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Activation of V1a vasopressin receptors facilitates neuronal excitability in the medial nucleus of central amygdala (CeM) V1a receptor activation excites about 80% CeM neurons by opening a cationic conductance and about 20% CeM neurons by suppressing an inwardly rectifying K+ (Kir) channels The cationic conductance activated by V1a receptors is identified as TRPC5 channels PLCβ-mediated depletion of PIP2 is involved in V1a receptor-elicited excitation of CeM neurons Intracellular Ca2+ release and PKC are unnecessary for V1a receptor-mediated excitation of CeM neurons ABSTRACT: Arginine vasopressin (AVP) serves as a hormone in the periphery to modulate water homeostasis and a neuromodulator in the brain to regulate a diverse range of functions including anxiety, social behaviors, cognitive activities and nociception. The amygdala is an essential brain region involved in modulating defensive and appetitive behaviors, pain, and alcohol use disorders. Whereas activation of V1a receptors in the medial nucleus of the central amygdala (CeM) increases neuronal excitability, the involved ionic and signaling mechanisms have not been determined. We found that activation of V1a receptors in the CeM facilitated neuronal excitability predominantly by opening TRPC5 channels, although AVP excited about one-fifth of the CeM neurons via suppressing an inwardly rectifying K+ (Kir) channel. G proteins and phospholipase Cβ (PLCβ) were required for AVP-elicited excitation of CeM neurons, whereas intracellular Ca2+ release and the activity of protein kinase C were unnecessary. Prevention of the depletion of phosphatidylinositol 4,5-bisphosphate (PIP2 ) blocked AVP-induced excitation of CeM neurons suggesting that PLCβ-mediated depletion of PIP2 is involved in AVP-mediated excitation of CeM neurons. Our results may provide a cellular and molecular mechanism to explain the anxiogenic effects of AVP in the amygdala. This article is protected by copyright. All rights reserved.

Key points Cartwheel neurons provide potent inhibition to fusiform neurons in the dorsal cochlear nucleus (DCN). Most cartwheel neurons fire action potentials spontaneously, but the ion channels responsible for this intrinsic activity are unknown. We investigated the ion channels responsible for the intrinsic firing of cartwheel neurons and the stable resting membrane potential found in a fraction of these neurons (quiet neurons). Among the ion channels controlling membrane potential of cartwheel neurons, the presence of open ATP-sensitive potassium channels (KATP ) is responsible for the existence of quiet neurons. Our results pinpoint KATP channel modulation as a critical factor controlling the firing of cartwheel neurons. Hence, it is a crucial channel influencing the balance of excitation and inhibition in the DCN. Abstract Cartwheel neurons from the dorsal cochlear nucleus (DCN) are glycinergic interneurons and the primary source of inhibition on the fusiform neurons, the DCN's principal excitatory neuron. Most cartwheel neurons present spontaneous firing (active neurons), producing a steady inhibitory tone on fusiform neurons. In contrast, a small fraction of these neurons do not fire spontaneously (quiet neurons). Hyperactivity of fusiform neurons is seen in animals with behavioural evidence of tinnitus. Because of its relevance in controlling the excitability of fusiform neurons, we investigated the ion channels responsible for the spontaneous firing of cartwheel neurons in DCN slices from rats. We found that quiet neurons presented an outward conductance not seen in active neurons, which generates a stable resting potential. This current was sensitive to tolbutamide, an ATP-sensitive potassium channel (KATP ) antagonist. After inhibition with tolbutamide, quiet neurons start to fire spontaneously, while the active neurons were not affected. On the other hand, in active neurons, KATP agonist diazoxide activated a conductance similar to quiet neurons' KATP conductance and stopped spontaneous firing. According to the effect of KATP channels on cartwheel neuron firing, glycinergic neurotransmission in DCN was increased by tolbutamide and decreased by diazoxide. Our results reveal a role of KATP channels in controlling the spontaneous firing of neurons not involved in fuel homeostasis.

Key points Some ion channels are known to behave as inductors and make up the parallel resonant circuit in the plasma membrane of neurons, which enables neurons to respond to current inputs with a specific frequency (so-called 'resonant properties'). Here, we report that heterologous expression of mouse Kv11 voltage-dependent K+ channels generate resonance and oscillation at depolarized membrane potentials in HEK293 cells; expressions of individual Kv11 subtypes generate resonance and oscillation with different frequency properties. Kv11.3-expressing HEK293 cells exhibited transient conductance changes that opposed the current changes induced by voltage steps; this probably enables Kv11 channels to behave like an inductor. The resonance and oscillation of inferior olivary neurons were impaired at the resting membrane potential in Kv11.3 knockout mice. This study helps to elucidate basic ion channel properties that are crucial for the frequency responses of neurons. Abstract The plasma membranes of some neurons preferentially respond to current inputs with a specific frequency, and output as large voltage changes. This property is called resonance, and is thought to be mediated by ion channels that show inductor-like behaviour. However, details of the candidate ion channels remain unclear. In this study, we mainly focused on the functional roles of Kv11 potassium (K+ ) channels, encoded by ether-a-go-go-related genes, in resonance in mouse inferior olivary (IO) neurons. We transfected HEK293 cells with long or short splice variants of Kv11.1 (Merg1a and Merg1b) or Kv11.3, and examined membrane properties using whole-cell recording. Transfection with Kv11 channels reproduced resonance at membrane potentials depolarized from the resting state. Frequency ranges of Kv11.3-, Kv11.1(Merg1b)- and Kv11.1(Merg1a)-expressing cells were 2-6 Hz, 2-4 Hz, and 0.6-0.8 Hz, respectively. Responses of Kv11.3 currents to step voltage changes were essentially similar to those of inductor currents in the resistor-inductor-capacitor circuit. Furthermore, Kv11 transfections generated membrane potential oscillations. We also confirmed the contribution of HCN1 channels as a major mediator of resonance at more hyperpolarized potentials by transfection into HEK293 cells. The Kv11 current kinetics and properties of Kv11-dependent resonance suggested that Kv11.3 mediated resonance in IO neurons. This finding was confirmed by the impairment of resonance and oscillation at -30 to -60 mV in Kcnh7 (Kv11.3) knockout mice. These results suggest that Kv11 channels have important roles in inducing frequency-dependent responses in a subtype-dependent manner from resting to depolarized membrane potentials.

