with significant clinical overlap with F448L. Since individuals with SCA13 have one wild type and one mutant allele of the Kv3.3 gene, we analysed the properties of tetrameric channels formed by mixtures of wild type and mutant subunits. We report that one R420H subunit and at least one R423H subunit can co-assemble with the wild type protein to form active channels. The functional properties of channels containing R420H and wild type subunits strongly resemble those of wild type alone. In contrast, channels containing R423H and wild type subunits show significantly altered gating, including a hyperpolarized shift in the voltage dependence of activation, slower activation, and modestly slower deactivation. Notably, these effects resemble the modified gating seen in channels containing a mixture of F448L and wild type subunits, although the F448L subunit slows deactivation more dramatically than the R423H subunit. Our results suggest that the clinical severity of R423H reflects its dual dominant negative and dominant gain of function effects. However, as shown by R420H, reducing current amplitude without altering gating does not result in infant onset disease. Therefore, our data strongly suggest that changes in Kv3.3 gating contribute significantly to an early age of onset in SCA13.

Human embryonic stem cells (hESCs) are an important cellular model for studying ion channel function in the context of a human cardiac cell and will provide a wealth of information about both heritable arrhythmias and acquired electrophysiological disorders. However, detailed electrophysiological characterization of the important cardiac ion channels has been so far overlooked. Because mutations in the gene for the I(Ks) α subunit, KCNQ1, constitute the majority of long QT syndrome (LQT-1) cases, we have carried out a detailed biophysical analysis of this channel expressed in hESCs to establish baseline I(Ks) channel biophysical properties in cardiac myocytes derived from hESCs (hESC-CMs). I(Ks) channels are heteromultimeric proteins consisting of four identical α-subunits (KCNQ1) assembled with auxiliary β-subunits (KCNE1). We found that the half-maximal I(Ks) activation voltage in hESC-CMs and in myocytes derived from human induced pluripotent stems cells (hiPSC-CMs) falls between that of KCNQ1 channels expressed alone and with full complement of KCNE1, the major KCNE subunit expressed in hESC-CMs as shown by qPCR analysis. Overexpression of KCNE1 by transfection of hESC-CMs markedly shifted and slowed native I(Ks) activation implying assembly of additional KCNE1 subunits with endogenous channels. Our results in hESC-CMs, which indicate an I(Ks) subunit stoichiometry that can be altered by variable KCNE1 expression, suggest the possibility for variable I(Ks) function in the developing heart, in different tissues in the heart, and in disease. This establishes a new baseline for I(Ks) channel properties in myocytes derived from pluripotent stem cells and will guide future studies in patient-specific hiPSCs.

A GABA(A) receptor α6 subunit mutation, R46W, was identified as a susceptibility gene that may contribute to the pathogenesis of childhood absence epilepsy (CAE), but the molecular basis for alteration of GABA(A) receptor function is unclear. The R46W mutation is located in a region homologous to a GABA(A) receptor γ2 subunit missense mutation, R82Q, that is associated with CAE and febrile seizures in humans. To determine how this mutation reduces GABAergic inhibition, we expressed wild-type (α6β2γ2L and α6β2δ) and mutant (α6(R46W)β2γ2L and α6(R46W)β2δ) receptors in HEK 293T cells and characterize their whole-cell and single-channel currents, and surface and total levels. We demonstrated that gating and assembly of both α6(R46W)β2γ2L and α6(R46W)β2δ receptors were impaired. Compared to wild-type currents, α6(R46W)β2γ2L and α6(R46W)β2δ receptors had a reduced current density, α6(R46W)β2γ2L currents desensitized to a greater extent and deactivated at a slower rate, α6(R46W)β2δ receptors did not desensitize but deactivated faster and both α6(R46W)β2γ2L and α6(R46W)β2δ single-channel current mean open times and burst durations were reduced. Surface levels of coexpressed α6(R46W), β2 and δ, but not γ2L, subunits were decreased. 'Heterozygous' coexpression of α6(R46W) and α6 subunits with β2 and γ2L subunits produced intermediate macroscopic current amplitudes by increasing incorporation of wild-type and decreasing incorporation of mutant subunits into receptors trafficked to the surface. Finally, these findings suggest that similar to the γ2(R82Q) mutation, the CAE-associated α6(R46W) mutation could cause neuronal disinhibition and thus increase susceptibility to generalized seizures through a reduction of αβγ and αβδ receptor function and expression.

