Modulation Of Cell Excitability Research Articles

Optogenetic polymerization and assembly of electrically functional polymers for modulation of single-neuron excitability.

Ionic conductivity and membrane capacitance are two foundational parameters that govern neuron excitability. Conventional optogenetics has emerged as a powerful tool to temporarily manipulate membrane ionic conductivity in intact biological systems. However, no analogous method exists for precisely manipulating cell membrane capacitance to enable long-lasting modulation of neuronal excitability. Genetically targetable chemical assembly of conductive and insulating polymers can modulate cell membrane capacitance, but further development of this technique has been hindered by poor spatiotemporal control of the polymer deposition and cytotoxicity from the widely diffused peroxide. We address these issues by harnessing genetically targetable photosensitizer proteins to assemble electrically functional polymers in neurons with precise spatiotemporal control. Using whole-cell patch-clamp recordings, we demonstrate that this optogenetic polymerization can achieve stepwise modulation of both neuron membrane capacitance and intrinsic excitability. Furthermore, cytotoxicity can be limited by controlling light exposure, demonstrating a promising new method for precisely modulating cell excitability.

Unraveling the Role of K2P Channels in Atrial Fibrillation.

Atrial fibrillation (AF) is a condition in which the electrical signals in the upper heart chambers (atria) are rapid and disorganized, producing an irregular and chaotical heartbeat. The sinus rhythm should be between 60 to 100 bpm at rest, while the heart rhythm in AF patients may be over 140 bpm. Either structural and electro-mechanical remodeling of the atrial tissue underlies the perpetuation and evolution of AF from the paroxysmal to persistent form. Unravelling the different pathological pathways involved in AF that lead to arrhythmogenesis and atrial remodeling is needed to discovery new and effective therapeutic approaches. A variety of drugs are available to convert and maintain the AF patient in a normal sinus rhythm; however, these strategies have limited chances of success or fail with the progression of AF to more persistent/permanent forms. Consequently, it is necessary to find new therapeutic targets for the relief of persistent or chronic AF forms, as well as the development of new and more effective pharmacological tools. The atrial specific two-pore domain K+ channels (K2P) constitute the background K+ current on atrial cardiomyocytes and modulate cell excitability emerging as novel targets in this disease and avoiding ventricle side effects. Moreover, several antiarrhythmic drugs used in AF treatment exert their mechanism of action in part by modulation of K2P channels. Thus far, TWIK-1, TREK-1, TASK-1, TASK-2 and TASK-3 channel have been identified as responsible for background currents IK2P current in atrial cells; however, it is not excluded that other K2PX subunits or subfamilies have physiological roles in atria. To date, a great diversity openers, activators and blockers of K2P channel have been identified, particularly those targeting TASK and TREK channels. Several studies have demonstrated that the expression of TWIK-1, TREK-1, TASK-1, TASK-2 and TASK-3 are dysregulated in AF and their pharmacology rescue could suppose a novel therapy in AF. The main objective is to examine the regulation of K2P channels and the current K2P channels pharmacological modulators for AF treatment.

Structures of Gi-bound metabotropic glutamate receptors mGlu2 and mGlu4.

The metabotropic glutamate receptors (mGlus) have key roles in modulating cell excitability and synaptic transmission in response to glutamate (the main excitatory neurotransmitter in the central nervous system)1. It has previously been suggested that only one receptor subunit within an mGlu homodimer is responsible for coupling to Gprotein during receptor activation2. However, the molecular mechanism that underlies the asymmetric signalling of mGlus remains unknown. Here we report two cryo-electron microscopy structures of human mGlu2 and mGlu4 bound to heterotrimeric Gi protein. The structures reveal a G-protein-binding site formed by three intracellular loops and helicesIII and IV that is distinct from the corresponding binding site in all ofthe other G-protein-coupled receptor (GPCR) structures. Furthermore, we observed an asymmetric dimer interface of the transmembrane domain of the receptor in the two mGlu-Gi structures. We confirmed that the asymmetric dimerization is crucial for receptor activation, which was supported by functional data; this dimerization may provide a molecular basis for the asymmetric signal transduction of mGlus. These findings offer insights into receptor signalling of classC GPCRs.