KEY POINTS: Medullo‐spinal CSF contacting neurones (CSF‐cNs) located around the central canal are conserved in all vertebrates and suggested to be a novel sensory system intrinsic to the CNS. CSF‐cNs receive GABAergic inhibitory synaptic inputs involving ionotropic GABA(A) receptors, but the contribution of metabotropic GABA(B) receptors (GABA(B)‐Rs) has not yet been studied. Here, we indicate that CSF‐cNs express functional GABA(B)‐Rs that inhibit postsynaptic calcium channels but fail to activate inhibitory potassium channel of the Kir3‐type. We further show that GABA(B)‐Rs localise presynaptically on GABAergic and glutamatergic synaptic inputs contacting CSF‐cNs, where they inhibit the release of GABA and glutamate. Our data are the first to address the function of GABA(B)‐Rs in CSF‐cNs and show that on the presynaptic side they exert a classical synaptic modulation whereas at the postsynaptic level they have an atypical action by modulating calcium signalling without inducing potassium‐dependent inhibition. ABSTRACT: Medullo‐spinal neurones that contact the cerebrospinal fluid (CSF‐cNs) are a population of evolutionary conserved cells located around the central canal. CSF‐cN activity has been shown to be regulated by inhibitory synaptic inputs involving ionotropic GABA(A) receptors, but the contribution of the G‐protein coupled GABA(B) receptors has not yet been studied. Here, we used a combination of immunofluorescence, electrophysiology and calcium imaging to investigate the expression and function of GABA(B)‐Rs in CSF‐cNs of the mouse brainstem. We found that CSF‐cNs express GABA(B)‐Rs, but their selective activation failed to induce G protein‐coupled inwardly rectifying potassium (GIRK) currents. Instead, CSF‐cNs express primarily N‐type voltage‐gated calcium (Ca(V)2.2) channels, and GABA(B)‐Rs recruit Gβγ subunits to inhibit Ca(V) channel activity induced by membrane voltage steps or under physiological conditions by action potentials. Moreover, using electrical stimulation, we indicate that GABAergic inhibitory (IPSCs) and excitatory glutamatergic (EPSCs) synaptic currents can be evoked in CSF‐cNs showing that mammalian CSF‐cNs are also under excitatory control by glutamatergic synaptic inputs. We further demonstrate that baclofen reversibly reduced the amplitudes of both IPSCs and EPSCs evoked in CSF‐cNs through a presynaptic mechanism of regulation. In summary, these results are the first to demonstrate the existence of functional postsynaptic GABA(B)‐Rs in medullar CSF‐cNs, as well as presynaptic GABA(B) auto‐ and heteroreceptors regulating the release of GABA and glutamate. Remarkably, postsynaptic GABA(B)‐Rs associate with Ca(V) but not GIRK channels, indicating that GABA(B)‐Rs function as a calcium signalling modulator without GIRK‐dependent inhibition in CSF‐cNs.

1. The unusual kinetics of human ether-à-go-go-related gene (HERG) K(+) channels are consistent with a role in the suppression of arrhythmias initiated by premature beats. Action potential clamp protocols were used to investigate the effect of premature stimulation on HERG K(+) channels, transfected in Chinese hamster ovary cells, at 37 degrees C. 2. HERG K(+) channel currents peaked during the terminal repolarization phase of normally paced action potential waveforms. However, the magnitude of the current and the time point at which conductance was maximal depended on the type of action potential waveform used (epicardial, endocardial, Purkinje fibre or atrial). 3. HERG K(+) channel currents recorded during premature action potentials consisted of an early transient outward current followed by a sustained outward current. The magnitude of the transient current component showed a biphasic dependence on the coupling interval between the normally paced and premature action potentials and was maximal at a coupling interval equivalent to 90 % repolarization (APD(90)) for ventricular action potentials. The largest transient current response occurred at shorter coupling intervals for Purkinje fibre (APD(90) - 20 ms) and atrial (APD(90) - 30 ms) action potentials. 4. The magnitude of the sustained current response following premature stimulation was similar to that recorded during the first action potential for ventricular action potential waveforms. However, for Purkinje and atrial action potentials the sustained current response was significantly larger during the premature action potential than during the normally paced action potential. 5. A Markov model that included three closed states, one open and one inactivated state with transitions permitted between the pre-open closed state and the inactivated state, successfully reproduced our results for the effects of premature stimuli, both during square pulse and action potential clamp waveforms. 6. These properties of HERG K(+) channels may help to suppress arrhythmias initiated by early afterdepolarizations and premature beats in the ventricles, Purkinje fibres or atria.