Ca(2+) influx via Ca(V)1/Ca(V)2 channels drives processes ranging from neurotransmission to muscle contraction. Association of a pore-forming α(1) and cytosolic β is necessary for trafficking Ca(V)1/Ca(V)2 channels to the cell surface through poorly understood mechanisms. A prevalent idea suggests β binds the α(1) intracellular I-II loop, masking an endoplasmic reticulum (ER) retention signal as the dominant mechanism for Ca(V)1/Ca(V)2 channel membrane trafficking. There are hints that other α(1) subunit cytoplasmic domains may play a significant role, but the nature of their potential contribution is unclear. We assessed the roles of all intracellular domains of Ca(V)1.2-α(1C) by generating chimeras featuring substitutions of all possible permutations of intracellular loops/termini of α(1C) into the β-independent Ca(V)3.1-α(1G) channel. Surprisingly, functional analyses demonstrated α(1C) I-II loop strongly increases channel surface density while other cytoplasmic domains had a competing opposing effect. Alanine-scanning mutagenesis identified an acidic-residue putative ER export motif responsible for the I-II loop-mediated increase in channel surface density. β-dependent increase in current arose as an emergent property requiring four α(1C) intracellular domains, with the I-II loop and C-terminus being essential. The results suggest β binding to the α(1C) I-II loop causes a C-terminus-dependent rearrangement of intracellular domains, shifting a balance of power between export signals on the I-II loop and retention signals elsewhere.

We investigated the effects of the overexpression of two enhanced green fluorescent protein (EGFP)-tagged α1sDHPR variants on Ca2+ currents (ICa), charge movements (Q) and SR Ca2+ release of muscle fibres isolated from adult mice. Flexor digitorum brevis (FDB)muscles were transfected by in vivo electroporation with plasmids encoding for EGFP-α1sDHPR-wt and EGFP-α1sDHPR-T935Y (an isradipine-insensitive mutant). Two-photon laser scanning microscopy (TPLSM) was used to study the subcellular localization of transgenic proteins, while ICa, Q and Ca2+ release were studied electrophysiologically and optically under voltage-clamp conditions. TPLSM images demonstrated that most of the transgenic α1sDHPR was correctly targeted to the transverse tubular system (TTS). Immunoblotting analysis of crude extracts of transfected fibres demonstrated the synthesis of bona fide transgenic EGFP-α1sDHPR-wt in quantities comparable to that of native α1sDHPR. Though expression of both transgenic variants of the alpha subunit of the dihydropyridine receptor (α1sDHPR) resulted in ∼50% increase in Q, they surprisingly had no effect on the maximal Ca2+ conductance (gCa) nor the SR Ca2+ release. Nonetheless, fibres expressing EGFP-α1sDHPR-T935Y exhibited up to 70% isradipine-insensitive ICa (ICa-ins) with a right-shifted voltage dependence compared to that in control fibres. Interestingly, Qand SRCa2+ release also displayed right-shifted voltage dependence in fibres expressing EGFP-α1sDHPR-T935Y. In contrast, the midpoints of the voltage dependence of gCa, Q and Ca2+ release were not different from those in control fibres and in fibres expressing EGFP-α1sDHPR-wt. Overall, our results suggest that transgenic α1sDHPRs are correctly trafficked and inserted in the TTS membrane, and that a substantial fraction of the mworks as conductive Ca2+ channels capable of physiologically controlling the release of Ca2+ from the SR. A plausible corollary of this work is that the expression of transgenic variants of the α1sDHPR leads to the replacement of native channels interacting with the ryanodine receptor 1 (RyR1), thus demonstrating the feasibility of molecular remodelling of the triads in adult skeletal muscle fibres.

Cerebral vascular smooth muscle contractility plays a crucial role in controlling arterial diameter and, thereby, blood flow regulation in the brain. A number of K(+) channels have been suggested to contribute to the regulation of diameter by controlling smooth muscle membrane potential (E(m)) and Ca(2+) influx. Previous studies indicate that stromatoxin (ScTx1)-sensitive, Kv2-containing channels contribute to the control of cerebral arterial diameter at 80 mmHg, but their precise role and molecular composition were not determined. Here, we tested if Kv2 subunits associate with 'silent' subunits from the Kv5, Kv6, Kv8 or Kv9 subfamilies to form heterotetrameric channels that contribute to control of diameter of rat middle cerebral arteries (RMCAs) over a range of intraluminal pressure from 10 to 100 mmHg. The predominant mRNAs expressed by RMCAs encode Kv2.1 and Kv9.3 subunits. Co-localization of Kv2.1 and Kv9.3 proteins at the plasma membrane of dissociated single RMCA myocytes was detected by proximity ligation assay. ScTx1-sensitive native current of RMCA myocytes and Kv2.1/Kv9.3 currents exhibited functional identity based on the similarity of their deactivation kinetics and voltage dependence of activation that were distinct from those of homomultimeric Kv2.1 channels. ScTx1 treatment enhanced the myogenic response of pressurized RMCAs between 40 and 100 mmHg, but this toxin also caused constriction between 10 and 40 mmHg that was not previously observed following inhibition of large conductance Ca(2+)-activated K(+) (BK(Ca)) and Kv1 channels. Taken together, this study defines the molecular basis of Kv2-containing channels and contributes to our understanding of the functional significance of their expression in cerebral vasculature. Specifically, our findings provide the first evidence of heteromultimeric Kv2.1/Kv9.3 channel expression in RMCA myocytes and their distinct contribution to control of cerebral arterial diameter over a wider range of E(m) and transmural pressure than Kv1 or BK(Ca) channels owing to their negative range of voltage-dependent activation.

