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The activity of many proteins depends on the phosphoinositide (PI) content of the membrane. E.g., dynamic changes of the concentration of PI(4,5)P2 are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5)P2. Yet, measuring affinities for endogenous PIs has not been possible directly, but has relied largely on the response to soluble analogs, which may not quantitatively reflect binding to native lipids. Voltage-sensitive phosphatases (VSPs) turn over PI(4,5)P2 to PI(4)P when activated by depolarization. In combination with voltage-clamp electrophysiology VSPs are useful tools for rapid and reversible depletion of PI(4,5)P2. Because cellular PI(4,5)P2 is resynthesized rapidly, steady state PI(4,5)P2 changes with the degree of VSP activation and thus depends on membrane potential. Here we show that titration of endogenous PI(4,5)P2 with Ci-VSP allows for the quantification of relative PI(4,5)P2 affinities of ion channels. The sensitivity of inward rectifier and voltage-gated K+ channels to Ci-VSP allowed for comparison of PI(4,5)P2 affinities within and across channel subfamilies and detected changes of affinity in mutant channels. The results also reveal that VSPs are useful only for PI effectors with high binding specificity among PI isoforms, because PI(4,5)P2 depletion occurs at constant overall PI level. Thus, Kir6.2, a channel activated by PI(4,5)P2 and PI(4)P was insensitive to VSP. Surprisingly, despite comparable PI(4,5)P2 affinity as determined by Ci-VSP, the Kv7 and Kir channel families strongly differed in their sensitivity to receptor-mediated depletion of PI(4,5)P2. While Kv7 members were highly sensitive to activation of PLC by Gq-coupled receptors, Kir channels were insensitive even when PI(4,5)P2 affinity was lowered by mutation. We hypothesize that different channels may be associated with distinct pools of PI(4,5)P2 that differ in their accessibility to PLC and VSPs. [ABSTRACT FROM AUTHOR]

Key points During the behavioural states of sleep and wakefulness thalamocortical relay neurons fire action potentials in high frequency bursts or tonic sequences, respectively., The modulation of specific K+ channel types, termed TASK and TREK, allows these neurons to switch between the two modes of activity., In this study we show that the signalling lipids phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG), which are components of their membrane environment, switch on and shut off TREK and TASK channels, respectively., These channel modulations contribute to a better understanding of the molecular basis of the effects of neurotransmitters such as ACh which are released by the brainstem arousal system., The present report introduces PIP2 and DAG as new elements of signal transduction in the thalamus., Abstract The activity of two-pore domain potassium channels (K2P) regulates the excitability and firing modes of thalamocortical (TC) neurons. In particular, the inhibition of two-pore domain weakly inwardly rectifying K+ channel (TWIK)-related acid-sensitive K+ (TASK) channels and TWIK-related K+ (TREK) channels, as a consequence of the stimulation of muscarinic ACh receptors (MAChRs) which are coupled to phosphoinositide-specific phospholipase C (PLCβ), induces a shift from burst to tonic firing. By using a whole cell patch-clamp approach, the contribution of the membrane-bound second messenger molecules phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) acting downstream of PLCβ was probed. The standing outward current ( ISO) was used to monitor the current through TASK and TREK channels in TC neurons. By exploiting different manoeuvres to change the intracellular PIP2 level in TC neurons, we here show that the scavenging of PIP2 (by neomycin) results in an increased muscarinic effect on ISO whereas increased availability of PIP2 (inclusion to the patch pipette; histone-based carrier) decreased muscarinic signalling. The degree of muscarinic inhibition specifically depends on phosphatidylinositol phosphate (PIP) and PIP2 but no other phospholipids (phosphatidic acid, phosphatidylserine). The use of specific blockers revealed that PIP2 is targeting TREK but not TASK channels. Furthermore, we demonstrate that the inhibition of TASK channels is induced by the application of the DAG analogue 1-oleoyl-2-acetyl- sn-glycerol (OAG). Under current clamp conditions the activation of MAChRs and PLCβ as well as the application of OAG resulted in membrane depolarization, while PIP2 application via histone carrier induced a hyperpolarization. These results demonstrate a differential role of PIP2 and DAG in K2P channel modulation in native neurons which allows a fine-tuned inhibition of TREK (via PIP2 depletion) and TASK (via DAG) channels following MAChR stimulation. [ABSTRACT FROM AUTHOR]

Modulation of [K.sub.ATP] channels by activation of metabotropic receptors and cell metabolism is an important pathway for regulation of cell excitability (l). A common feature of these regulatory effects is [...], Adenosine triphosphate (ATP)-sensitive potassium ([K.sub.ATP]) channels couple electrical activity to cellular metabolism through their inhibition by intracellular ATP. ATP inhibition of [K.sub.ATP] channels varies among tissues and is affected by the metabolic and regulatory state of individual cells, suggesting involvement of endogenous factors. It is reported here that phosphatidylinositol-4,5-bisphosphate ([PIP.sub.2]) and phosphatidylinositol-4-phosphate (PIP) controlled ATP inhibition of cloned [K.sub.ATP] channels ([K.sub.ir] 6.2 and SUR1). These phospholipids acted on the [K.sub.ir] 6.2 subunit and shifted ATP sensitivity by several orders of magnitude. Receptor-mediated activation of phospholipase C resulted in inhibition of [K.sub.ATP]-mediated currents. These results represent a mechanism for control of excitability through phospholipids.

