Differential contributions of voltage-gated potassium channel subunits in enhancing temporal coding in the bushy cells of the ventral cochlear nucleus.

Temporal coding precision of bushy cells in the ventral cochlear nucleus (VCN), critical for sound localization and communication, depends on the generation of rapid and temporally precise action potentials (APs). Voltage-gated potassium (Kv) channels are critically involved in this. The bushy cells in rat VCN express Kv1.1, 1.2, 1.3, 1.6, 3.1, 4.2, and 4.3 subunits. The Kv1.1 subunit contributes to the generation of a temporally precise single AP. However, the understanding of the functions of other Kv subunits expressed in the bushy cells is limited. Here, we investigated the functional diversity of Kv subunits concerning their contributions to temporal coding. We characterized the electrophysiological properties of the Kv channels with different subunits using whole cell patch-clamp recording and pharmacological methods. The neuronal firing pattern changed from single to multiple APs only when the Kv1.1 subunit was blocked. The Kv subunits, including the Kv1.1, 1.2, 1.6, or 3.1, were involved in enhancing temporal coding by lowering membrane excitability, shortening AP latencies, reducing jitter, and regulating AP kinetics. Meanwhile, all the Kv subunits contributed to rapid repolarization and sharpening peaks by narrowing half-width and accelerating fall rate, and the Kv1.1 subunit also affected the depolarization of AP. The Kv1.1, 1.2, and 1.6 subunits endowed bushy cells with a rapid time constant and a low input resistance of membrane for enhancing spike timing precision. The present results indicate that the Kv channels differentially affect intrinsic membrane properties to optimize the generation of rapid and reliable APs for temporal coding.NEW & NOTEWORTHY This study investigates the roles of Kv channels in effecting precision using electrophysiological and pharmacological methods in bushy cells. Different Kv channels have varying electrophysiological characteristics, which contribute to the interplay between changes in the membrane properties and regulation of neuronal excitability which then improve temporal coding. We conclude that the Kv channels are specialized to promote the precise and rapid coding of acoustic input by optimizing the generation of reliable APs.

Coarse-grained simulations of the gating current in the voltage-activated Kv1.2 channel.

Quantitative structure-based modeling of voltage activation of ion channels is very challenging. For example, it is very hard to reach converging results, by microscopic simulations while macroscopic treatments involve major uncertainties regarding key features. The current work overcomes some of the above challenges by using our recently developed coarse-grained (CG) model in simulating the activation of the Kv1.2 channel. The CG model has allowed us to explore problems that cannot be fully addressed at present by microscopic simulations, while providing insights on some features that are not usually considered in continuum models, including the distribution of the electrolytes between the membrane and the electrodes during the activation process and thus the physical nature of the gating current. Here, we demonstrate that the CG model yields realistic gating charges and free energy landscapes that allow us to simulate the fluctuating gating current in the activation processes. Our ability to simulate the time dependence of the fast gating current allows us to reproduce the observed trend and provides a clear description of its relationship to the landscape involved in the activation process.

Moving gating charges through the gating pore in a Kv channel voltage sensor.

Voltage sensor domains (VSDs) regulate ion channels and enzymes by transporting electrically charged residues across a hydrophobic VSD constriction called the gating pore or hydrophobic plug. How the gating pore controls the gating charge movement presently remains debated. Here, using saturation mutagenesis and detailed analysis of gating currents from gating pore mutations in the Shaker Kv channel, we identified statistically highly significant correlations between VSD function and physicochemical properties of gating pore residues. A necessary small residue at position S240 in S1 creates a "steric gap" that enables an intracellular access pathway for the transport of the S4 Arg residues. In addition, the stabilization of the depolarized VSD conformation, a hallmark for most Kv channels, requires large side chains at positions F290 in S2 and F244 in S1 acting as "molecular clamps," and a hydrophobic side chain at position I237 in S1 acting as a local intracellular hydrophobic barrier. Finally, both size and hydrophobicity of I287 are important to control the main VSD energy barrier underlying transitions between resting and active states. Taken together, our study emphasizes the contribution of several gating pore residues to catalyze the gating charge transfer. This work paves the way toward understanding physicochemical principles underlying conformational dynamics in voltage sensors.

Kv1.2 mediates heterosynaptic modulation of direct cortical synaptic inputs in CA3 pyramidal cells.