TTX-resistant sodium currents in medial prefrontal cortex pyramidal neurons depend on extracellular Ca2+ concentration

Background: Previous reports reported the presence of TTX-resistant Nav1.8 and Nav1.9 sodium chan­nels in the cortex pyramidal neurons. A characteristic feature of Nav1.9 channels is activation at voltages close to — 70 mV. Therefore, they do not participate directly in the action potential but contribute to the regulation of the resting membrane potential. Their physiological role is modulation of cell excitability. The aim of the study was to investigate, with the use of patch clamp technique, the dependence of the activation thresholds of TTX–resistant sodium currents on the concentration of extracellular calcium in medial prefrontal cortex pyramidal neurons in rats. Results: The recorded values of the threshold of the voltage-dependent sodium currents were in the range of -65 mV to -75 mV. This suggests that the sodium currents may result from the presence of Nav1.9 channels in the rat pyramidal neurons. The threshold for the activation of sodium currents depended on the concentration of Ca2+. Increasing the concentration of calcium ions in the extracellular solution by 5 mM caused the depolarizing shift in the activation potential by about 10 mV. The effect of calcium ion concentration on the potential of TTX-resistant currents activation suggests that Nav1.9 channels are modulated by extracellular Ca2+ concentration. Conclusions: The study has experimentally confirmed that TTX-resistant channels are present in the cell membrane of the rat prefrontal cortex pyramidal neurons and may, therefore, take a physiological role in the conductivity of sodium currents in a manner dependent on the concentration of extracellular ions.

Trophic modulation of gamma oscillations: The key role of processing protease for Neuregulin-1 and BDNF precursors

Gamma oscillations within the cerebral cortex and hippocampus are associated with cognitive processes, including attention, sensory perception, and memory formation; a deficit in gamma regulation is a common symptom of neurologic and psychiatric disorders. Accumulating evidence has suggested that gamma oscillations result from the synchronized activity of cell assemblies coordinated mainly by parvalbumin-positive inhibitory interneurons. The modulator molecules for parvalbumin-positive interneurons are major research targets and have the potential to control the specific oscillatory rhythm and behavior originating from neural coordination. Neuregulin-1 and brain-derived neurotrophic factor have been focused on as synaptic trophic factors that are associated with gamma oscillations. Synaptic activity converts precursor trophic factors into their biologically active forms by proteolytic cleavage, which could, in turn, modulate cell excitability and synaptic plasticity through each receptor's signaling. From these findings, the processing of trophic factors by proteases in a synaptic microenvironment might involve gamma oscillations during cognition. Here, we review the trophic modulation of gamma oscillations through extracellular proteolysis and its implications in neuronal diseases.

Styrax blocks inward and outward current of Kir2.1 channel

ABSTRACTKir2.1 plays key roles in setting rest membrane potential and modulation of cell excitability. Mutations of Kir2.1, such as D172N or E299V, inducing gain-of-function, can cause type3 short QT syndrome (SQT3) due to the enlarged outward currents. So far, there is no clinical drug target to block the currents of Kir2.1. Here, we identified a novel blocker of Kir2.1, styrax, which is a kind of natural compound selected from traditional Chinese medicine. Our data show that styrax can abolish the inward and outward currents of Kir2.1. The IC50 of styrax for WT, D172N and E299V are 0.0113 ± 0.00075, 0.0204 ± 0.0048 and 0.0122 ± 0.0012 (in volume), respectively. The results indicate that styrax can serve as a novel blocker for Kir2.1.

Modulation of the input-output function by GABAA receptor-mediated currents in rat oculomotor nucleus motoneurons.

