Kv Channel Activity Research Articles

Characterization of a novel high-potency positive modulator of Kv7 channels

Kv7 channel activators decrease neuronal excitability and might potentially treat neuronal hyperexcitability disorders like epilepsy and mania. Here we introduce NS15370 ((2-(3,5-difluorophenyl)-N-[6-[(4-fluorophenyl)methylamino]-2-morpholino-3-pyridyl]acetamide)hydrochloride, an in vitro high-potency chemical analogue of retigabine, without effects on GABAA receptors. NS15370 activates recombinant homo- and heteromeric Kv7.2–Kv7.5 channels in HEK293 cells at sub-micromolar concentrations (EC50∼100nM, as quantified by a fluorescence based Tl+-influx assay). In voltage clamp experiments NS15370 exhibits a complex, concentration-dependent mode-of-action: At low concentrations it accelerates voltage-dependent activation rates, slows deactivations, and increases steady-state current amplitudes. Quantified by the peak-tail current method, the V½ value of the steady-state activation curve is shifted towards hyperpolarized potentials at concentrations ∼100 times lower than retigabine. However, in contrast to retigabine, NS15370 also introduces a distinct time-dependent current decrease, which eventually, at higher concentrations, causes suppression of the current at depolarized potentials, and an apparent “cross-over“ of the voltage-activation curve. In brain slices, NS15370 hyperpolarizes and increases spike frequency adaptation of hippocampal CA1 neurons and the compound reduces the autonomous firing of dopaminergic neurons in the substantia-nigra pars compacta. NS15370 is effective in rodent models of hyperexcitability: (i) it yields full protection against mouse 6Hz seizures and rat amygdala kindling discharges, two models of partial epilepsia; (ii) it reduces (+)-MK-801 hydrogen maleate (MK-801)-induced hyperactivity as well as chlordiazepoxide (CDP)+d-amphetamine (AMP)-induced hyperactivity, models sensitive to classic anti-psychotic and anti-manic treatments, respectively. Our findings with NS15370 consolidate neuronal Kv7 channels as targets for anti-epileptic and psychiatric drug development.

Functional implications of Kv7 channels expression in guinea pig urinary bladder smooth muscle contractility

The physiological roles of Kv7 channel family have been well studied in various tissues. However, knowledge about the functional role of Kv7 channels in the urinary bladder smooth muscle (UBSM) is limited. We investigated Kv7 channels molecular expression in guinea pig UBSM and further assessed their functional role using isometric DSM tension recordings and Kv7 channels modulators. RT‐PCR experiments revealed mRNA expression for Kv7.1, Kv7.2, Kv7.3, Kv7.4, and Kv7.5 channel subunits in guinea pig UBSM tissue. Quantitative PCR analysis showed that in UBSM tissue, Kv7 channel subunits relative mRNA expression level was as follows: Kv7.1 ≈ Kv7.2 >; Kv7.3 ≈ Kv7.5 >; Kv7.4. Retigabine, a Kv7 channel activator, and L‐364,373, a selective Kv7.1 channel activator, induced relaxation of spontaneous phasic contraction amplitude, force integral, duration, and frequency as well as the 10 Hz electrical field stimulation (EFS)‐induced contractions amplitude and force of UBSM isolated strips (IC50 = 3 – 6 μM). Linopiridine and XE991, two well‐established Kv7 channel inhibitors, increased the spontaneous phasic contraction amplitude and force as well as the 10 Hz EFS‐induced contractions amplitude and force of UBSM isolated strips (IC50 = 3 – 4 μM). In summary, we revealed the expression and the regulatory role of Kv7 channels in the spontaneous and nerve‐evoked contractions of guinea pig UBSM. Supported by NIH DK084284 to GVP.