KEY POINTS: We investigated the cellular mechanisms underlying mossy fibre-induced heterosynaptic long-term potentiation of perforant path (PP) inputs to CA3 pyramidal cells. Here we show that this heterosynaptic potentiation is mediated by downregulation of Kv1.2 channels. The downregulation of Kv1.2 preferentially enhanced PP-evoked EPSPs which occur at distal apical dendrites. Such enhancement of PP-EPSPs required activation of dendritic Na(+) channels, and its threshold was lowered by downregulation of Kv1.2. Our results may provide new insights into the long-standing question of how mossy fibre inputs constrain the CA3 network to sparsely represent direct cortical inputs. ABSTRACT: A short high frequency stimulation of mossy fibres (MFs) induces long-term potentiation (LTP) of direct cortical or perforant path (PP) synaptic inputs in hippocampal CA3 pyramidal cells (CA3-PCs). However, the cellular mechanism underlying this heterosynaptic modulation remains elusive. Previously, we reported that repetitive somatic firing at 10 Hz downregulates Kv1.2 in the CA3-PCs. Here, we show that MF inputs induce similar somatic firing and downregulation of Kv1.2 in the CA3-PCs. The effect of Kv1.2 downregulation was specific to PP synaptic inputs that arrive at distal apical dendrites. We found that the somatodendritic expression of Kv1.2 is polarized to distal apical dendrites. Compartmental simulations based on this finding suggested that passive normalization of synaptic inputs and polarized distributions of dendritic ionic channels may facilitate the activation of dendritic Na(+) channels preferentially at distal apical dendrites. Indeed, partial block of dendritic Na(+) channels using 10 nm tetrodotoxin brought back the enhanced PP-evoked excitatory postsynaptic potentials (PP-EPSPs) to the baseline level. These results indicate that activity-dependent downregulation of Kv1.2 in CA3-PCs mediates MF-induced heterosynaptic LTP of PP-EPSPs by facilitating activation of Na(+) channels at distal apical dendrites.

Noncoding RNAs: new players in chronic pain.

Chronic pain, a common clinical symptom, is often treated inadequately or ineffectively in part due to the incomplete understanding of molecular mechanisms that initiate and maintain this disorder. Newly identified noncoding RNAs govern gene expression. Recent studies have shown that peripheral noxious stimuli drive expressional changes in noncoding RNAs and that these changes are associated with pain hypersensitivity under chronic pain conditions. This review first presents current evidence for the peripheral inflammation/nerve injury-induced change in the expression of two types of noncoding RNAs, microRNAs, and Kcna2 antisense RNA, in pain-related regions, particularly in the dorsal root ganglion. The authors then discuss how peripheral noxious stimuli induce such changes. The authors finally explore potential mechanisms of how expressional changes in dorsal root ganglion microRNAs and Kcna2 antisense RNA contribute to the development and maintenance of chronic pain. An understanding of these mechanisms may propose novel therapeutic strategies for preventing and/or treating chronic pain.

Intermediate states of the Kv1.2 voltage sensor from atomistic molecular dynamics simulations.

The response of a membrane-bound Kv1.2 ion channel to an applied transmembrane potential has been studied using molecular dynamics simulations. Channel deactivation is shown to involve three intermediate states of the voltage sensor domain (VSD), and concomitant movement of helix S4 charges 10-15 Å along the bilayer normal; the latter being enabled by zipper-like sequential pairing of S4 basic residues with neighboring VSD acidic residues and membrane-lipid head groups. During the observed sequential transitions S4 basic residues pass through the recently discovered charge transfer center with its conserved phenylalanine residue, F(233). Analysis indicates that the local electric field within the VSD is focused near the F(233) residue and that it remains essentially unaltered during the entire process. Overall, the present computations provide an atomistic description of VSD response to hyperpolarization, add support to the sliding helix model, and capture essential features inferred from a variety of recent experiments.

Molecular recognition and self-assembly special feature: Calix[4]arene-based conical-shaped ligands for voltage-dependent potassium channels.

Potassium channels are among the core functional elements of life because they underpin essential cellular functions including excitability, homeostasis, and secretion. We present here a series of multivalent calix[4]arene ligands that bind to the surface of voltage-dependent potassium channels (K(v)1.x) in a reversible manner. Molecular modeling correctly predicts the best candidates with a conical C(4) symmetry for optimal binding, and the effects on channel function are assessed electrophysiologically. Reversible inhibition was observed, without noticeable damage of the oocytes, for tetraacylguanidinium or tetraarginine members of the series with small lower rim O-substituents. Apparent binding constants were in the low micromolar range and had Hill coefficients of 1, consistent with a single site of binding. Suppression of current amplitude was accompanied by a positive shift in the voltage dependence of gating and slowing of both voltage sensor motion and channel opening. These effects are in keeping with expectations for docking in the central pore and interaction with the pore domain "turret."