The neuronal input–output function depends on recruitment threshold and gain of the firing frequency–current (f–I) relationship. These two parameters are positively correlated in ocular motoneurons (MNs) recorded in alert preparation and inhibitory inputs could contribute to this correlation. Phasic inhibition mediated by γ-amino butyric acid (GABA) occurs when a high concentration of GABA at the synaptic cleft activates postsynaptic GABAA receptors, allowing neuronal information transfer. In some neuronal populations, low concentrations of GABA activate non-synaptic GABAA receptors and generate a tonic inhibition, which modulates cell excitability. This study determined how ambient GABA concentrations modulate the input–output relationship of rat oculomotor nucleus MNs. Superfusion of brain slices with GABA (100 μm) produced a GABAA receptor-mediated current that reduced the input resistance, increased the recruitment threshold and shifted the f–I relationship rightward without any change in gain. These modifications did not depend on MN size. In absence of exogenous GABA, gabazine (20 μm; antagonist of GABAA receptors) abolished spontaneous inhibitory postsynaptic currents and revealed a tonic current in MNs. Gabazine increased input resistance and decreased recruitment threshold mainly in larger MNs. The f–I relationship shifted to the left, without any change in gain. Gabazine effects were chiefly due to MN tonic inhibition because tonic current amplitude was five-fold greater than phasic. This study demonstrates a tonic inhibition in ocular MNs that modulates cell excitability depending on cell size. We suggest that GABAA tonic inhibition acting concurrently with glutamate receptors activation could reproduce the positive covariation between threshold and gain reported in alert preparation.

Potent and selective inhibitors of the TASK-1 potassium channel through chemical optimization of a bis-amide scaffold

TASK-1 is a two-pore domain potassium channel that is important to modulating cell excitability, most notably in the context of neuronal pathways. In order to leverage TASK-1 for therapeutic benefit, its physiological role needs better characterization; however, designing selective inhibitors that avoid the closely related TASK-3 channel has been challenging. In this study, a series of bis-amide derived compounds were found to demonstrate improved TASK-1 selectivity over TASK-3 compared to reported inhibitors. Optimization of a marginally selective hit led to analog 35 which displays a TASK-1 IC50=16nM with 62-fold selectivity over TASK-3 in an orthogonal electrophysiology assay.

Disulfide Cross-linking of Transport and Trimerization Domains of a Neuronal Glutamate Transporter Restricts the Role of the Substrate to the Gating of the Anion Conductance

Excitatory amino acid transporters remove synaptically released glutamate and maintain its concentrations below neurotoxic levels. EAATs also mediate a thermodynamically uncoupled substrate-gated anion conductance that may modulate cell excitability. A structure of an archeal homologue, which reflects an early intermediate on the proposed substrate translocation path, has been suggested to be similar to an anion conducting conformation. To probe this idea by functional studies, we have introduced two cysteine residues in the neuronal glutamate transporter EAAC1 at positions predicted to be close enough to form a disulfide bond only in outward-facing and early intermediate conformations of the homologue. Upon treatment of Xenopus laevis oocytes expressing the W441C/K269C double mutant with dithiothreitol, radioactive transport was stimulated >2-fold but potently inhibited by low micromolar concentrations of the oxidizing reagent copper(II)(1,10-phenanthroline)3. The substrate-induced currents by the untreated double mutant, reversed at approximately -20 mV, close to the reversal potential of chloride, but treatment with dithiothreitol resulted in transport currents with the same voltage dependence as the wild type. It appears therefore that in the oocyte expression system the introduced cysteine residues in many of the mutant transporters are already cross-linked and are only capable of mediating the substrate-gated anion conductance. Reduction of the disulfide bond now allows these transporters to execute the full transport cycle. Our functional data support the idea that the anion conducting conformation of the neuronal glutamate transporter is associated with an early step of the transport cycle.