GABAergic interneurones are diverse in their morphological and functional properties. Perisomatic inhibitory cells show fast spiking during sustained current injection, whereas dendritic inhibitory cells fire action potentials with lower frequency. We examined functional and molecular properties of K(+) channels in interneurones with horizontal dendrites in stratum oriens-alveus (OA) of the hippocampal CA1 region, which mainly comprise somatostatin-positive dendritic inhibitory cells. Voltage-gated K(+) currents in nucleated patches isolated from OA interneurones consisted of three major components: a fast delayed rectifier K(+) current component that was highly sensitive to external 4-aminopyridine (4-AP) and tetraethylammonium (TEA) (half-maximal inhibitory concentrations0.1 mM for both blockers), a slow delayed rectifier K(+) current component that was sensitive to high concentrations of TEA, but insensitive to 4-AP, and a rapidly inactivating A-type K(+) current component that was blocked by high concentrations of 4-AP, but resistant to TEA. The relative contributions of these components to the macroscopic K(+) current were estimated as 57 +/- 5, 25 +/- 6, and 19 +/- 2 %, respectively. Dendrotoxin, a selective blocker of Kv1 channels had only minimal effects on K(+) currents in nucleated patches. Coapplication of the membrane-permeant cAMP analogue 8-(4-chlorophenylthio)-adenosine 3':5'-cyclic monophosphate (cpt-cAMP) and the phosphodiesterase blocker isobutyl-methylxanthine (IBMX) resulted in a selective inhibition of the fast delayed rectifier K(+) current component. This inhibition was absent in the presence of the protein kinase A (PKA) inhibitor H-89, implying the involvement of PKA-mediated phosphorylation. Single-cell reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed a high abundance of Kv3.2 mRNA in OA interneurones, whereas the expression level of Kv3.1 mRNA was markedly lower. Similarly, RT-PCR analysis showed a high abundance of Kv4.3 mRNA, whereas Kv4.2 mRNA was undetectable. This suggests that the fast delayed rectifier K(+) current and the A-type K(+) current component are mediated predominantly by homomeric Kv3.2 and Kv4.3 channels. Selective modulation of Kv3.2 channels in OA interneurones by cAMP is likely to be an important factor regulating the activity of dendritic inhibitory cells in principal neurone-interneurone microcircuits.

In this issue of The Journal of Physiology the properties and somatodendritic distribution of voltage-gated K+ channels in pyramidal cells of neocortex are systematically analysed in three papers: two companion papers by John Bekkers (JB) in Canberra (Bekkers, 2000a,b) and one paper by Alon Korngreen and Bert Sakmann (K&S) in Heidelberg (Korngreen & Sakmann, 2000). Both laboratories focus on K+ channels in one of the most remarkable cell types in the mammalian brain: the large layer V (L5) pyramidal cells. These are primary output cells of the cortex, with long apical dendrites that rise through most of the cortical thickness, receiving thousands of synaptic contacts and reaching lengths of nearly 1 mm in the rat (Fig. 1A).

Activation of both small-conductance (SKCa) and intermediate-conductance (IKCa) Ca2+-activated K+ channels in endothelial cells leads to vascular smooth muscle hyperpolarization and relaxation in rat mesenteric arteries. The contribution that each endothelial K+ channel type makes to the smooth muscle hyperpolarization is unknown. In the presence of a nitric oxide (NO) synthase inhibitor, ACh evoked endothelium and concentration-dependent smooth muscle hyperpolarization, increasing the resting potential (approx. -53 mV) by around 20 mV at 3 microM. Similar hyperpolarization was evoked with cyclopiazonic acid (10 microM, an inhibitor of sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA)) while 1-EBIO (300 microM, an IKCa activator) only increased the potential by a few millivolts. Hyperpolarization in response to either ACh or CPA was abolished with apamin (50 nM, an SKCa blocker) but was unaltered by 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (1 microM TRAM-34, an IKCa blocker). During depolarization and contraction in response to phenylephrine (PE), ACh still increased the membrane potential to around -70 mV, but with apamin present the membrane potential only increased just beyond the original resting potential (circa -58 mV). TRAM-34 alone did not affect hyperpolarization to ACh but, in combination with apamin, ACh-evoked hyperpolarization was completely abolished. These data suggest that true endothelium-dependent hyperpolarization of smooth muscle cells in response to ACh is attributable to SKCa channels, whereas IKCa channels play an important role during the ACh-mediated repolarization phase only observed following depolarization.