Independent and cooperative motions of the Kv1.2 channel: voltage sensing and gating.

Voltage-gated potassium (Kv) channels, such as Kv1.2, are involved in the generation and propagation of action potentials. The Kv channel is a homotetramer, and each monomer is composed of a voltage-sensing domain (VSD) and a pore domain (PD). We analyzed the fluctuations of a model structure of Kv1.2 using elastic network models. The analysis suggested a network of coupled fluctuations of eight rigid structural units and seven hinges that may control the transition between the active and inactive states of the channel. For the most part, the network is composed of amino acids that are known to affect channel activity. The results suggested allosteric interactions and cooperativity between the subunits in the coupling between the motion of the VSD and the selectivity filter of the PD, in accordance with recent empirical data. There are no direct contacts between the VSDs of the four subunits, and the contacts between these and the PDs are loose, suggesting that the VSDs are capable of functioning independently. Indeed, they manifest many inherent fluctuations that are decoupled from the rest of the structure. In general, the analysis suggests that the two domains contribute to the channel function both individually and cooperatively.

Policing the ball: a new potassium channel subunit determines inactivation rate.

Inactivation of potassium currents during maintained firing results in a progressive increase in action potential width and neuronal excitability. In Kv1.1 channels, inactivation has attributed to a beta subunit that blocks the pore of the channel shortly after channel opening. In this issue of Neuron, Shulte and colleagues have identified a novel channel subunit whose interaction with Kv1.1 and the beta subunit prevents such inactivation. Mutations in this subunit lead to temporal lobe epilepsy.

The epilepsy-linked Lgi1 protein assembles into presynaptic Kv1 channels and inhibits inactivation by Kvbeta1.

The voltage-gated potassium (Kv) channel subunit Kv1.1 is a major constituent of presynaptic A-type channels that modulate synaptic transmission in CNS neurons. Here, we show that Kv1.1-containing channels are complexed with Lgi1, the functionally unassigned product of the leucine-rich glioma inactivated gene 1 (LGI1), which is causative for an autosomal dominant form of lateral temporal lobe epilepsy (ADLTE). In the hippocampal formation, both Kv1.1 and Lgi1 are coassembled with Kv1.4 and Kvbeta1 in axonal terminals. In A-type channels composed of these subunits, Lgi1 selectively prevents N-type inactivation mediated by the Kvbeta1 subunit. In contrast, defective Lgi1 molecules identified in ADLTE patients fail to exert this effect resulting in channels with rapid inactivation kinetics. The results establish Lgi1 as a novel subunit of Kv1.1-associated protein complexes and suggest that changes in inactivation gating of presynaptic A-type channels may promote epileptic activity.

Identification of amino acids in the pore region of Kv1.2 potassium channel that regulate its glycosylation and cell surface expression.

The Kv1.4 potassium channel is reported to exhibit higher cell surface expression than the Kv1.1 potassium channel when expressed as a homomer in cell lines. Kv1.4 also shows highly efficient trans-Golgi glycosylation whereas Kv1.1 is not glycosylated. The surface expression and glycosylation of Kv1.2 is intermediate between those of Kv1.1 and Kv1.4. Amino acid determinants controlling the surface expression of Kv1 channels were localized to the highly conserved pore region and both positive and negative determinants of Kv1.1 and Kv1.4 trafficking have been reported. In this study, we analyzed the effect of substituting amino acids in the pore region of Kv1.2 with the corresponding amino acid present in Kv1.1 or Kv1.4 on glycosylation and trafficking of Kv1.2. Mutations in the outer pore region of Kv1.2 of Arg(354) to Pro (corresponding to Kv1.4) and to Ala (corresponding to Kv1.1) enhanced and reduced, respectively, cell surface expression of Kv1.2. Mutations in a different outer pore region of Val(381) to Lys (Kv1.4) and Tyr (Kv1.1) both reduced the cell surface expression. In contrast, mutation in the deep pore region of Ser(371) to Thr (Kv1.4) markedly enhanced cell surface expression. These results suggest that the cell surface expression of Kv1.2 is regulated by specific amino acids in the pore region in a similar manner to Kv1.1 and Kv1.4, and that the cell surface expression of Kv1.2, a channel intermediate between Kv1.1 and Kv1.4, can be attributed to these specific residues.