Reductions in External Divalent Cations Evoke Novel Voltage-Gated Currents in Sensory Neurons

It has long been recognized that divalent cations modulate cell excitability. Sensory nerve excitability is of critical importance to peripheral diseases associated with pain, sensory dysfunction and evoked reflexes. Thus we have studied the role these cations play on dissociated sensory nerve activity. Withdrawal of both Mg2+ and Ca2+ from external solutions activates over 90% of dissociated mouse sensory neurons. Imaging studies demonstrate a Na+ influx that then causes depolarization-mediated activation of voltage-gated Ca2+ channels (CaV), which allows Ca2+ influx upon divalent re-introduction. Inhibition of CaV (ω-conotoxin, nifedipine) or NaV (tetrodotoxin, lidocaine) fails to reduce the Na+ influx. The Ca2+ influx is inhibited by CaV inhibitors but not by TRPM7 inhibition (spermine) or store-operated channel inhibition (SKF96365). Withdrawal of either Mg2+ or Ca2+ alone fails to evoke cation influxes in vagal sensory neurons. In electrophysiological studies of dissociated mouse vagal sensory neurons, withdrawal of both Mg2+ and Ca2+ from external solutions evokes a large slowly-inactivating voltage-gated current (IDF) that cannot be accounted for by an increased negative surface potential. Withdrawal of Ca2+ alone fails to evoke IDF. Evidence suggests IDF is a non-selective cation current. The IDF is not reduced by inhibition of NaV (lidocaine, riluzole), CaV (cilnidipine, nifedipine), KV (tetraethylammonium, 4-aminopyridine) or TRPM7 channels (spermine). In summary, sensory neurons express a novel voltage-gated cation channel that is inhibited by external Ca2+ (IC50∼0.5 µM) or Mg2+ (IC50∼3 µM). Activation of this putative channel evokes substantial cation fluxes in sensory neurons.

Ion Channel Screening: Advances in Technologies and Analysis

EDITORIAL article Front. Pharmacol., 07 May 2012Sec. Pharmacology of Ion Channels and Channelopathies Volume 3 - 2012 | https://doi.org/10.3389/fphar.2012.00086

Identification and characterization of alternative splice variants of the mouse Trek2/Kcnk10 gene

Two-pore domain K(+) (K(2P)) channels underlie leak or background potassium conductances in many cells. The Trek subfamily of K(2P) channels, which includes Trek1/Kcnk2 and Trek2/Kcnk10 and has been implicated in depression, nociception, and cognition, exhibits complex regulation and can modulate cell excitability in response to a wide array of stimuli. While alternative translation initiation and alternative splicing contribute to the structural and functional diversity of Trek1, the impact of post-transcriptional modifications on the expression and function of Trek2 is unclear. Here, we characterized two novel splice isoforms of the mouse Trek2 gene. One variant is a truncated form of Trek2 that possesses two transmembrane segments and one pore domain (Trek2-1p), while the other (Trek2b) differs from two known mouse Trek2 isoforms (Trek2a and Trek2c) at the extreme amino terminus. Both Trek2-1p and Trek2b, and Trek2a and Trek2c, showed prominent expression in the mouse CNS. Expression patterns of the Trek2 variants within the CNS were largely overlapping, though some isoform-specific differences were noted. Heterologous expression of Trek2-1p yielded no novel whole-cell currents in transfected human embryonic kidney (HEK) 293 cells. In contrast, expression of Trek2b correlated with robust K(+) currents that were ~fivefold larger than currents measured in cells expressing Trek2a or Trek2c, a difference mirrored by significantly higher levels of Trek2b found at the plasma membrane. This study provides new insights into the molecular diversity of Trek channels and suggests a potential role for the Trek2 amino terminus in channel trafficking and/or stability.