Voltage‐activated K+ channel activity and its effects on bronchoconstriction in rat airways

Asthma and other airway diseases are often classified by symptoms that include airway hyperresponsiveness (AHR), the excessive contraction of airway smooth muscle cells (ASMCs) in response to a variety of stimuli. The hypercontraction of ASMCs may result from membrane depolarization followed by activation of voltage‐sensitive Ca2+ channels (VSCCs). K+ channels have a high open probability in the absence of ASMC hypercontraction and therefore are thought to oppose membrane depolarization. Using image analysis techniques to measure airway constriction in rat precision cut lung slices (PCLS), we found that blocking voltage‐activated K+ channels using 2mM 4‐aminopyridine (4‐AP) resulted in only 6.3 % constriction of rat airways whereas 230nM methacholine (MeCh) gave rise to 41% constriction. Both responses were blocked by 10μM verapamil. We hypothesized that in the presence of MeCh, K+ channel blockers would cause amplified constriction of the airways when compared to MeCh alone. However, on average, we found that blocking voltage‐gated K+ channels using 2mM 4‐AP in the presence of 230nM MeCh caused significantly less constriction than with MeCh alone in rat PCLS. These findings suggest that Kv channel activity is essential for optimal MeCh‐induced bronchoconstriction. Further work is required to elucidate the roles of Kv channels in bronchoconstriction. (Research supported by Loyola University Chicago.)

The effect of PI3 kinase inhibitor LY294002 on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells

AimsWe examined the effect of LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, on voltage-dependent K+ (Kv) channels. Main methodsElectrophysiological recordings were performed in freshly isolated rabbit coronary arterial smooth muscle cells. Key findingsThe Kv current amplitude was inhibited by LY294002 in a dose-dependent manner, with a Kd value of 1.48μM. Without alteration of the kinetics of activation, LY294002 accelerated the decay rate of Kv channel inactivation. The rate constants of association and dissociation for LY294002 were 1.83±0.01μM−1s−1 and 2.59±0.14s−1, respectively. Application of LY294002 had no significant impact on the steady-state activation or inactivation curves. In the presence of LY294002, the recovery time constant from inactivation was increased, and Kv channel inhibition increased under train pulses (1 or 2Hz). This indicates that LY294002-induced Kv channel inhibition is use-dependent. Furthermore, pretreatment with another PI3K inhibitor, wortmannin (10μM), did not affect the Kv current, and did not change the inhibitory effect of LY294002. SignificanceBased on these results, we suggest that LY294002 directly blocks Kv current irrespective of PI3K inhibition.

Activation of axonal Kv7 channels in human peripheral nerve by flupirtine but not placebo - therapeutic potential for peripheral neuropathies: results of a randomised controlled trial

BackgroundFlupirtine is an analgesic with muscle-relaxing properties that activates Kv7 potassium channels. Kv7 channels are expressed along myelinated and unmyelinated peripheral axons where their activation is expected to reduce axonal excitability and potentially contribute to flupirtine’s clinical profile.Trial designTo investigate the electrical excitability of peripheral myelinated axons following orally administered flupirtine, in-vitro experiments on isolated peripheral nerve segments were combined with a randomised, double-blind, placebo-controlled, phase I clinical trial (RCT).MethodsThreshold tracking was used to assess the electrical excitability of myelinated axons in isolated segments of human sural nerve in vitro and motoneurones to abductor pollicis brevis (APB) in situ in healthy subjects. In addition, the effect of flupirtine on ectopic action potential generation in myelinated axons was examined using ischemia of the lower arm.ResultsFlupirtine (3-30 μM) shortened the relative refractory period and increased post-conditioned superexcitability in human myelinated axons in vitro. Similarly, in healthy subjects the relative refractory period of motoneurones to APB was reduced 2 hours after oral flupirtine but not following placebo. Whether this effect was due to a direct action of flupirtine on peripheral axons or temperature could not be resolved. Flupirtine (200 mg p.o.) also reduced ectopic axonal activity induced by 10 minutes of lower arm ischemia. In particular, high frequency (ca. 200 Hz) components of EMG were reduced in the post-ischemic period. Finally, visual analogue scale ratings of sensations perceived during the post-ischemic period were reduced following flupirtine (200 mg p.o.).ConclusionsClinical doses of flupirtine reduce the excitability of peripheral myelinated axons.Trial registrationClinicalTrials registration is NCT01450865.