From the gating charge response to pore domain movement: initial motions of Kv1.2 dynamics under physiological voltage changes.

Recent structures of the potassium channel provide an essential beginning point for explaining how the pore is gated between open and closed conformations by changes in membrane voltage. Yet, the molecular details of this process and the connections to transmembrane gradients are not understood. To begin addressing how changes within a membrane environment lead to the channel's ability to sense shifts in membrane voltage and to gate, we performed double-bilayer simulations of the Kv1.2 channel. These double-bilayer simulations enable us to simulate realistic voltage drops from resting potential conditions to depolarized conditions by changes in the bath conditions on each side of the bilayer. Our results show how the voltage sensor domain movement responds to differences in transmembrane potential. The initial voltage sensor domain movement, S4 in particular, is modulated by the gating charge response to changes in voltage and is initially stabilized by the lipid headgroups. We show this response is directly coupled to the initial stages of pore domain motion. Results presented here provide a molecular model for how the pre-gating process occurs in sequential steps: Gating charge response, movement and stabilization of the S4 voltage sensor domain, and movement near the base of the S5 region to close the pore domain.

Potassium channel opening: a subtle two-step.

Voltage-gated K(+) channels undergo a voltage-dependent conductance change that plays a key role in modulating cellular excitability. While the Open state is captured in crystal structures of Kv1.2 and a chimeric Kv1.2/Kv2.1 channel, the Close state and the mechanism of this transition are still a subject of debate. Here, we propose a model based on mutagenesis combined with measurements of both ionic and gating currents which is consistent with the idea that the Open state is the default state, the energy of the electric field being used to keep the channel closed. Our model incorporates an 'Activated state' where the bulk of sensor movement is completed without channel opening. The model accounts for the well characterized electrophysiology of the 'V2' and 'ILT' mutations in Shaker, where sensor movement and channel opening occur over distinct voltage ranges. Moreover, the model proposes relatively small protein rearrangements in going from the Activated to the Open state, consistent with the rapid transitions observed in single channel records of Shaker type channels at zero millivolts.

Sound Localization in Preweanling Mice Was More Severely Affected by Deleting the Kcna1 Gene Compared to Deleting Kcna2, and a Curious Inverted-U Course of Development That Appeared to Exceed Adult Performance Was Observed in All Groups.

The submillisecond acuity for detecting rapid spatial and temporal fluctuations in acoustic stimuli observed in humans and laboratory animals depends in part on select groups of auditory neurons that preserve synchrony from the ears to the binaural nuclei in the brainstem. These fibers have specialized synapses and axons that use a low-threshold voltage-activated outward current, IKL, conducted through Kv1 potassium ion channels. These are in turn coupled with HCN channels that express a mixed cation inward mixed current, IH, to support precise synchronized firing. The behavioral evidence is that their respective Kcna1 or HCN1 genes are absent in adult mice; the results are weak startle reflexes, slow responding to noise offsets, and poor sound localization. The present behavioral experiments were motivated by an in vitro study reporting increased IKL in an auditory nucleus in Kcna2-/- mice lacking the Kv1.2 subunit, suggesting that Kcna2-/- mice might perform better than Kcna2+/+ mice. Because Kcna2-/- mice have only a 17-18-day lifespan, we compared both preweanling Kcna2-/- vs. Kcna2+/+ mice and Kcna1-/- vs. Kcna1+/+ mice at P12-P17/18; then, the remaining mice were tested at P23/P25. Both null mutant strains had a stunted physique, but the Kcna1-/- mice had severe behavioral deficits while those in Kcna2-/- mice were relatively few and minor. The in vitro increase of IKL could have resulted from Kv1.1 subunits substituting for Kv1.2 units and the loss of the inhibitory "managerial" effect of Kv1.2 on Kv1.1. However, any increased neuronal synchronicity that accompanies increased IKL may not have been enough to affect behavior. All mice performed unusually well on the early spatial tests, but then, they fell towards adult levels. This unexpected effect may reflect a shift from summated independent monaural pathways to integrated binaural processing, as has been suggested for similar observations for human infants.