Cannabinoids modulate spontaneous synaptic activity in retinal ganglion cells

The endocannabinoid (ECB) system has been found throughout the central nervous system and modulates cell excitability in various forms of short-term plasticity. ECBs and their receptors have also been localized to all retinal cells, and cannabinoid receptor activation has been shown to alter voltage-dependent conductances in several different retinal cell types, suggesting a possible role for cannabinoids in retinal processing. Their effects on synaptic transmission in the mammalian retina, however, have not been previously investigated. Here, we show that exogenous cannabinoids alter spontaneous synaptic transmission onto retinal ganglion cells (RGCs). Using whole-cell voltage-clamp recordings in whole-mount retinas, we measured spontaneous postsynaptic currents (SPSCs) in RGCs in adult and young (P14-P21) mice. We found that the addition of an exogenous cannabinoid agonist, WIN55212-2 (5 μM), caused a significant reversible reduction in the frequency of SPSCs. This change, however, did not alter the kinetics of the SPSCs, indicating a presynaptic locus of action. Using blockers to isolate inhibitory or excitatory currents, we found that cannabinoids significantly reduced the release probability of both GABA and glutamate, respectively. While the addition of cannabinoids reduced the frequency of both GABAergic and glutamatergic SPSCs in both young and adult mice, we found that the largest effect was on GABA-mediated currents in young mice. These results suggest that the ECB system may potentially be involved in the modulation of signal transmission in the retina. Furthermore, they suggest that it might play a role in the developmental maturation of synaptic circuits, and that exogenous cannabinoids are likely able to disrupt retinal processing and consequently alter vision.

KCa1.1 channel contributes to cell excitability in unmyelinated but not myelinated rat vagal afferents

High conductance calcium-activated potassium (BK(Ca)) channels can modulate cell excitability and neurotransmitter release at synaptic and afferent terminals. BK(Ca) channels are present in primary afferents of most, if not, all internal organs and are an intriguing target for pharmacological manipulation of visceral sensation. Our laboratory has a long-standing interest in the neurophysiological differences between myelinated and unmyelinated visceral afferent function. Here, we seek to determine whether there is a differential distribution of BK(Ca) channels in myelinated and unmyelinated vagal afferents. Immunocytochemistry studies with double staining for the BK-type K(Ca)1.1 channel protein and isolectin B4 (IB4), a reliable marker of unmyelinated peripheral afferents, reveal a pattern of IB4 labeling that strongly correlates with the expression of the K(Ca)1.1 channel protein. Measures of cell size and immunostaining intensity for K(Ca)1.1 and IB4 cluster into two statistically distinct (P < 0.05) populations of cells. Smaller diameter neurons most often presented with strong IB4 labeling and are presumed to be unmyelinated (n = 1,390) vagal afferents. Larger diameter neurons most often lacked or exhibited a very weak IB4 labeling and are presumed to be myelinated (n = 58) vagal afferents. Complimentary electrophysiological studies reveal that the BK(Ca) channel blockers charybdotoxin (ChTX) and iberiotoxin (IbTX) bring about a comparable elevation in excitability and action potential widening in unmyelinated neurons but had no effect on the excitability of myelinated vagal afferents. This study is the first to demonstrate using combined immunohistochemical and electrophysiological techniques that K(Ca)1.1 channels are uniquely expressed in unmyelinated C-type vagal afferents and do not contribute to the dynamic discharge characteristics of myelinated A-type vagal afferents. This unique functional distribution of BK-type K(Ca) channels may provide an opportunity for afferent selective pharmacological intervention across a wide range of visceral pathophysiologies, particularly those with a reflexogenic etiology and pain.

Activation of TREK Currents by the Neuroprotective Agent Riluzole in Mouse Sympathetic Neurons