A Novel Interaction in the Selectivity Filter of Voltage-Gated Potassium Channels Crucial for Slow Inactivation

Voltage-gated potassium (Kv) channels undergo a process termed slow inactivation in response to prolonged depolarizations. This process is thought to occur in response to structural rearrangements near the selectivity filter, which ultimately result in nonconducting (inactivated) channels. Although substantial efforts have been made to understand this process, the details of slow inactivation at the atomic level have yet to be unraveled in eukaryotic Kv channels. Based on information gleaned from crystal structures, the indole nitrogen of a highly conserved Trp, Trp435 in Shaker potassium channels, had been proposed to form a hydrogen bond with Tyr445 in the GYGD selectivity filter signature sequence, thus contributing to open pore stability and likely slow inactivation. Functionally, however, we find that the indole nitrogen of Trp435 does not contribute to slow inactivation as mutations to Phe or Tyr at this site do not affect slow inactivation. In contrast, removal of the hydroxyl group from Tyr445 by Phe substitution results in a rapidly inactivating phenotype, and this could be slowed by application of external TEA, thus suggesting a role for Tyr445 in slow inactivation. A closer examination of the available structural information of side chains in close physical proximity of Tyr445 pointed towards a possible role of Thr439, a highly conserved side chain in the pore helix. Indeed, Thr439Val channels were non-conducting and behaved similar to the permanently inactivated Trp434Phe phenotype. Re-introduction of a hydroxyl moiety in position 439 via the Thr439Ser mutation led to a WT-like conductance-voltage relationship and only a modestly increased inactivation rate compared to WT. Together, these results suggest a novel and energetically significant hydroxyl-hydroxyl interaction between Tyr445 and Thr439 that regulates slow inactivation in Kv channels.

Inhibition of insulin secretion from rat pancreatic islets by dexmedetomidine and medetomidine, two sedatives frequently used in clinical settings

The aim of this study was to determine whether dexmedetomidine (DEX) and medetomidine (MED), α2-adrenergic agonists clinically used as sedatives, influence insulin secretion from rat pancreatic islets. Islets were isolated from adult male Wistar rats after collagenase digestion. Static incubation was used to determine effects of DEX or MED on insulin secretion and ionic-channel currents of β-cells. Results indicate that both drugs dose-dependently inhibit insulin secretion, DEX more potently than MED. The inhibitory effects were attenuated by addition of yohimbine or by pretreatment of rats with pertussis toxin (PTX). 10 nM DEX decreased the current amplitude of voltage-dependent Ca2+ channels, but this did not occur when the N-type Ca2+ channel blocker ω-conotoxin was added. In the presence of tetraethylammonium, a classical voltage-gated K+ channel (Kv channel) blocker, the magnitude of inhibition of insulin secretion by MED was reduced. However, when tolbutamide, a specific blocker of the ATP-sensitive K+ channel (KATP channel), was present, the magnitude of MED inhibition of insulin secretion was not influenced, suggesting that Kv-channel activity alteration, but not that of KATP channels, is involved in MED-associated insulin secretory inhibition. The Kv-channel currents were increased during 1 nM MED exposure at membrane potentials ranging from -30 mV to -10 mV, where action potentials were generated in response to glucose stimulation. These results indicate that DEX and MED inhibit insulin secretion through an α2-adrenoceptor and PTX-sensitive GTP-binding protein pathway that eventually involves Kv channel activation and Ca2+ channel inhibition.

A Common Structural Component for β-Subunit Mediated Modulation of Slow Inactivation in Different KV Channels

Background/Aims: Potassium channels are tetrameric proteins providing potassium selective passage through lipid embedded proteinaceous pores with highest fidelity. The selectivity results from binding to discrete potassium binding sites and stabilization of a hydrated potassium ion in a central internal cavity. The four potassium binding sites, generated by the conserved TTxGYGD signature sequence are formed by the backbone carbonyls of the amino acids TXGYG. Residues K_V1.5-Val481, K_V4.3-Leu368 and K_V7.1- Ile 313 represent the amino acids in the X position of the respective channels. Methods: Here, we study the impact of these residues on ion selectivity, permeation and inactivation kinetics as well as the modulation by β-subunits using site-specific mutagenesis, electrophysiological analyses and molecular dynamics simulations. Results: We identify this position as key in modulation of slow inactivation by structurally dissimilar β-subunits in different K_V channels. Conclusion: We propose a model in which structural changes accompanying activation and β-subunit modulation allosterically constrain the backbone carbonyl oxygen atoms via the side chain of the respective X-residue in the signature sequence to reduce conductance during slow inactivation.