Lumen formation is a critical event in biological tube formation, yet its molecular mechanisms remain poorly understood. Specifically, how lumen expansion is coordinated with other processes of tubulogenesis is not well known, and the role of membrane transporters in tubulogenesis during development has not been adequately addressed. Here we identify a solute carrier 26 (Slc26) family protein as an essential regulator of tubulogenesis using the notochord of the invertebrate chordate Ciona intestinalis as a model. Ci-Slc26aα is indispensable for lumen formation and expansion, but not for apical/luminal membrane formation and lumen connection. Ci-Slc26aα acts as an anion transporter, mediating the electrogenic exchange of sulfate or oxalate for chloride or bicarbonate and electroneutral chloride:bicarbonate exchange. Mutant rescue assays show that this transport activity is essential for Ci-Slc26aα's in vivo function. Our work reveals the consequences and relationships of several key processes in lumen formation, and establishes an in vivo assay for studying the molecular basis of the transport properties of SLC26 family transporters and their related diseases. [ABSTRACT FROM AUTHOR]

Nematocytes, the stinging cells of Hydrozoa, can be considered as prototypic mechanosensory hair cells bearing a concentric hair bundle, the cnidocil apparatus. These cells produce typical mechanoreceptor potentials in response to deflection of their cnidocil. Here we show that mechanosensory signals are relayed to neighbouring nematocytes via chemical neurotransmission and that nematocytes receive synaptic input from surrounding nematocytes, hair cells and probably from epithelial cells. Intracellular voltage recordings from stenotele nematocytes of capitate hydroid polyps showed two distinct types of responses when other nematocytes within the same tentacle were mechanically stimulated: (i) graded depolarizations of variable duration ('T-potentials'), and (ii) uniform impulse-like, often repetitive depolarizations ('T-potentials') that occurred in correlation with contractions of epitheliomuscular cells. Voltage clamp experiments showed that despite the stereotyped time course of T- potentials, their generation did not involve electrically excitable conductances. Instead, time course, post-stimulus delay, susceptibility to blockers of neurotransmission and gap junctions, and induction by electrical stimulation of other nematocytes indicate that L- and T-potentials are postsynaptic, most likely glutamatergic potentials. Both result from different presynaptic pathways: T-potentials are induced monosynaptically by presynaptic receptor potentials, T-potentials are most likely triggered by presynaptic action potentials propagating through the ectodermal epithelium via gap junctions. Moreover, contact-chemosensory (phospholipid) stimulation of the presynaptic nematocyte is a positive modulator of the nematocyte's afferent synaptic efficacy and of cnidocyst discharge, both triggered by mechanoreceptor potentials. The results reveal that hydrozoan nematocytes act as bimodal sensory cells, signalling coincident chemical and mechanical stimuli indicative of prey, and receive signals from other nematocytes and sensory cells. [ABSTRACT FROM AUTHOR]

Large-conductance voltage- and Ca2+-activated K+ channels (BKCa) are involved in shaping spiking patterns in many neurons. Less is known about their role in mammalian inner hair cells (IHCs), mechanosensory cells with unusually large BKCa currents. These currents may be involved in shaping the receptor potential, implying crucial importance for the properties of afferent auditory signals. We addressed the function of BKCa by recording sound-induced responses of afferent auditory nerve (AN) fibers from mice with a targeted deletion of the pore-forming α-subunit of BKCa (BKα-/-) and comparing these with voltage responses of current-clamped IHCs. BKCa-mediated currents in IHCs were selectively abolished in BKα-/-, whereas cochlear physiology was essentially normal with respect to cochlear sensitivity and frequency tuning. BKα-/- AN fibers showed deteriorated precision of spike timing, measured as an increased variance of first spike latency in response to tone bursts. This impairment could be explained by a slowed voltage response in the presynaptic IHC resulting from the reduced K+ conductance in the absence of BKCa. Maximum spike rates of AN fibers were reduced nearly two-fold in BKα-/-, contrasting with increased voltage responses of IHCs. In addition to presynaptic changes, which may be secondary to a modest depolarization of BKα-/- IHCs, this reduction in AN rates suggests a role of BKCa in postsynaptic AN neurons, which was supported by increased refractory periods. In summary, our results indicate an essential role of IHC BKCa channels for precise timing of high-frequency cochlear signaling as well as a function of BKCa in the primary afferent neuron. [ABSTRACT FROM AUTHOR]