Background K2P channels play a key role in stabilizing the resting membrane potential, thereby modulating cell excitability in the central and peripheral somatic nervous system. Whole-cell experiments revealed a riluzole-activated current (I(RIL)), transported by potassium, in mouse superior cervical ganglion (mSCG) neurons. The activation of this current by riluzole, linoleic acid, membrane stretch, and internal acidification, its open rectification and insensitivity to most classic potassium channel blockers, indicated that I(RIL) flows through channels of the TREK [two-pore domain weak inwardly rectifying K channel (TWIK)-related K channel] subfamily. Whole-ganglia and single-cell reverse transcription-PCR demonstrated the presence of TREK-1, TREK-2, and TRAAK (TWIK-related arachidonic acid-activated K(+) channel) mRNA, and the expression of these three proteins was confirmed by immunocytochemistry in mSCG neurons. I(RIL) was enhanced by zinc, inhibited by barium and fluoxetine, but unaffected by quinine and ruthenium red, strongly suggesting that it was carried through TREK-1/2 channels. Consistently, a channel with properties identical with the heterologously expressed TREK-2 was recorded in most (75%) cell-attached patches. These results provide the first evidence for the expression of K2P channels in the mammalian autonomic nervous system, and they extend the impact of these channels to the entire nervous system.

Galantamine inhibits slowly inactivating K + currents with a dual dose–response relationship in differentiated N1E-115 cells and in CA1 neurones

Galantamine, one of the major drugs used in Alzheimer's disease therapy, is a relatively weak acetylcholinesterase inhibitor and an allosteric potentiating ligand of nicotinic acetylcholine receptors. However, a role in the control of excitability has also been attributed to galantamine via modulation of K + currents in central neurones. To further investigate the effect of galantamine on voltage-activated K + currents, we performed whole-cell voltage-clamp recordings in differentiated neuroblastoma N1E-115 cells and in dissociated rat CA1 neurones. In both cell models, one can identify two main voltage-activated K + current components: a relatively fast inactivating component ( Ifast; time constant ≈ hundred milliseconds) and a slowly inactivating one ( Islow; time constant ≈ 1 s). We show that galantamine (1 pM–300 µM) inhibits selectively Islow, exhibiting a dual dose–response relationship, in both differentiated N1E-115 cells and CA1 neurones. We also demonstrate that, in contrast with what was previously reported, galantamine-induced inhibition is not due to a shift on the steady-state inactivation and activation curves. Additionally, we characterized a methodological artefact that affects voltage-dependence as a function of time in whole-cell configuration, observed in both cell models. By resolving an inhibitory role on K + currents in a non-central neuronal system and in hippocampal neurones, we are attributing a widespread role of galantamine on the modulation of cell excitability. The present results are relevant in the clinical context, since the effects at low dosages suggest that galantamine-induced K + current inhibition may contribute to the efficiency of galantamine in the treatment of Alzheimer's disease.

Inhibition of hyperpolarization-activated cation currents by phencyclidine and some sigma ligands in rat hippocampal CA1 pyramidal neurons in vitro

Using whole-cell voltage-clamp recordings, hyperpolarization-activated cation currents ( I h) were elicited with hyperpolarizing voltage jumps in CA1 pyramidal neurons of rat hippocampal slices, and the effects of phencyclidine (PCP) and some sigma ligands on I h were studied. PCP concentration-dependently (0.1–100 μM) suppressed I h and shifted the activation curve of I h to the negative direction. d-3-(2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP, 20 μM) and MK-801 (30 μM), competitive and non-competitive NMDA blockers, respectively, failed to mimic the inhibitory effect of PCP on I h, and suppression of I h by PCP was unaffected in the presence of these blockers. To explore the involvement of sigma 1 receptors in the reduction of I h, the effects of representative sigma 1 ligands were studied. SKF10047 (100 μM), a sigma 1 agonist, attenuated the maximal I h and shifted the half-activation potential of I h to the hyperpolarized direction. In the presence of the sigma 1 antagonist NE-100 (1 μM), which alone did not affect I h, the effect of SKF10047 on I h was unaltered. By contrast, a higher concentration of NE-100 (10 μM) mimicked the effect of SKF10047. Again, no antagonism of I h suppression by SKF10047 was obtained with rimcazole (100 μM), a sigma 1 receptor antagonist that is structurally distinct from NE-100. This concentration of rimcazole alone resulted in a slight but significant reduction of I h. Thus these major sigama 1 ligands appear to suppress I h independently of their agonistic or antagonistic properties. The results of this study suggest that PCP and some sigma ligands could modulate cell excitability partly through their action on I h.