Interaction between Soluble and Membrane-Embedded Potassium Channel Peptides Monitored by Fourier Transform Infrared Spectroscopy

Recent studies have explored the utility of Fourier transform infrared spectroscopy (FTIR) in dynamic monitoring of soluble protein-protein interactions. Here, we investigated the applicability of FTIR to detect interaction between synthetic soluble and phospholipid-embedded peptides corresponding to, respectively, a voltage-gated potassium (Kv) channel inactivation domain (ID) and S4–S6 of the Shaker Kv channel (KV1; including the S4–S5 linker “pre-inactivation” ID binding site). KV1 was predominantly α-helical at 30°C when incorporated into dimyristoyl-l-α-phosphatidylcholine (DMPC) bilayers. Cooling to induce a shift in DMPC from liquid crystalline to gel phase reversibly decreased KV1 helicity, and was previously shown to partially extrude a synthetic S4 peptide. While no interaction was detected in liquid crystalline DMPC, upon cooling to induce the DMPC gel phase a reversible amide I peak (1633 cm−1) consistent with novel hydrogen bond formation was detected. This spectral shift was not observed for KV1 in the absence of ID (or vice versa), nor when the non-inactivating mutant V7E ID was applied to KV1 under similar conditions. Alteration of salt or redox conditions affected KV1-ID hydrogen bonding in a manner suggesting electrostatic KV1-ID interaction favored by a hairpin conformation for the ID and requiring extrusion of one or more KV1 domains from DMPC, consistent with ID binding to S4–S5. These findings support the utility of FTIR in detecting reversible interactions between soluble and membrane-embedded proteins, with lipid state-sensitivity of the conformation of the latter facilitating control of the interaction.

Novel Activation of Voltage-gated K+ Channels by Sevoflurane

Halogenated inhaled anesthetics modulate voltage-gated ion channels by unknown mechanisms. Biophysical analyses revealed novel activation of K(v) channels by the inhaled anesthetic sevoflurane. K(v) channel activation by sevoflurane results from the positive allosteric modulation of activation gating. The unique activation of K(v) channels by sevoflurane demonstrates novel anesthetic specificity and offers new insights into allosteric modulation of channel gating. Voltage-gated ion channels are modulated by halogenated inhaled general anesthetics, but the underlying molecular mechanisms are not understood. Alkanols and halogenated inhaled anesthetics such as halothane and isoflurane inhibit the archetypical voltage-gated Kv3 channel homolog K-Shaw2 by stabilizing the resting/closed states. By contrast, sevoflurane, a more heavily fluorinated ether commonly used in general anesthesia, specifically activates K-Shaw2 currents at relevant concentrations (0.05-1 mM) in a rapid and reversible manner. The concentration dependence of this modulation is consistent with the presence of high and low affinity interactions (K(D) = 0.06 and 4 mM, respectively). Sevoflurane (<1 mM) induces a negative shift in the conductance-voltage relation and increases the maximum conductance. Furthermore, suggesting possible roles in general anesthesia, mammalian Kv1.2 and Kv1.5 channels display similar changes. Quantitative description of the observations by an economical allosteric model indicates that sevoflurane binding favors activation gating and eliminates an unstable inactivated state outside the activation pathway. This study casts light on the mechanism of the novel sevoflurane-dependent activation of Kv channels, which helps explain how closely related inhaled anesthetics achieve specific actions and suggests strategies to develop novel Kv channel activators.

Modeling-independent elucidation of inactivation pathways in recombinant and native A-type Kv channels

A-type voltage-gated K+ (Kv) channels self-regulate their activity by inactivating directly from the open state (open-state inactivation [OSI]) or by inactivating before they open (closed-state inactivation [CSI]). To determine the inactivation pathways, it is often necessary to apply several pulse protocols, pore blockers, single-channel recording, and kinetic modeling. However, intrinsic hurdles may preclude the standardized application of these methods. Here, we implemented a simple method inspired by earlier studies of Na+ channels to analyze macroscopic inactivation and conclusively deduce the pathways of inactivation of recombinant and native A-type Kv channels. We investigated two distinct A-type Kv channels expressed heterologously (Kv3.4 and Kv4.2 with accessory subunits) and their native counterparts in dorsal root ganglion and cerebellar granule neurons. This approach applies two conventional pulse protocols to examine inactivation induced by (a) a simple step (single-pulse inactivation) and (b) a conditioning step (double-pulse inactivation). Consistent with OSI, the rate of Kv3.4 inactivation (i.e., the negative first derivative of double-pulse inactivation) precisely superimposes on the profile of the Kv3.4 current evoked by a single pulse because the channels must open to inactivate. In contrast, the rate of Kv4.2 inactivation is asynchronous, already changing at earlier times relative to the profile of the Kv4.2 current evoked by a single pulse. Thus, Kv4.2 inactivation occurs uncoupled from channel opening, indicating CSI. Furthermore, the inactivation time constant versus voltage relation of Kv3.4 decreases monotonically with depolarization and levels off, whereas that of Kv4.2 exhibits a J-shape profile. We also manipulated the inactivation phenotype by changing the subunit composition and show how CSI and CSI combined with OSI might affect spiking properties in a full computational model of the hippocampal CA1 neuron. This work unambiguously elucidates contrasting inactivation pathways in neuronal A-type Kv channels and demonstrates how distinct pathways might impact neurophysiological activity.