The defining characteristic of large-conductance Ca2+- and voltage-activated K+ channels (BKCa) is their allosteric activation by two distinct stimuli, membrane depolarization and cytosolic Ca2+ ions. In this allosteric gating, increasing cytosolic Ca2+ concentration ([Ca2+]i) shifts the depolarization required for channel opening into the physiological voltage range. In fact, according to present knowledge, elevation of [Ca2+]i to micromolar levels is the only means to activate BKCa at membrane potentials below 0 mV. We recorded BKCa-mediated currents from auditory inner hair cells (IHCs) in acutely isolated organs of Corti using the patch-clamp technique in whole-cell and excised patch configuration. In inside-out and outside-out patches, activation of BKCa channels from IHCs showed the prototypic sensitivity to increased [Ca2+]i. However, channel activation at 0 [Ca2+]i occurred at unusually negative potentials (half-maximal activation ( Vh) around 0 mV), indicating that a large fraction of the channels can be activated at physiological voltages without elevated [Ca2+]i. In intact IHCs, the activation curve of BKCa currents recorded in whole-cell configuration exhibited a Vh of −42 mV together with a high voltage dependence (slope factor of 10 mV) and submillisecond onset of current. Surprisingly, this activation was independent of changes in local [Ca2+]i as shown by experiments that interfered with Ca2+ influx through voltage-gated Ca2+ (Cav) channels, release of Ca2+ from internal stores, or intracellular buffer capacity. This behaviour is not due to β-subunits of BKCa (BKβ), as genetic inactivation of the β-subunit expressed in IHCs, KCNMB1, did not affect BKCa gating. We conclude that the BKCa channel protein in IHCs may be modified in order to rapidly activate and deactivate at resting [Ca2+]i. Our results suggest that BKCa may function as a purely voltage-gated K+ channel with exceptionally rapid activation kinetics, challenging the view that both increased cytosolic Ca2+ and depolarization are generally required for activation of BKCa. [ABSTRACT FROM AUTHOR]

Voltage-gated potassium (Kv) channels control action potential repolarization, interspike membrane potential, and action potential frequency in excitable cells. It is thought that the combinatorial association between distinct α and β subunits determines whether Kv channels function as non-inactivating delayed rectifiers or as rapidly inactivating A-type channels. We show that membrane lipids can convert A-type channels into delayed rectifiers and vice versa. Phosphoinositides remove N-type inactivation from A-type channels by immobilizing the inactivation domains. Conversely, arachidonic acid and its amide anandamide endow delayed rectifiers with rapid voltage-dependent inactivation. The bidirectional control of Kv channel gating by lipids may provide a mechanism for the dynamic regulation of electrical signaling in the nervous system. [ABSTRACT FROM AUTHOR]

Studies the contribution of the membrane protein prestin to the electromotility of the outer hair cells (OHC) of the mammalian cochlea. Characterization of the prestin molecule; Possibility that the voltage sensor may be a charged particle extrinsic to the protein; Elimination of voltage-dependent motility of OHC after removal of chloride.

Investigates reciprocal electromechanical properties of the rat homologue of prestin, a protein with similarity to pendrin-related anion transporters. Unique property of outer hair cells of the mammalian cohclea; Mechanical force generated by prestin in response to electrical stimulation; Mechanoelectrical properties of prestin.

Examines the structural and functional properties of the inward-rectifier potassium channels. Types of cells responsible for setting the resting membrane potential; Cause of the inward-rectification of potassium channels; Block of the channel pore by intracellular polyamines and magnesium.

Our hearing ability is exquisite in terms of sensitivity, dynamic range, and frequency discrimination. However, this acuity comes at the price of high vulnerability to a range of adverse influences—including sustained loud sounds. One may therefore expect that the ear is equipped with an alert system that can help prevent hearing damage, akin to the nociceptive sensory system that safeguards us from undergoing tissue injury. Here, Ceriani et al reveal a complex signaling interplay of sensory and non‐sensory cells that helps setting up such a system in development. [ABSTRACT FROM AUTHOR]