KCNQ gene expression was previously shown in various rodent blood vessels, where the products of KCNQ4 and KCNQ5, Kv7.4 and Kv7.5 potassium channel subunits, respectively, have an influence on vascular reactivity. The aim of this study was to determine if small cerebral resistance arteries of the rat express KCNQ genes and whether Kv7 channels participate in the regulation of myogenic control of diameter. Quantitative reverse transcription polymerase chain reaction (QPCR) was undertaken using RNA isolated from rat middle cerebral arteries (RMCAs) and immunocytochemistry was performed using Kv7 subunit-specific antibodies and freshly isolated RMCA myocytes. KCNQ4 message was more abundant than KCNQ5 = KCNQ1, but KCNQ2 and KCNQ3 message levels were negligible. Kv7.1, Kv7.4 and Kv7.5 immunoreactivity was present at the sarcolemma of freshly isolated RMCA myocytes. Linopirdine (1 microm) partially depressed, whereas the Kv7 activator S-1 (3 and/or 20 microm) enhanced whole-cell Kv7.4 (in HEK 293 cells), as well as native RMCA myocyte Kv current amplitude. The effects of S-1 were voltage-dependent, with progressive loss of stimulation at potentials of >15 mV. At the concentrations employed linopirdine and S-1 did not alter currents due to recombinant Kv1.2/Kv1.5 or Kv2.1/Kv9.3 channels (in HEK 293 cells) that are also expressed by RMCA myocytes. In contrast, another widely used Kv7 blocker, XE991 (10 microm), significantly attenuated native Kv current and also reduced Kv1.2/Kv1.5 and Kv2.1/Kv9.3 currents. Pressurized arterial myography was performed using RMCAs exposed to intravascular pressures of 10-100 mmHg. Linopirdine (1 microm) enhanced the myogenic response at 20 mmHg, whereas the activation of Kv7 channels with S-1 (20 microm) inhibited myogenic constriction at >20 mmHg and reversed the increased myogenic response produced by suppression of Kv2-containing channels with 30 nm stromatoxin (ScTx1). These data reveal a novel contribution of KCNQ gene products to the regulation of myogenic control of cerebral arterial diameter and suggest that Kv7 channel activating drugs may be appropriate candidates for the development of an effective therapy to ameliorate cerebral vasospasm.

Kainate receptor subunits can form functional channels as homomers of GluK1, GluK2 or GluK3, or as heteromeric combinations with each other or incorporating GluK4 or GluK5 subunits. However, GluK4 and GluK5 cannot form functional channels by themselves. Incorporation of GluK4 or GluK5 into a heteromeric complex increases glutamate apparent affinity and also enables receptor activation by the agonist AMPA. Utilizing two-electrode voltage clamp of Xenopus oocytes injected with cRNA encoding kainate receptor subunits, we have observed that heteromeric channels composed of GluK2/GluK4 and GluK2/GluK5 have steady state concentration-response curves that were bell-shaped in response to either glutamate or AMPA. By contrast, homomeric GluK2 channels exhibited a monophasic steady state concentration-response curve that simply plateaued at high glutamate concentrations. By fitting several specific Markov models to GluK2/GluK4 heteromeric and GluK2 homomeric concentration-response data, we have determined that: (a) two strikingly different agonist binding affinities exist; (b) the high-affinity binding site leads to channel opening; and (c) the low-affinity agonist binding site leads to strong desensitization after agonist binding. Model parameters also approximate the onset and recovery kinetics of desensitization observed for macroscopic currents measured from HEK-293 cells expressing GluK2 and GluK4 subunits. The GluK2(E738D) mutation lowers the steady state apparent affinity for glutamate by 9000-fold in comparison to GluK2 homomeric wildtype receptors. When this mutant subunit was expressed with GluK4, the rising phase of the glutamate steady state concentration-response curve overlapped with the wildtype curve, whereas the declining phase was right-shifted toward lower affinity. Taken together, these data are consistent with a scheme whereby high-affinity agonist binding to a non-desensitizing GluK4 subunit opens the heteromeric channel, whereas low-affinity agonist binding to GluK2 desensitizes the whole channel complex.

Voltage-gated proton channels have been described in different cells and organisms since the early '80s, but the first member of the family, Hv1, was cloned only recently. The Hv1 channel was found to contain a voltage-sensing domain (VSD), similar to those of voltage-gated sodium, potassium and calcium channels. All these other channels also contain a pore domain, which forms a central pore at the interface of the four subunits. The pore domain is missing in Hv1. This raised several questions on the location of the proton pore and on the mechanism of gating. Here, we briefly review our effort to understand the structural organization of Hv1 channels and discuss the relationship between the gating of Hv1 and the gating of ion-conducting pores recently discovered in the VSDs of mutant voltage-gated potassium and sodium channels.