Of receptors, channels, and watching the red cell center lose hold

Of receptors, channels, and watching the red cell center lose hold

Nitrite- and nitroxyl-induced relaxation in porcine coronary (micro-) arteries: Underlying mechanisms and role as endothelium-derived hyperpolarizing factor(s)

To investigate the vasorelaxant efficacy of nitrite and nitroxyl (HNO) in porcine coronary (micro)arteries (PC(M)As), evaluating their role as endothelium-derived hyperpolarizing factors (EDHFs), preconstricted PCAs and PCMAs were exposed to UV light (a well-known inductor of nitrite; wave-length: 350–370nm), nitrite, the HNO donor Angeli's salt, or bradykinin. UV light-induced relaxation of PCAs increased identically after endothelium removal and endothelial nitric oxide (NO) synthase (eNOS) blockade. UV light-induced relaxation diminished during Na+-K+-ATPase inhibition and S-nitrosothiol-depletion, and disappeared during NO scavenging with hydroxocobalamin or soluble guanylyl cyclase (sGC) inhibition with ODQ. Nitrite-induced relaxation of PCAs required millimolar levels, i.e., >1000 times endogenous vascular nitrite. Angeli's salt relaxed PCMAs more potently than PCAs, and this was due to the fact that HNO directly activated sGC in PCMAs, whereas in PCAs this occurred following its conversion to NO only. sGC activation by NO/HNO resulted in Na+-K+-ATPase stimulation and Kv channel activation. The HNO scavenger l-cysteine blocked bradykinin-induced relaxation in PCAs, and potentiated it in PCMAs. The latter did not occur in the presence of hydroxocobalamin, suggesting that it depended on l-cysteine-induced generation of vasorelaxant S-nitrosothiols. In all experimental setups, incubation with red wine extract mimicked the effects of ODQ. In conclusion, nitrite, via its conversion to NO and S-nitrosothiols, and HNO, either directly, or via its conversion to NO, mediate relaxant effects involving the sGC-cGMP pathway, Na+-K+-ATPase and/or Kv channels. Red wine extract counteracts these beneficial effects. NO blocks nitrite activation, and HNO, but not nitrite, may act as EDHF in the coronary vascular bed.

Atypical Reactive Center Kunitz-Type Inhibitor from the Sea Anemone Heteractis crispa

The primary structure of a new Kunitz-type protease inhibitor InhVJ from the sea anemone Heteractis crispa (Radianthus macrodactylus) was determined by protein sequencing and cDNA cloning. InhVJ amino acid sequence was shown to share high sequence identity (up to 98%) with the other known Kunitz-type sea anemones sequences. It was determined that the P1 Thr at the reactive site resulted in a decrease of the Ki of InhVJ to trypsin and α-chymotrypsin (7.38 × 10−8 M and 9.93 × 10−7 M, respectively). By structure modeling the functional importance of amino acids at the reactive site as well as at the weak contact site were determined. The significant role of Glu45 for the orientation and stabilization of the InhVJ-trypsin complex was elucidated. We can suggest that there has been an adaptive evolution of the P1 residue at the inhibitor reactive site providing specialization or functional diversification of the paralogs. The appearance of a key so-called P1 Thr residue instead of Lys might lead to refinement of inhibitor specificity in the direction of subfamilies of serine proteases. The absence of Kv channel and TRPV1-receptor modulation activity was confirmed by electrophysiological screening tests.