Inhibition of Kv2.1 Potassium Channels by MiDCA1, A Pre-Synaptically Active PLA2-Type Toxin from Micrurus dumerilii carinicauda Coral Snake Venom.

MiDCA1, a phospholipase A2 (PLA2) neurotoxin isolated from Micrurus dumerilii carinicauda coral snake venom, inhibited a major component of voltage-activated potassium (Kv) currents (41 ± 3% inhibition with 1 µM toxin) in mouse cultured dorsal root ganglion (DRG) neurons. In addition, the selective Kv2.1 channel blocker guangxitoxin (GxTx-1E) and MiDCA1 competitively inhibited the outward potassium current in DRG neurons. MiDCA1 (1 µM) reversibly inhibited the Kv2.1 current by 55 ± 8.9% in a Xenopus oocyte heterologous system. The toxin showed selectivity for Kv2.1 channels over all the other Kv channels tested in this study. We propose that Kv2.1 channel blockade by MiDCA1 underlies the toxin's action on acetylcholine release at mammalian neuromuscular junctions.

The sigma-1 receptor behaves as an atypical auxiliary subunit to modulate the functional characteristics of Kv1.2 channels expressed in HEK293 cells.

Expression of Kv1.2 within Kv1.x potassium channel complexes is critical in maintaining appropriate neuronal excitability and determining the threshold for action potential firing. This is attributed to the interaction of Kv1.2 with a hitherto unidentified protein that confers bimodal channel activation gating, allowing neurons to adapt to repetitive trains of stimulation and protecting against hyperexcitability. One potential protein candidate is the sigma-1 receptor (Sig-1R), which regulates other members of the Kv1.x channel family; however, the biophysical nature of the interaction between Sig-1R and Kv1.2 has not been elucidated. We hypothesized that Sig-1R may regulate Kv1.2 and may further act as the unidentified modulator of Kv1.2 activation. In transiently transfected HEK293 cells, we found that ligand activation of the Sig-1R modulates Kv1.2 current amplitude. More importantly, Sig-1R interacts with Kv1.2 in baseline conditions to influence bimodal activation gating. These effects are abolished in the presence of the auxiliary subunit Kvß2 and when the Sig-1R mutation underlying ALS16 (Sig-1R-E102Q), is expressed. These data suggest that Kvß2 occludes the interaction of Sig-1R with Kv1.2, and that E102 may be a residue critical for Sig-1R modulation of Kv1.2. The results of this investigation describe an important new role for Sig-1R in the regulation of neuronal excitability and introduce a novel mechanism of pathophysiology in Sig-1R dysfunction.

Expression of Kv1.2 in microglia and its putative roles in modulating production of proinflammatory cytokines and reactive oxygen species.

Microglial cells are endowed with different potassium ion channels but their expression and specific functions have remained to be fully clarified. This study has shown Kv1.2 expression in the amoeboid microglia in the rat brain between 1 (P1) and 10 (P10) days of age. Kv1.2 expression was localized in the ramified microglia at P14 and was hardly detected at P21. In postnatal rats exposed to hypoxia, Kv1.2 immunoreactivity in microglia was markedly enhanced. Quantitative RT-PCR analysis confirmed Kv1.2 mRNA expression in microglial cells in vitro. It was further shown that Kv1.2 and protein expression coupled with that of interleukin 1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) was significantly increased when the cells were subjected to hypoxia. The same increase was observed in cells exposed to adenosine 5'-triphosphate (ATP) and lipopolysaccharide (LPS). Concomitantly, the intracellular potassium concentration decreased significantly. Blockade of Kv1.2 channel with rTityustoxin-Kalpha (TsTx) resulted in partial recovery of intracellular potassium concentration accompanied by a reduced expression of IL-1beta and TNF-alpha mRNA and protein expression and intracellular reactive oxygen species (ROS) production. We conclude that Kv1.2 in microglia modulates IL-1beta and TNF-alpha expression and ROS production probably by regulating the intracellular potassium concentration.

Voltage gated K+ channel expression in arteries of Wistar-Kyoto and spontaneously hypertensive rats.