As a major inhibitory neurotransmitter, GABA plays a vital role in the brain by controlling the extent of neuronal excitation. This widespread role is reflected by the ubiquitous distribution of GABA(A) receptors throughout the central nervous system. To regulate the level of neuronal inhibition requires some endogenous control over the release of GABA and/or its postsynaptic response. In this context, Ca(2+) ions are often used as primary or secondary messengers frequently resulting in the activation of protein kinases and phosphatases. One such kinase, Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), can target the GABA(A) receptor to cause its phosphorylation. Evidence is now emerging, which is reviewed here, that GABA(A) receptors are indeed substrates for CaMKII and that this covalent modification alters the expression of cell surface receptors and their function. This type of regulation can also feature at inhibitory synapses leading to long-term inhibitory synaptic plasticity. Most recently, CaMKII has now been proposed to differentially phosphorylate particular isoforms of GABA(A) receptors in a synapse-specific context.

Dopaminergic (DAergic) neuronal activity in the ventral tegmental area (VTA) is thought to contribute generally to pleasure, reward, and drug reinforcement and has been implicated in nicotine dependence. nAChRs expressed in the VTA exhibit diverse subunit compositions, but the functional and pharmacological properties are largely unknown. Here, using patch-clamp recordings in single DAergic neurons freshly dissociated from rat VTA, we clarified three functional subtypes of nAChRs (termed ID, IID and IIID receptors) based on whole-cell current kinetics and pharmacology. Kinetic analysis demonstrated that comparing to ID, IID receptor-mediated current had faster activation and decay constant and IIID receptor-mediated current had larger current density. Pharmacologically, ID receptor-mediated current was sensitive to the alpha4beta2-nAChR agonist RJR-2403 and antagonist dihydro-beta-erythroidine (DHbetaE); IID receptor-mediated current was sensitive to the selective alpha7-nAChR agonist choline and antagonist methyllycaconitine (MLA); while IIID receptor-mediated current was sensitive to the beta4-containing nAChR agonist cytisine and antagonist mecamylamine (MEC). The agonist concentration-response relationships demonstrated that IID receptor-mediated current exhibited the highest EC(50) value compared to ID and IIID receptors, suggesting a relatively low agonist affinity of type IID receptors. These results suggest that the type ID, IID and IIID nAChR-mediated currents are predominately mediated by activation of alpha4beta2-nAChR, alpha7-nAChR and a novel nAChR subtype(s), respectively. Collectively, these findings indicate that the VTA DAergic neurons express diversity and multiplicity of functional nAChR subtypes. Interestingly, each DAergic neuron predominantly expresses only one particularly functional nAChR subtype, which may have distinct but important roles in regulation of VTA DA neuronal function, DA transmission and nicotine dependence.

The NMDA-type glutamate receptor is a heteromeric complex composed of the NR1 and at least one of the NR2 subunits. Switching from the NR2B to the NR2A subunit is thought to underlie functional alteration of the NMDA receptor during synaptic maturation, and it is generally believed that it results in preferential localization of NR2A subunits on the synaptic site and that of NR2B subunits on the extracellular site in the mature brain. It has also been proposed that activation of the NR2A and NR2B subunits results in long-term potentiation (LTP) and long-term depression (LTD), respectively. Furthermore, recent reports suggest that synaptic and extrasynaptic receptors may have distinct roles in synaptic plasticity as well as in gene expression associated with neuronal death. Here, we have investigated whether NR2B subunit-containing receptors are present and functional at mature synapses in the lateral nucleus of the amygdala (LA) and the CA1 region of the hippocampus, comparing their properties between the two brain regions. We have found, in contrast to the above hypotheses, that the NR2B subunit significantly contributes to synaptic transmission as well as LTP induction. Furthermore, its contribution is greater in the LA than in the CA1 region, and biophysical properties of NMDA receptors and the NR2B/NR2A ratio are different between the two brain regions. These results indicate that NR2B subunit-containing NMDA receptors accumulate on the synaptic site and are responsible for the unique properties of synaptic function and plasticity in the amygdala.