1. The hypothesis that inward rectifier K(+) channels are involved in the vasodilatation of small coronary and cerebral arteries (100-200 microm diameter) in response to elevated [K+]o was tested. The diameters and membrane potentials of pressurized arteries from rat were measured using a video-imaging system and conventional microelectrodes, respectively. 2. Elevation of [K+]o from 6 to 16 mM caused the membrane potential of pressurized (60 mmHg) arteries to hyperpolarize by 12-14 mV. Extracellular Ba(2+) (Ba2+(o)) blocked K(+)-induced membrane potential hyperpolarizations at concentrations (IC(50), 6 microM) that block inward rectifier K(+) currents in smooth muscle cells isolated from these arteries. 3. Elevation of [K+]o from 6 to 16 mM caused sustained dilatations of pressurized coronary and cerebral arteries with diameters increasing from 125 to 192 microm and 110 to 180 microm in coronary and cerebral arteries, respectively. Ba2+(o) blocked K(+)-induced dilatations of pressurized coronary and cerebral arteries (IC50, 3-8 microM). 4. Elevated [K+]o-induced vasodilatation was not prevented by blockers of other types of K(+) channels (1 mM 4-aminopyridine, 1 mM TEA+, and 10 mu M glibenclamide), and blockers of Na(+)-K(+)-ATPase. Elevated [K+]o-induced vasodilatation was unaffected by removal of the endothelium. 5. These findings suggest that K+(o) dilates small rat coronary and cerebral arteries through activation of inward rectifier K(+) channels. Furthermore, these results support the hypothesis that inward rectifier K(+) channels may be involved in metabolic regulation of coronary and cerebral blood flow in response to changes in [K+]o.

We made whole-cell recordings from CA1 pyramidal cells of hippocampal slices in combination with brief dendritic glutamate pulses to study the role of constitutive inwardly rectifying K+ channels (IRK, Kir2.0) and G-protein-activated inwardly rectifying K+ channels (GIRK, Kir3.0) in the processing of excitatory inputs. Phasic activation of GIRK channels by baclofen (20 microM) produced a reversible reduction of glutamate-evoked postsynaptic potentials (GPSPs), our equivalent of EPSPs, by about one-third. Conversely, tertiapin (30 nM), a selective inhibitor of GIRK channels, and Ba2+ (200 microM), a non-selective blocker of inwardly rectifying K+ channels, enhanced GPSPs and, in voltage-clamp experiments, reduced the underlying K+ conductances, indicating a functionally significant background GIRK conductance, in addition to constitutive IRK channel activity. When examined after suppression of endogenous adenosinergic inhibition, using either adenosine deaminase or the selective A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine, tertiapin failed to influence either the GPSPs or the inwardly rectifying K+ conductance. Voltage-clamp recordings from acutely isolated CA1 pyramidal cells not exposed to ambient adenosine exhibited no response to tertiapin, whereas Ba2+ was still capable of reducing hyperpolarizing inward rectification. Our data indicate that in hippocampal pyramidal cells, two components of the inwardly rectifying K+ conductance can be identified, which together exert a tonic modulation of excitatory synaptic input: one arises from constitutive putative IRK channels, the other is mediated by the background activity of GIRK channels that results from the tonic activation of A1 receptors by ambient adenosine.