Brain‐derived neurotrophic factor modulates cell excitability in the mouse medial nucleus of the trapezoid body

Neurotrophins are a large class of trophic factors located throughout the central nervous system. While the role of neurotrophins in neuronal survival and axon guidance is well known, their secondary role in modulating synaptic transmission and cell firing properties is largely unexplored. In this study we examined the expression of neurotrophins in the mouse medial nucleus of the trapezoid body (MNTB) and investigated the effect of exogenous brain-derived neurotrophic factor (BDNF) application on the firing properties of MNTB principal cells. The expression levels of nerve growth factor, BDNF, neurotrophin-3, neurotrophin-4/5 and major receptor tyrosine kinase B was found to be moderate to high at postnatal day 12, indicating that the neurotrophins may have a role following synaptogenesis. A 2-h exposure to exogenous BDNF (100 ng/mL) had a significant effect on principal cell firing properties and voltage-gated potassium currents. Importantly, preincubation in BDNF increased the incidence of multifiring and rebounding cells, and significantly increased the number of action potentials fired in response to a single depolarizing step. BDNF exposure also significantly decreased underlying voltage-gated potassium currents, including both the low- and high-voltage-activated components. Our data show that the neurotrophins, specifically BDNF, may have a novel role in modulating cell excitability in the auditory brainstem.

Variable Kainate Receptor Distributions of Oriens Interneurons

Interneuron kainate receptor (KAR) activation regulates normal network activity and modulates cell excitability. As a result, determining the subcellular distribution of KARs in a cell-specific manner is a necessary step toward understanding their role in network function. We have functionally mapped synaptic and extrasynaptic dendritic KARs on hippocampal oriens interneurons using local photolysis of caged glutamate. We find that the majority of trilaminar and oriens lacunosum-moleculare (O-LM) cells have uniform and continuous current densities along the lengths of their dendrites. However, there is a subpopulation of interneurons that have no KAR currents or currents exclusively at "hot spots" on the soma and dendrites. Finally, bistratified cells have KAR currents on all dendrites except those extending into the stratum radiatum. Thus KARs are functionally distributed in a cell-specific and cell-independent manner that may reflect the physiologically distinct roles they play in the hippocampal network.

Modulation of Kv2.1 channel gating and TEA sensitivity by distinct domains of SNAP-25

Distinct domains within the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins, STX1A (syntaxin 1A) and SNAP-25 (synaptosome-associated protein-25 kDa), regulate hormone secretion by their actions on the cell's exocytotic machinery, as well as voltage-gated Ca2+ and K+ channels. We examined the action of distinct domains within SNAP-25 on Kv2.1 (voltage gated K+ 2.1) channel gating. Dialysis of N-terminal SNAP-25 domains, S197 (SNAP-25(1-197)) and S180 (SNAP-25(1-180)), but not S206 (full-length SNAP-25(1-206)) increased the rate of Kv2.1 channel activation and slowed channel inactivation. Remarkably, these N-terminal SNAP-25 domains, acting on the Kv2.1 cytoplasmic N-terminus, potentiated the external TEA (tetraethylammonium)-mediated block of Kv2.1. To further examine whether these are effects of the channel pore domain, internal K+ was replaced with Na+ and external K+ was decreased from 4 to 1 mM, which decreased the IC50 of the TEA block from 6.8+/-0.9 mM to >100 mM. Under these conditions S180 completely restored TEA sensitivity (7.9+/-1.5 mM). SNAP-25 C-terminal domains, SNAP-25(198-206) and SNAP-25(181-197), had no effect on Kv2.1 gating kinetics. We conclude that different domains within SNAP-25 can form distinct complexes with Kv2.1 to execute a fine allosteric regulation of channel gating and the architecture of the outer pore structure in order to modulate cell excitability.