Inwardly rectifying potassium (Kir) channels are gated by the interaction of their cytoplasmic regions with membrane-bound phosphatidylinositol-4,5-bisphosphate (PIP(2)). In the present study, we examined how PIP(2) interaction regulates channel availability and channel openings to various subconductance levels (sublevels) as well as the fully open state in the strong inward rectifier Kir2.1 channel. Various Kir2.1 channel constructs were expressed in Xenopus oocytes and single channel or macroscopic currents were recorded from inside-out patches. The wild-type (WT) channel rarely visited the subconductance levels under control conditions. However, upon reducing Kir2.1 channel interaction with PIP(2) by a variety of interventions, including PIP(2) antibodies, screening PIP(2) with neomycin, or mutating PIP(2) binding sites (e.g. K188Q), visitation to the sublevels was markedly increased before channels were converted to an unavailable mode in which they did not open. No channel activity was detected in channels with the double mutation K188A/R189A, a mutant which exhibits extremely weak interaction with PIP(2). By linking subunits together in tandem dimers or tetramers containing mixtures of WT and K188A/R189A subunits, we demonstrate that one functional PIP(2)-interacting WT subunit is sufficient to convert channels from the unavailable to the available mode with a high open probability dominated by the fully open state, with similar kinetics as tetrameric WT channels. Occasional openings to sublevels become progressively less frequent as the number of WT subunits increases. Quantitative analysis reveals that the interaction of PIP(2) with WT subunits exerts strong positive cooperativity in both converting the channels from the unavailable to the available mode, and in promoting the fully open state over sublevels. We conclude that the interaction of PIP(2) with only one Kir2.1 subunit is sufficient for the channel to become available and to open to its full conductance state. Interaction with additional subunits exerts positive cooperativity at multiple levels to further enhance channel availability and promote the fully open state.

Reconciling ion channel alpha-subunit expression with native ionic currents and their pharmacological sensitivity in target organs has proved difficult. In native tissue, many K(+) channel alpha-subunits co-assemble with ancillary subunits, which can profoundly affect physiological parameters including gating kinetics and pharmacological interactions. In this review, we examine the link between voltage-gated potassium ion channel pharmacology and the biophysics of ancillary subunits. We propose that ancillary subunits can modify the interaction between pore blockers and ion channels by three distinct mechanisms: changes in (1) binding site accessibility; (2) orientation of pore-lining residues; (3) the ability of the channel to undergo post-binding conformational changes. Each of these subunit-induced changes has implications for gating, drug affinity and use dependence of their respective channel complexes. A single subunit may modulate its associated alpha-subunit by more than one of these mechanisms. Voltage-gated potassium channels are the site of action of many therapeutic drugs. In addition, potassium channels interact with drugs whose primary target is another channel, e.g. the calcium channel blocker nifedipine, the sodium channel blocker quinidine, etc. Even when K(+) channel block is the intended mode of action, block of related channels in non-target organs, e.g. the heart, can result in major and potentially lethal side-effects. Understanding factors that determine specificity, use dependence and other properties of K(+) channel drug binding are therefore of vital clinical importance. Ancillary subunits play a key role in determining these properties in native tissue, and so understanding channel-subunit interactions is vital to understanding clinical pharmacology.

Long-term depression (LTD) of excitatory transmission at cerebellar parallel fibre-Purkinje cell synapses is a form of synaptic plasticity crucial for cerebellar motor learning. Around the postsynaptic membrane of these synapses, B-type gamma-aminobutyric acid receptor (GABABR), a Gi/o protein-coupled receptor for the inhibitory transmitter GABA is concentrated and closely associated with type-1 metabotropic glutamate receptors (mGluR1) whose signalling is a key factor for inducing LTD. We found that in cultured Purkinje cells, GABABR activation enhanced LTD of a glutamate-evoked current (LTDglu), increasing the magnitude of depression. It has been reported that parallel fibre-Purkinje cell synapses receive a micromolar level of GABA spilled over from the synaptic terminals of the neighbouring GABAergic interneurons. This level of GABA was able to enhance LTDglu. Our pharmacological analyses revealed that the betagamma subunits but not the alpha subunit of Gi/o protein mediated GABABR-mediated LTDglu enhancement. Gi/o protein activation was sufficient to enhance LTDglu. In this respect, LTDglu enhancement is clearly distinguished from the previously reported GABABR-mediated augmentation of an mGluR1-coupled slow excitatory postsynaptic potential. Baclofen application for only the induction period of LTDglu was sufficient to enhance LTDglu, suggesting that GABABR signalling may modulate mechanisms underlying LTDglu induction. Baclofen augmented mGluR1-coupled Ca2+ release from the intracellular stores in a Gi/o protein-dependent manner. Therefore, GABABR-mediated LTDglu enhancement is likely to result from augmentation of mGluR1 signalling. Furthermore, pharmacological inhibition of GABABR reduced the magnitude of LTD at parallel fibre-Purkinje cell synapses in cerebellar slices. These findings demonstrate a novel mechanism that would facilitate cerebellar motor learning.