The molecular identity of the ion channel underlying the slow cardiac delayed rectifier K+ current, IKs, is a heteromultimeric complex, composed of the 6-transmembrane α-subunit KCNQ1 (Wang et al. 1996; Yang et al. 1997), which is structurally similar to Shaker channels, and a small, 1-transmembrane β-subunit, KCNE1 (Takumi et al. 1988; Barhanin et al. 1996; Sanguinetti et al. 1996). KCNQ1 is the first member of a relatively recently identified gene family so far comprising up to five members (Wang et al. 1996; Singh et al. 1998; Schroeder et al. 1998; Kubisch et al. 1999; Lerche et al. 2000; for review see Jentsch, 2000), whereas KCNE1 (also called IsK or minK) was expression cloned as far back as 12 years ago (Takumi et al. 1988). The expression cloning was possible because injection of KCNE1 RNA alone induces slowly activating K+ currents in Xenopus oocytes, probably because it forms functional channels with endogenous α-subunits (Sanguinetti et al. 1996). More recently, homologues of KCNE1 have also been identified (Abbott et al. 1999; Schroeder et al. 2000). One of these forms heteromers with and alters the functional properties of HERG potassium channels (encoded by the human ether-a-go-go-related gene) (Abbott et al. 1999). Another associates with KCNQ1, in a similar way to KCNE1, in epithelial cells of kidney, intestine and colon - but not in cardiac myocytes and the inner ear - probably forming a basolateral, constitutively open K+ channel (Schroeder et al. 2000). Mutations in the genes coding for KCNQ1 and KCNE1 can lead to the dominant long-QT syndrome or to the recessive Jervell Lange Nielssen syndrome, a cardiac abnormality that is also associated with deafness (see Schulze-Bahr et al. 1999, for review). These genetic data strongly support a physiologically relevant association of KCNQ1 with KCNE1 in the heart. KCNQ1 can be heterologously expressed as a homomeric, probably tetrameric, channel with properties that are quite different from those of ‘classic’ K+ channels. Gating of homomeric KCNQ1 channels is characterised by rather slow activation kinetics and a delayed, incomplete and intrinsically voltage-independent inactivation (Barhanin et al. 1996; Sanguinetti et al. 1996; Pusch et al. 1998; Tristani-Firouzi & Sanguinetti, 1998). The open channel has a low single-channel conductance (in the 1 pS range) and displays a pronounced flicker in the high-frequency range (Pusch, 1998; Yang & Sigworth, 1998; Sesti & Goldstein, 1998; Pusch et al. 2000). Many, if not all of these properties are changed by the association of KCNQ1 with KCNE1: activation becomes extremely slow, inactivation appears to be abolished, the single-channel conductance is increased, and other open channel properties also seem to be altered (Barhanin et al. 1996; Sanguinetti et al. 1996; Tristani-Firouzi & Sanguinetti, 1998; Tai & Goldstein, 1998; Pusch et al. 1998; Pusch, 1998, 2000; Yang & Sigworth, 1998; Sesti & Goldstein, 1998). Despite extensive efforts, the mechanisms underlying the unusual properties of homomeric KCNQ and especially the drastic effects of the association with KCNE1 are still obscure. Here we investigate the block of homomeric KCNQ1 and heteromeric KCNQ1/KCNE1 channels by intracellular Na+. Intracellular Na+ block is a common feature of K+ channels (Bezanilla & Armstrong, 1972; French & Wells, 1977; Yellen, 1984a; Kiss et al. 1998; Heginbotham et al. 1999; Thompson & Begenisich, 2000). In view of the structure of the bacterial KscA channel (Doyle et al. 1998), intracellular Na+ and other poorly permeable small cations block the outward flow through the pore. They probably do this by residing in the cavity just in front of the selectivity filter, where they are not allowed to enter, and impede the flow of K+ ions in the outward direction (Thompson & Begenisich, 1999). Our detailed analysis of the properties of the Na+ block of homomeric KCNQ1 proves the existence of (at least) two kinetically distinct open states with different propensities to be blocked by Na+. Furthermore, the temporal development of the Na+ block indicates that the rate-limiting step of the gating of homomers is the transition between the open states. The Na+ block of heteromers, in contrast, indicates that the much slower activation of this physiologically relevant complex is rate limited by the entry into the first open state.

ATP-sensitive K+ (KATP) channels activated by glucose-free anoxia close immediately upon reoxygenation in single guinea-pig ventricular myocytes, while KATP channels open persistently during reperfusion in coronary-perfused guinea-pig ventricular myocardium. To investigate the reasons behind this discrepancy, we investigated whether protein kinase C (PKC) modulates the opening of KATP channels during anoxia-reoxygenation and ischaemia-reperfusion. Exposure of guinea-pig ventricular cells to glucose-free anoxia shortened the action potential duration at 90% repolarisation (APD90) and evoked the glibenclamide-sensitive robust outward current (IK,ATP). Subsequent reoxygenation caused an immediate prolongation of APD90 and a decrease in IK,ATP within approximately 20 s. When the novel (Ca2+-independent) PKC was activated by applying 1,2-dioctanoyl-sn-glycerol (1,2DOG, 20 M) with EGTA (20 mM) in the pipette, the APD90 restored gradually after reoxygenation and the extent of recovery was appoximately 80% of the pre-anoxic value. Moreover, IK,ATP decreased slowly and remained opened for up to approximately 4 min after reoxygenation. These results suggest persistent opening of KATP channels during reoxygenation. The persistent activation of KATP channels was augmented when both novel and conventional (Ca2+-dependent) isoforms of PKC were activated by applying 1,2DOG without EGTA in the pipette. In coronary-perfused right ventricular myocardium, APD90 remained shortened for up to approximately 30 min of reperfusion. The gradual restoration of APD90 after ischaemia-reperfusion was facilitated by the KATP channel blocker glibenclamide and by the potent PKC inhibitor chelerythrine. Our results provide the first evidence that PKC activation contributes to the persistent opening of KATP channels during reoxygenation and reperfusion. We also conclude that both novel and conventional PKC isoforms co-operatively modulate the opening of KATP channels during the early phase of reoxygenation.

The present experiments were performed to assess the role of K+ channels in hormonal stimulation of the Na+-K+ pump and to determine the contribution of Na+-K+ pumps to the recovery of excitability and contractility in depolarized skeletal muscle. In soleus muscle, Ba2+ (0.02 and 1 mM) was found to inhibit 42K+ efflux and 42K+ influx. Both in the absence and the presence of Ba2+ (1 mM), salbutamol and calcitonin gene-related peptide (CGRP) induced a marked decrease in intracellular Na+ and stimulation of 42K+ uptake. In soleus muscles Ba2+ (0.1 and 1.0 mM) decreased twitch and tetanic force. Subsequent stimulation of the Na+-K+ pumps by salbutamol, CGRP or repeated electrical stimulation produced a highly significant restoration of force development, which was suppressed by ouabain, but not by glibenclamide. Also, in extensor digitorum longus muscles Ba2+ (0.1 mM) produced a considerable force decline, which was partly restored by salbutamol and CGRP. The area of compound action potentials (M-waves) elicited by indirect stimulation was decreased by Ba2+ (0.1 mM). This was associated with a concomitant decrease in tetanic force and depolarization. Salbutamol, CGRP or repeated electrical stimulation all elicited marked recovery of M-wave area, force and membrane potential. All recordings showed close correlations between these three parameters. The data add further support to the concept that due to its electrogenic nature and large transport capacity, the Na+-K+ pump is a rapid and efficient mechanism for the maintenance of excitability in skeletal muscle, acting independently of Ba2+- or ATP-sensitive K+ channel function.