BACKGROUND: We have previously demonstrated differences in the gene expression of voltage-gated K v1.X channel alpha-subunits in arteries from Wistar-Kyoto rats (WKYs) and spontaneously hypertensive rats (SHRs). The purpose of this study was to test the hypothesis that these differences are also present at the protein level. METHODS: Proteins were isolated from the aorta, mesenteric (MAs) and tail arteries (TAs) of 12- to 15-week-old male WKY and SHR, and analyzed by immunoblotting. K(v) currents were recorded from MA myocytes by patch clamp methods. RESULTS: Expression of Kv1.2, Kv1.5, and Kv2.1 was higher in MAs but was not different in aortas of SHRs as compared to WKYs. In the TA, expression of Kv1.2 and Kv1.5 was higher while that of Kv2.1 was lower in SHR compared to WKY. In the MA, the larger expression of an 80 kDa species of Kv1.2 in SHRs was associated with a lower expression of a 60 kDa species. Kv2.1 gene expression was larger in MAs from SHRs but not different in TAs. K(v) currents associated with Kv1.X and Kv2.1 channels were both larger in MA myocytes from SHRs but less than expected based upon differences in K(v) alpha-subunit protein expression. CONCLUSIONS: For the MA, K(v) protein expression and current components between WKYs and SHRs were qualitatively consistent, but differences in gene and protein expression were not closely correlated. The higher expression of K(v) subunits in small mesenteric arteries (SMAs) of SHR would tend to maintain normal myogenic activity and vasoconstrictor reserve, and could be viewed as a form of homeostatic remodeling.

An activation gating switch in Kv1.2 is localized to a threonine residue in the S2-S3 linker.

The activation properties of Kv1.2 channels are highly variable, with reported half-activation (V((1/2))) values ranging from approximately -40 mV to approximately +30 mV. Here we show that this arises because Kv1.2 channels occupy two distinct gating modes ("fast" and "slow"). "Slow" gating (tau(act) = 90 +/- 6 ms at +35 mV) was associated with a V((1/2)) of activation of +16.6 +/- 1.1 mV, whereas "fast" gating (tau(act) = 4.5 +/- 1.7 ms at +35 mV) was associated with a V((1/2)) of activation of -18.8 +/- 2.3 mV. It was possible to switch between gating modes by applying a prepulse, which suggested that channels activate to a single open state along separate "fast" and "slow" activation pathways. Using chimeras and point mutants between Kv1.2 and Kv1.5 channels, we determined that introduction of a positive charge at or around threonine 252 in the S2-S3 linker of Kv1.2 abolished "slow" activation gating. Furthermore, dialysis of the cytoplasm or excision of cell-attached patches from cells expressing Kv1.2 channels switched gating from "slow" to "fast", suggesting involvement of cytoplasmic regulators. Collectively, these results demonstrate two modes of activation gating in Kv1.2 and specific residues in the S2-S3 linker that act as a switch between these modes.

Kv1.1/1.2 channels are downstream effectors of nitric oxide on synaptic GABA release to preautonomic neurons in the paraventricular nucleus.

The paraventricular nucleus (PVN) of the hypothalamus is important for the neural regulation of cardiovascular function. Nitric oxide (NO) increases synaptic GABA release to presympathetic PVN neurons through the cyclic guanosine monophosphate (cGMP)/protein kinase G signaling pathway. However, the downstream signaling mechanisms underlying the effect of NO on synaptic GABA release remain unclear. In this study, whole-cell voltage-clamp recordings were performed on retrograde-labeled spinally projecting PVN neurons in rat brain slices. Bath application of the NO precursor l-arginine or the NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly increased the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in labeled PVN neurons. A specific antagonist of cyclic ADP ribose, 8-bromo-cyclic ADP ribose (8-Br-cADPR), had no significant effect on l-arginine-induced potentiation of mIPSCs. Surprisingly, blocking of voltage-gated potassium channels (Kv) with 4-aminopyridine or alpha-dendrotoxin eliminated the effect of l-arginine on mIPSCs in all labeled PVN neurons tested. The membrane permeable cGMP analog mimicked the effect of l-arginine on mIPSCs, and this effect was blocked by alpha-dendrotoxin. Furthermore, the specific Kv channel blocker for Kv1.1 (dendrotoxin-K) or Kv1.2 (tityustoxin-Kalpha) abolished the effect of l-arginine on mIPSCs in all neurons tested. SNAP failed to inhibit the firing activity of labeled PVN neurons in the presence of dendrotoxin-K, Kalpha. Additionally, the immunoreactivity of Kv1.1 and Kv1.2 subunits was colocalized extensively with synaptophysin in the PVN. These findings suggest that NO increases GABAergic input to PVN presympathetic neurons through a downstream mechanism involving the Kv1.1 and Kv1.2 channels at the nerve terminals.