ATP-sensitive potassium (K(ATP)) channels comprise four pore-forming Kir6 and four regulatory sulphonylurea receptor (SUR) subunits. SUR, an ATP-binding cassette protein, associates with Kir6 through its N-terminal transmembrane domain (TMD0). TMD0 connects to the core domain of SUR through a cytosolic linker (L0). The intrinsic gating of Kir6.2 is greatly altered by SUR. It has been hypothesized that these changes are conferred by TMD0. Exploiting the fact that the pancreatic (SUR1/Kir6.2) and the cardiac (SUR2A/Kir6.2) K(ATP) channels show different gating behaviours, we have tested this hypothesis by comparing the intrinsic gating of Kir6.2 with the last 26 residues deleted (Kir6.2Delta26) co-expressed with SUR1, S1-TMD0, SUR2A and S2-TMD0 at -40 and -100 mV (S is an abbreviation for SUR; TMD0/Kir6.2Delta26, but not TMD0/Kir6.2, can exit the endoplastic reticulum and reach the cell membrane). Single-channel kinetic analyses revealed that the mean burst and interburst durations are shorter for TMD0/Kir6.2Delta26 than for the corresponding SUR channels. No differences were found between the two TMD0 channels. We further demonstrated that in isolation even TMD0-L0 (SUR truncated after L0) cannot confer the wild-type intrinsic gating to Kir6.2Delta26 and that swapping L0 (SUR truncated after L0)between SUR1 and SUR2A only partially exchanges their different intrinsic gating. Therefore, in addition to TMD0, L0 and the core domain also participate in determining the intrinsic gating of Kir6.2. However, TMD0 and L0 are responsible for the different gating patterns of full-length SUR1 and SUR2A channels. A kinetic model with one open and four closed states is presented to explain our results in a mechanistic context.

Slow-channel congenital myasthenic syndrome, caused by mutations in subunits of the endplate ACh receptor (AChR), results in prolonged synaptic currents and excitotoxic injury of the postsynaptic region by Ca2+ overloading. The Ca2+ overloading could be due entirely to the prolonged openings of the AChR channel or could be abetted by enhanced Ca2+ permeability of the mutant channels. We therefore measured the fractional Ca2+ current, defined as the percentage of the total ACh-evoked current carried by Ca2+ ions (Pf), for AChRs harbouring the alphaG153S or the alphaV249F slow-channel mutation, and for wild-type human AChRs in which Pf has not yet been determined. Experiments were performed in transiently transfected GH4C1 cells and human myotubes with simultaneous recording of ACh-evoked whole-cell currents and fura-2 fluorescence signals. We found that the Pf of the wild-type human endplate AChR was unexpectedly high (Pf approximately 7%), but neither the alphaV249F nor the alphaG153S mutation altered Pf. Fetal human AChRs containing either the wild-type or the mutated alpha subunit had a much lower Pf (2-3%). We conclude that the Ca2+ permeability of human endplate AChRs is higher than that reported for any other human nicotinic AChR, with the exception of alpha7-containing AChRs (Pf > 10%); and that neither the alphaG153S nor the alphaV249F mutations affect the Pf of fetal or adult endplate AChRs. However, the intrinsically high Ca2+ permeability of human AChRs probably predisposes to development of the endplate myopathy when opening events of the AChR channel are prolonged by altered AChR-channel kinetics.

Heterotrimeric guanine-nucleotide-binding proteins (G proteins) act as molecular switches in signaling pathways by coupling the activation of heptahelical receptors at the cell surface to intracellular responses. In the resting state, the G-protein alpha subunit (Galpha) binds GDP and Gbetagamma. Receptors activate G proteins by catalyzing GTP for GDP exchange on Galpha, leading to a structural change in the Galpha(GTP) and Gbetagamma subunits that allows the activation of a variety of downstream effector proteins. The G protein returns to the resting conformation following GTP hydrolysis and subunit re-association. As the G-protein cycle progresses, the Galpha subunit traverses through a series of conformational changes. Crystallographic studies of G proteins in many of these conformations have provided substantial insight into the structures of these proteins, the GTP-induced structural changes in Galpha, how these changes may lead to subunit dissociation and allow Galpha and Gbetagamma to activate effector proteins, as well as the mechanism of GTP hydrolysis. However, relatively little is known about the receptor-G protein complex and how this interaction leads to GDP release from Galpha. This article reviews the structural determinants of the function of heterotrimeric G proteins in mammalian systems at each point in the G-protein cycle with special emphasis on the mechanism of receptor-mediated G-protein activation. The receptor-G protein complex has proven to be a difficult target for crystallography, and several biophysical and computational approaches are discussed that complement the currently available structural information to improve models of this interaction. Additionally, these approaches enable the study of G-protein dynamics in solution, which is becoming an increasingly appreciated component of all aspects of G-protein signaling.

Synchrony within the thalamocortical system is regulated in part by intranuclear synaptic inhibition within the reticular nucleus (RTN). Inhibitory postsynaptic currents (IPSCs) in RTN neurons are largely characterized by slow decay kinetics that result in powerful and prolonged suppression of spikes. Here we show that some individual RTN neurons are characterized by highly variable mixtures of fast, slow and mixed IPSCs. Heterogeneity arose largely through differences in the contribution of an initial decay component (tau(D) approximately 10 ms) which was insensitive to loreclezole, suggesting involvement of the GABA(A) receptor beta(1) subunit. Single-cell RT-PCR revealed the presence of beta(1) subunit mRNA only in those neurons whose IPSCs were dominated by a rapid and prominent initial decay phase. These data show that brief, beta(1)-dependent, loreclezole-insensitive IPSCs are present in a subpopulation of RTN neurons, and suggest that striking differences in IPSC heterogeneity within single neurons can result from of the presence or absence of a single GABA(A) receptor subunit.