Receptor-mediated modulation of ion channels generally involves G-proteins, phosphorylation, or both in combination. The sigma receptor, which modulates voltage-gated K+ channels, is a novel protein with no homology to other receptors known to modulate ion channels. In the present study patch clamp and photolabelling techniques were used to investigate the mechanism by which sigma receptors modulate K+ channels in peptidergic nerve terminals. The sigma receptor photoprobe iodoazidococaine labelled a protein with the same molecular mass (26 kDa) as the sigma receptor protein identified by cloning. The sigma receptor ligands pentazocine and SKF10047 modulated K+ channels, despite intra-terminal perfusion with GTP-free solutions, a G-protein inhibitor (GDPβS), a G-protein activator (GTPγS) or a non-hydrolysable ATP analogue (AMPPcP). Channels in excised outside-out patches were modulated by ligand, indicating that soluble cytoplasmic factors are not required. In contrast, channels within cell-attached patches were not modulated by ligand outside a patch, indicating that receptors and channels must be in close proximity for functional interactions. Channels expressed in oocytes without receptors were unresponsive to sigma receptor agonists, ruling out inhibition through a direct drug interaction with channels. These experiments indicate that sigma receptor-mediated signal transduction is membrane delimited, and requires neither G-protein activation nor protein phosphorylation. This novel transduction mechanism is mediated by membrane proteins in close proximity, possibly through direct interactions between the receptor and channel. This would allow for more rapid signal transduction than other ion channel modulation mechanisms, which in the present case of neurohypophysial nerve terminals would lead to the enhancement of neuropeptide release. Sigma receptors modulate the excitability of peptidergic nerve terminals in the neurohypophysis by inhibiting voltage-dependent K+ channels (IK) (Wilke et al. 1999a). The activation of sigma receptors by a variety of ligands reduces current flow through two distinct K+ channel types, the A-current channel (IA) and the Ca2+-activated K+ channel (IBK). Current is reduced by the same proportion over the entire accessible voltage range, with no shift in the voltage dependence of activation or inactivation. Furthermore, the residual unblocked currents inactivate with very similar rates, indicating that sigma receptor modulation entails a shutting down of function rather than a modification of gating behaviour (Wilke et al. 1998, 1999a,b). A comparison of the concentration dependence of IA reduction with that of IBK reduction indicated that the sigma receptor ligand PPHT inhibits both of these channels with a very similar EC50 (Wilke et al. 1998); similar results were obtained with haloperidol (Wilke et al. 1999a). Both IA and IK were reduced proportionally by a large number of sigma receptor ligands (including ditolylguanidine, SKF10047, pentazocine, haloperidol, PPHT, U101958, and apomorphine), suggesting that in the rat the same receptor is coupled to two types of K+ channels. In D2, D3 and D4 dopamine receptor-deficient mice, sigma receptor ligands reduced IK as effectively as in wild-type mice, indicating that the responses are not mediated by dopamine receptor subtypes known to interact with some sigma receptor ligands (Wilke et al. 1999a). Many candidate endogenous ligands were tested, including neuropeptides, catecholamines, and steroids, and none altered IK in this preparation. Furthermore, although the posterior pituitary contains κ-opioid receptors, which modulate Ca2+ channels (Rusin et al. 1997), K+ channels are not modulated by dynorphin in this preparation (authors’ unpublished observations), indicating that the reduction of IK by these ligands is not mediated by opioid receptors. The sigma receptor binding site was first described over two decades ago. Originally thought to be a novel opioid receptor (Martin et al. 1976), subsequent studies demonstrated that the sigma receptor binding site is a distinct pharmacological entity distinguished by unusually promiscuous binding properties (Su, 1993; de Costa & He, 1994). Recent molecular characterization has shown that the sigma receptor is a novel protein with a molecular mass of 26 kDa. Hydropathy analysis has indicated that this protein has a single putative membrane-spanning segment (Hanner et al. 1996; Kekuda et al. 1996; Seth et al. 1997). The deduced amino acid sequence of the sigma receptor has no homology with known mammalian proteins, but a weak homology with fungal sterol isomerase has led some investigators to speculate that sigma receptors may be involved in steroid hormone biosynthesis (Jbilo et al. 1997; Moebius et al. 1997). Sigma receptors are ubiquitously distributed in both brain and peripheral tissue. Their binding to many different kinds of drugs has made it difficult to determine their function, but sigma receptors have been implicated in a variety of functions, including motor control, psychosis, and a wide range of endocrine and immune processes (Su, 1993; Bowen, 1993). The unique molecular structure of the sigma receptor has raised intriguing questions about its mechanism of signal transduction. As novel proteins, sigma receptors fall outside the large families of membrane signalling molecules that have been identified in the past two decades. The sigma receptor does not have seven putative transmembrane domains, and so it would appear that this protein lacks the essential structural elements necessary for an interaction with G-proteins. At a topological level, the single putative transmembrane segment of the sigma receptor is reminiscent of many growth factor receptors with protein kinase activity, but at the sequence level no homology was found. There is some evidence that sigma receptor activation stimulates GTPase activity in mouse and rat brain (Itzhak, 1989; Tokuyama et al. 1997). GTP analogues have been reported to influence the binding of sigma receptor ligands (Beart et al. 1989), but a number of other binding studies led to different conclusions (Bowen, 1994). The modulation of IK by sigma receptor ligands was abolished by reagents that inactivate G-proteins in some studies (Nakazawa et al. 1995; Soriani et al. 1998, 1999), but in other studies G-protein perturbation had no effect (Morio et al. 1994; Wilke et al. 1999b). These questions and conflicting results prompted us to use patch clamp techniques to investigate the mechanism by which sigma receptors inhibit IK in nerve terminals of slices prepared from the posterior pituitary gland. Reagents known to block G-protein and phosphorylation-mediated signal transduction pathways failed to prevent IK modulation. Sigma receptor ligands modulated channel function in excised outside-out patches in the absence of soluble cytoplasmic factors. In contrast, channels within cell-attached patches could not be modulated by drug application to adjacent membrane outside the pipette tip. These results indicate that sigma receptors modulate membrane function by a novel membrane-delimited mechanism requiring close proximity between receptors and channels.