The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. The family shares a 7 + 7 transmembrane segments inverted repeat architecture with the SLC4 and SLC23 families, but holds a regulatory STAS domain in addition. While the only experimental SLC26 structure is monomeric, SLC26 proteins form structural and functional dimers in the lipid membrane. Here we resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize its functional relevance by combining PELDOR/DEER distance measurements and biochemical studies with MD simulations and spin-label ensemble refinement. Our structural model reveals a unique interface different from the SLC4 and SLC23 families. The functionally relevant STAS domain is no prerequisite for dimerization. Characterization of heterodimers indicates that protomers in the dimer functionally interact. The combined structural and functional data define the framework for a mechanistic understanding of functional cooperativity in SLC26 dimers. The SLC26 family of transporters maintains anion equilibria in all kingdoms of life. Here, the authors resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize it by PELDOR/DEER distance measurements, biochemical studies with MD simulations and spin-label ensemble refinement. [ABSTRACT FROM AUTHOR]

Voltage-gated K+ (KV) channels govern K+ ion flux across cell membranes in response to changes in membrane potential. They are formed by the assembly of four subunits, typically from the same family. Electrically silent KV channels (KVS), however, are unable to conduct currents on their own. It has been assumed that these KVS must obligatorily assemble with subunits from the KV2 family into heterotetrameric channels, thereby giving rise to currents distinct from those of homomeric KV2 channels. Herein, we show that KVS subunits indeed also modulate the activity, biophysical properties and surface expression of recombinant KV7 isoforms in a subunit-specific manner. Employing co-immunoprecipitation, and proximity labelling, we unveil the spatial coexistence of KVS and KV7 within a single protein complex. Electrophysiological experiments further indicate functional interaction and probably heterotetramer formation. Finally, single-cell transcriptomic analyses identify native cell types in which this KVS and KV7 interaction may occur. Our findings demonstrate that KV cross-family interaction is much more versatile than previously thought—possibly serving nature to shape potassium conductance to the needs of individual cell types. [ABSTRACT FROM AUTHOR]

The sensitivity of many ion channels to phosphatidylinositol-4,5-bisphosphate (PIP2) levels in the cell membrane suggests that PIP2 fluctuations are important and general signals modulating neuronal excitability. Yet the PIP2 dynamics of central neurons in their native environment remained largely unexplored. Here, we examined the behavior of PIP2 concentrations in response to activation of Gq-coupled neurotransmitter receptors in rat CA1 hippocampal neurons in situ in acute brain slices. Confocal microscopy of the PIP2-selective molecular sensors tubbyCT-GFP and PLCδ1-PH-GFP showed that pharmacological activation of muscarinic acetylcholine (mAChR) or group I metabotropic glutamate (mGluRI) receptors induces transient depletion of PIP2 in the soma as well as in the dendritic tree. The observed PIP2 dynamics were receptor-specific, with mAChR activation inducing stronger PIP2 depletion than mGluRI, whereas agonists of other Gαq-coupled receptors expressed in CA1 neurons did not induce measureable PIP2 depletion. Furthermore, the data show for the first time neuronal receptor-induced oscillations of membrane PIP2 concentrations. Oscillatory behavior indicated that neurons can rapidly restore PIP2 levels during persistent activation of Gq and PLC. Electrophysiological responses to receptor activation resembled PIP2 dynamics in terms of time course and receptor specificity. Our findings support a physiological function of PIP2 in regulating electrical activity. [ABSTRACT FROM AUTHOR]

The phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] acts as a signaling lipid at the plasma membrane (PM) with pleiotropic regulatory actions on multiple cellular processes. Signaling specificity might result from spatiotemporal compartmentalization of the lipid and from combinatorial binding of PI(4,5)P2 effector proteins to additional membrane components. Here, we analyzed the spatial distribution of tubbyCT, a paradigmatic PI(4,5)P2-binding domain, in live mammalian cells by total internal reflection fluorescence (TIRF) microscopy and molecular dynamics simulations. We found that unlike other well-characterized PI(4,5)P2 recognition domains, tubbyCT segregates into distinct domains within the PM. TubbyCT enrichment occurred at contact sites between PM and endoplasmic reticulum (ER) (i.e. at ER–PM junctions) as shown by colocalization with ER–PM markers. Localization to these sites was mediated in a combinatorial manner by binding to PI(4,5)P2 and by interaction with a cytosolic domain of extended synaptotagmin 3 (E-Syt3), but not other E-Syt isoforms. Selective localization to these structures suggests that tubbyCT is a novel selective reporter for a ER–PM junctional pool of PI(4,5)P2. Finally, we found that association with ER–PM junctions is a conserved feature of tubby-like proteins (TULPs), suggesting an as-yet-unknown function of TULPs [ABSTRACT FROM AUTHOR]

The article talks about Phosphoinositides (PIs) that have multifaceted signaling functions, acting as a cellular membrane identity code and providing instructive molecular signals in various pathways and membrane dynamics. Mentions, the impact of PIs on cellular processes is mediated by the binding of proteins to the membrane via PI recognition domains including pleckstrin homology (PH), phox (PX), Fab1-YOTB-Vac1- EEA1 (FYVE), and epsin N-terminal homology (ENTH) domains among others.