The erg1a (HERG) K+ channel subunit and its N-terminal splice variant erg1b are coexpressed in several tissues and both isoforms have been shown to form heteromultimeric erg channels in heart and brain. The reduction of erg1a current by thyrotropin-releasing hormone (TRH) is well studied, but no comparable data exist for erg1b. Since TRH and TRH receptors are widely expressed in the brain, we have now studied the different TRH effects on the biophysical properties of homomeric rat erg1b as well as heteromeric rat erg1a/1b channels. The erg channels were overexpressed in the clonal somatomammotroph pituitary cell line GH3/B6, which contains TRH receptors and endogenous erg channels. Compared to rerg1a, homomeric rerg1b channels exhibited not only faster deactivation kinetics, but also considerably less steady-state inactivation, and half-maximal activation occurred at about 10 mV more positive potentials. Coexpression of both isoforms resulted in erg currents with intermediate properties concerning the deactivation kinetics, whereas rerg1a dominated the voltage dependence of activation and rerg1b strongly influenced steady-state inactivation. Application of TRH induced a reduction of maximal erg conductance for all tested erg1 currents without effects on the voltage dependence of steady-state inactivation. Nevertheless, homomeric rerg1b channels significantly differed in their response to TRH from rerg1a channels. The TRH-induced shift in the activation curve to more positive potentials, the dramatic slowing of activation and the acceleration of deactivation typical for rerg1a modulation were absent in rerg1b channels. Surprisingly, most effects of TRH on heteromeric rerg1 channels were dominated by the rerg1b subunit.

Adrenal medullary (AM) cells are exposed to high concentrations of cortical hormones, one of which is a ouabain-like substance. Thus, the effects of ouabain on catecholamine secretion and distribution of Na+,K+-ATPase alpha and beta subunits in rat and guinea-pig AM cells were examined using amperometry and immunological techniques. While exposure to 1 microm ouabain did not have a marked effect on resting secretion, it induced an increase in secretion due to mobilization of Ca2+ ions that were stored during a 4 min interval between muscarine applications. Immunocytochemistry revealed that Na+,K+-ATPase alpha1 subunit-like and beta3 subunit-like immunoreactive (IR) materials were distributed ubiquitously at the cell periphery, whereas alpha2- and beta2-like IR materials were present in restricted parts of the cell periphery. The alpha1 and alpha2 subunits were mainly immunoprecipitated from AM preparations by anti-beta3 and anti-beta2 antisera, respectively. Peripheral BODIPY-FL-InsP3 binding sites were localized below membrane domains with alpha2- and beta2-like IR materials. The results indicate that in AM cells, alpha1beta3 isozymes of Na+,K+-ATPase were present ubiquitously in the plasma membrane, while alpha2beta2 isozymes were in the membrane domain closely associated with peripheral Ca2+ store sites. This close association of the alpha2beta2 isozyme with peripheral Ca2+ store sites may account for the facilitation of mobilization-dependent secretion in the presence of 1 microm ouabain.

KCNQ1 (Kv 7.1) alpha-subunits and KCNE1 beta-subunits co-assemble to form channels that conduct the slow delayed rectifier K+ current (IKs) in the heart. Mutations in either subunit cause long QT syndrome (LQTS), an inherited disorder of cardiac repolarization. Here, the functional consequences of the LQTS-associated missense mutation V310I and several nearby residues were determined. Val310 is located at the base of the pore helix of KCNQ1, two residues below the TIGYG signature sequence that defines the K+ selectivity filter. Channels were heterologously expressed in Xenopus laevis oocytes and currents were recorded using the two-microelectrode voltage-clamp technique. V310I KCNQ1 reduced IKs amplitude when co-expressed with wild-type KCNQ1 and KCNE1 subunits. Val310 was also mutated to Gly, Ala or Leu to explore the importance of amino acid side chain volume at this position. Like V310I, V310L KCNQ1 channels gated normally. Unexpectedly, V310G and V310A KCNQ1 channels inactivated strongly and did not close normally in response to membrane hyperpolarization. Based on a homology model of the KCNQ1 channel pore, we speculate that the side group of residue 310 can interact with specific residues in the S5 and S6 domains to alter channel gating. When volume of the side chain is small, the stability of the closed state is disrupted and the extent of channel inactivation is enhanced. We mutated putative interacting residues in S5 and S6 and found that mutant Leu273 and Phe340 channels also can disrupt close states and modify inactivation. Together these findings indicate the importance of a putative pore helix-S5-S6 interaction for normal KCNQ1 channel deactivation and confirm its role in KCNQ1 inactivation. Disturbance of these interactions might underly LQTS associated with KCNQ1 mutant channels.