1. 12-Lipoxygenase produces 12-hydroperoxy acid from arachidonic acid released from membrane phospholipids. To elucidate the role of the enzyme in neuronal functions, mouse neuroblastoma x rat glioma hybrid NG108-15 cells were permanently transfected with the cDNA for human 12-lipoxygenase. 2. The number of action potentials evoked by depolarizing current steps in a current-clamp mode was strikingly increased in 12-lipoxygenase-expressing NG108-15 cells as compared with the wild-type cells which hardly had the enzyme activity. 3. In the transformed cells, the M-type voltage-dependent K+ current was significantly reduced with little or no change in other ion channel currents. 4. Treatment of the transformed cells with a 12-lipoxygenase inhibitor recovered the action potential frequency and the M-current amplitude to the control level. 5. These results indicate that 12-lipoxygenase and/or its metabolites target K+ channels and upregulate the membrane excitability, and thereby modulate neuronal signalling.

1. G protein-gated inwardly rectifying K+ (GIRK) channels are activated independently by Gbetagamma and internal Na+ via mechanisms requiring phosphatidylinositol phosphates. An aspartate (Asp) at position 226 in GIRK2 is crucial for Na+-dependent activation of GIRK1-GIRK2 heteromeric channels. We expressed wild-type and mutant GIRK1-GIRK2 channels in Xenopus oocytes and tested the effects of Na+ and neutralizing Asp226 on the functional interactions of the channels with phosphatidylinositol 4, 5-bisphosphate (PIP2). 2. The rate of inhibition of GIRK1-GIRK2 currents by application of anti-PIP2 antibody to inside-out membrane patches was slowed > 2-fold by the D226N mutation in GIRK2 and by increasing internal [Na+]. The reverse mutation in GIRK1 (N217D) increased the rate of inhibition. 3. The dose-response relationship for activation by purified PIP2 was shifted to lower concentrations in the presence of 20 mM Na+. 4. Three synthetic isoforms of PIP2, PI(4,5)P2, PI(3,4)P2 and PI(3,5)P2, activated GIRK channels with similar potencies. 5. We conclude that Na+ directly interacts with Asp226 of GIRK2 to reduce the negative electrostatic potential and promote the functional interaction of the channels with PIP2.

1. The role of large-conductance Ca2+-dependent K+ channels (BK-channels; also known as maxi-K- or slo-channels) in spike broadening during repetitive firing was studied in CA1 pyramidal cells, using sharp electrode intracellular recordings in rat hippocampal slices, and computer modelling. 2. Trains of action potentials elicited by depolarizing current pulses showed a progressive, frequency-dependent spike broadening, reflecting a reduced rate of repolarization. During a 50 ms long 5 spike train, the spike duration increased by 63.6 +/- 3.4 % from the 1st to the 3rd spike. The amplitude of the fast after-hyperpolarization (fAHP) also rapidly declined during each train. 3. Suppression of BK-channel activity with (a) the selective BK-channel blocker iberiotoxin (IbTX, 60 nM), (b) the non-peptidergic BK-channel blocker paxilline (2-10 microM), or (c) calcium-free medium, broadened the 1st spike to a similar degree ( approximately 60 %). BK-channel suppression also caused a similar change in spike waveform as observed during repetitive firing, and eliminated (occluded) most of the spike broadening during repetitive firing. 4. Computer simulations using a reduced compartmental model with transient BK-channel current and 10 other active ionic currents, produced an activity-dependent spike broadening that was strongly reduced when the BK-channel inactivation mechanism was removed. 5. These results, which are supported by recent voltage-clamp data, strongly suggest that in CA1 pyramidal cells, fast inactivation of a transient BK-channel current (ICT), substantially contributes to frequency-dependent spike broadening during repetitive firing.

1. Nitric oxide (NO) has been shown to modulate neuropeptide secretion from the posterior pituitary. Here we show that NO activates large-conductance Ca2+-activated K+ (BK) channels in posterior pituitary nerve terminals. 2. NO, generated either by the photolysis of caged-NO or with chemical donors, irreversibly enhanced the component of whole-terminal K+ current due to BK channels and increased the activity of BK channels in excised patches. NO also inhibited the transient A-current. The time courses of these effects on K+ current were very different; activation of BK channels developed slowly over several minutes whereas inhibition of A-current immediately followed NO uncaging. 3. Activation of BK channels by NO occurred in the presence of guanylyl cyclase inhibitors and after removal of ATP or GTP from the pipette solution, suggesting a cGMP-independent signalling pathway. 4. The sulfhydryl alkylating agent N-ethyl maleimide (NEM) increased BK channel activity. Pretreatment with NEM occluded NO activation. 5. NO activation of BK channels occurred independently of voltage and cytoplasmic Ca2+ concentration. In addition, NO removed the strict Ca2+ requirement for channel activation, rendering channels highly active even at nanomolar Ca2+ levels. 6. These results suggest that NO, or a reactive nitrogen byproduct, chemically modifies nerve terminal BK channels or a closely associated protein and thereby produces an increase in channel activity. Such activation is likely to inhibit impulse activity in posterior pituitary nerve terminals and this may explain the inhibitory action of NO on secretion.

1. Using the perforated patch-clamp or whole-cell clamp technique, we investigated the contribution of Ca2+-activated K+ current (IK(Ca)) and non-selective cation currents (INSC) to the membrane potential in small pulmonary arterial smooth muscle cells of the rabbit. 2. The resting membrane potential (Vm) was -39.2 +/- 0.9 mV (n = 72). It did not stay at a constant level, but hyperpolarized irregularly, showing spontaneous transient hyperpolarizations (STHPs). The mean frequency and amplitude of the STHPs was 5.6 +/- 1. 1 Hz and -7.7 +/- 0.7 mV (n = 12), respectively. In the voltage-clamp mode, spontaneous transient outward currents (STOCs) were recorded with similar frequency and irregularity. 3. Intracellular application of BAPTA or extracellular application of TEA or charybdotoxin suppressed both the STHPs and STOCs. The depletion of intracellular Ca2+ stores by caffeine or ryanodine, and the removal of extracellular Ca2+ also abolished STHPs and STOCs. 4. Replacement of extracellular Na+ with NMDG+ caused hyperpolarization Vm of without affecting STHPs. Removal of extracellular Ca2+ induced a marked depolarization of Vm along with the disappearance of STHPs. 5. The ionic nature of the background inward current was identified. The permeability ratio of K+ : Cs+ : Na+ : Li+ was 1.7 : 1.3 : 1 : 0. 9, indicating that it is a non-selective cation current (INSC). The reversal potential of this current in control conditions was calculated to be -13.9 mV. The current was blocked by millimolar concentrations of extracellular Ca2+ and Mg2+. 6. From these results, it was concluded that (i) hyperpolarizing currents are mainly contributed by Ca2+-activated K+ (KCa) channels, and thus STOCs result in transient membrane hyperpolarization, and (ii) depolarizing currents are carried through NSC channels.

1. Using the whole-cell recording mode we have characterized two non-conducting states in mammalian Shaker-related voltage-gated K+ channels induced by the removal of extracellular potassium, K+o. 2. In the absence of K+o, current through Kv1.4 was almost completely abolished due to the presence of a charged lysine residue at position 533 at the entrance to the pore. Removal of K+o had a similar effect on current through Kv1.3 when the histidine at the homologous position (H404) was protonated (pH 6.0). Channels containing uncharged residues at the corresponding position (Kv1.1: Y; Kv1.2: V) did not exhibit this behaviour. 3. To characterize the nature of the interaction between Kv1.3 and K+o concentration ([K+]o), we replaced H404 with amino acids of different character, size and charge. Substitution of hydrophobic residues (A, V and L) either in all four subunits or in only two subunits in the tetramer made the channel insensitive to the removal of K+o, possibly by stabilizing the channel complex. Replacement of H404 with the charged residue arginine, or the polar residue asparagine, enhanced the sensitivity of the channel to 0 mM K+o, possibly by making the channel unstable in the absence of K+o. Mutation at a neighbouring position (400) had a similar effect. 4. The effect of removing K+o on current amplitude does not seem to be correlated with the rate of C-type inactivation since the slowly inactivating G380F mutant channel exhibited a similar [K+]o dependence as the wild-type Kv1.3 channel. 5. CP-339,818, a drug that recognizes only the inactivated conformation of Kv1.3, could not block current in the absence of K+o unless the channels were inactivated through depolarizing pulses. 6. We conclude that removal of K+o induces the Kv1.3 channel to transition to a non-conducting 'closed' state which can switch into a non-conducting 'inactivated' state upon depolarization.