NR2A and NR2B are the predominant NR2 NMDA receptor subunits expressed in cortex and hippocampus. The relative expression level of NR2A and NR2B is regulated developmentally and these two subunits have been suggested to play distinct roles in long-term synaptic plasticity. We have used patch-clamp recording of recombinant NMDA receptors expressed in HEK293 cells to characterize the activation properties of both NR1/NR2A and NR1/NR2B receptors. Recordings from outside-out patches that contain a single active channel show that NR2A-containing receptors have a higher probability of opening at least once in response to a brief synaptic-like pulse of glutamate than NR2B-containing receptors (NR2A, 0.80; NR2B, 0.56), a higher peak open probability (NR2A, 0.50; NR2B, 0.12), and a higher open probability within an activation (NR2A, 0.67; NR2B, 0.37). Analysis of the sequence of single-channel open and closed intervals shows that both NR2A- and NR2B-containing receptors undergo multiple conformational changes prior to opening of the channel, with at least one of these steps being faster for NR2A than NR2B. These distinct properties produce profoundly different temporal signalling profiles for NR2A- and NR2B-containing receptors. Simulations of synaptic responses demonstrate that at low frequencies typically used to induce long-term depression (LTD; 1 Hz), NR1/NR2B makes a larger contribution to total charge transfer and therefore calcium influx than NR1/NR2A. However, under high-frequency tetanic stimulation (100 Hz; > 100 ms) typically used to induce long-term potentiation (LTP), the charge transfer mediated by NR1/NR2A considerably exceeds that of NR1/NR2B.

The GABA(A) receptors show a large degree of structural heterogeneity, with seven different subunit families, and 16 different subtypes in mammalian species. The alpha family is the largest, with six different subtypes. The alpha1 and alpha6 subtypes are among the most diverse within this family and confer distinct pharmacological properties to recombinant and neuronal receptors. To determine whether different single channel and macroscopic kinetic properties were also associated with these subtypes, the alpha1 or alpha6 subunit was expressed in mammalian cells along with beta3 and gamma2L subunits and the kinetic properties examined with outside-out patch recordings. The alpha1 beta3 gamma2L receptors responded to GABA with long-duration openings organized into multi-opening bursts. In contrast, channel openings of the alpha6 beta3 gamma2L receptors were predominately short in duration and occurred as isolated, single openings. The subunit subtype also affected the deactivation rate of the receptor, which was almost 2-fold slower for alpha6 beta3 gamma2L, compared with the alpha1 beta3 gamma2L isoform. Onset of fast desensitization did not differ between the isoforms. To determine the structural domains responsible for these differences in kinetic properties, we constructed six chimeric subunits, combining different regions of the alpha1 and alpha6 subunits. The properties of the chimeric subunits indicated that structures within the third transmembrane domain (TM3) and the TM3-TM4 intracellular loop conferred differences in single channel gating kinetics that subsequently affected the deactivation rate and GABA EC50. The effect of agonist concentration on the rise time of the current showed that the extracellular N-terminal domain was largely responsible for binding characteristics, while the transmembrane domains determined the activation rate at saturating GABA concentrations. This suggests that subunit structures outside of the agonist binding and pore-lining domains are responsible for the kinetic differences conferred by the alpha1 and alpha6 subtypes. Structural heterogeneity within these transmembrane and intracellular regions can therefore influence the characteristics of the postsynaptic response of GABA(A) receptors with different subunit composition.

Though all in vitro models of gamma frequency network oscillations are critically dependent on GABAA receptor-mediated synaptic transmission little is known about the specific role played by different subtypes of GABAA receptor. Strong expression of the alpha5 subunit of the GABAA receptor is restricted to few brain regions, amongst them the hippocampal dendritic layers. Receptors containing this subunit may be expressed on the extrasynaptic membrane of principal cells and can mediate a tonic GABAA conductance. Using hippocampal slices of wild-type (WT) and alpha5-/- mice we investigated the role of alpha5 subunits in the generation of kainate-induced gamma frequency oscillations (20-80 Hz). The change in power of the oscillations evoked in CA3 by increasing network drive (kainate, 50-400 nm) was significantly greater in alpha5-/- than in WT slices. However, the change in frequency of gamma oscillations with increasing network drive seen in WT slices was absent in alpha5-/- slices. Raising the concentration of extracellular GABA by bathing slices in the GABA transaminase inhibitor vigabatrin and blocking uptake with tiagabine reduced the power of gamma oscillations more in WT slices than alpha5-/- slices (43%versus 15%). The data suggest that loss of this GABAA receptor subunit alters the dynamic profile of gamma oscillations to changes in network drive, possibly via actions of GABA at extrasynaptic receptors.