Preclinical efficacy and safety of Kv7 potassium channel openers in the treatment of cerebral vasospasm

Cerebral vasospasm is a sequel to subarachnoid hemorrhage (SAH) that leads to death or disability due to cerebral ischemia. The persistent constriction of the conduit arteries in the brain is due to sustained depolarization of myocytes lining the arteries, resulting in continuous influx of calcium (Ca2+) through voltage‐gated Ca2+ channels. Ca2+ channel blockers (CCB) or potassium (K+) channel openers that oppose membrane depolarization are expected to dilate the arteries and reduce ischemia. CCBs have limited efficacy because of heterogenous expression of Ca2+ channel subunits in myocytes and their propensity to induce hypotension. We recently reported that Kv7channels are expressed in rat basilar artery myocytes and Kv7 channel openers reverse the constriction of rat basilar artery produced by spasmogens — endothelin, serotonin and vasopressin. Here we demonstrate that Kv7 channel activators, retigabine and celecoxib significantly attenuate basilar artery spasm in a rat SAH model, when administered intraperitoneally every 12 hours for 48 hours. Both Kv7 channel openers induced hypothermia, an effect expected to limit neuronal damage. They also induced a transient drop in blood pressure (BP) generally lasting 30–60 min after each administration, but mean 24‐h BP measured using radio‐telemetry in normal rats was not changed. These results indicate that Kv7 channel openers can attenuate vasospasm without sustained hypotension.

Involvement of vascular Kv7 potassium channels in the pathogenesis of cerebral vasospasm

Cerebral vasospasm is a grave complication after subarachnoid hemorrhage characterized by persistent intense constriction of large conducting arteries in brain. Spastic contraction of the arterial myocytes is associated with membrane depolarization implicating calcium entry through voltage‐gated calcium channels. Endothelin, serotonin and vasopressin are spasmogens involved in the initiation and maintenance of vasospasm. We recently reported that Kv7 potassium channels are expressed in rat basilar artery myocytes and potassium conductance through the channels determine resting membrane potential and contractile status of basilar artery. Here we use patch‐clamp electrophysiology and pressure myography to test the involvement of Kv7 channels in the pathogenesis of cerebral vasospasm. Spasmogens suppressed Kv7 currents and produced membrane depolarization of basilar artery myocytes. The effects were reversed by Kv7 channel activator retigabine. Additivity experiments using pressure myography reveal the involvement of Kv7 channel suppression in the constriction produced by spasmogens. Activated PKC, a signal transduction intermediate of Gq‐coupled spasmogen receptor stimulation, suppressed Kv7 currents in myocytes and constricted basilar artery. These results indicate suppression of Kv7 currents as a mechanism for membrane depolarization and constriction of arteries by spasmogens resulting in vasospasm.

Voltage-Gating in the Hv1 Proton Channel: Clues from Atomistic Molecular Dynamics Simulations

The voltage-gated proton channel, Hv1, is homologous to the voltage-sensing domain (VSD) of voltage-gated potassium (Kv) channels, but uniquely lacks a central pore domain (Ramsey et al., Nature 440, 1213, 2006; Sasaki et al., Science 312, 589, 2006). To begin to understand voltage-dependent activation in the Hv1 channel, we perform molecular dynamics simulations of a homology model for the transmembrane region of the human Hv1 in a hydrated lipid bilayer under a membrane potential. Under a depolarizing potential (positive on the intracellular side), the channel pathway exhibits a robust transmembrane hydrogen bonded water wire connecting the intracellular and extracellular sides. Under a hyperpolarizing potential, a cluster of hydrophobic side chains occludes the central region of the channel and prevents water wire formation. Assuming that a water wire plays a role in proton conduction by Hv1, these observations are consistent with experiments showing that the channel opens at depolarizing potentials. The basic side chains in the VSD S4 segment carry most of the gating charge during the activation of Kv channels. We observe translocation of the third arginine in the Hv1 S4 segment (Arg211) relative to a highly conserved phenylalanine side chain (Phe150) located at the center of the putative channel permeation pathway. Phe150 is homologous to a highly conserved Phe side chain in Kv channels, which is thought to modulate channel activation (Tao et al., Science 328, 67, 2010; Lacroix and Bezanilla, PNAS 108, 12313, 2011). Thus, our simulations suggest that these residues may be involved in voltage activation of Hv1. This work is supported by grants from the NIH (GM74637, GM86685 and GM72507) and NSF (CHE-0750175), and Teragrid resources provided by the NSF-supported NICS.

The Role of PIP2 in the Voltage-Dependent Activation of Kv7.1

Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important regulator of ion channel activity. While PIP2 modulation of voltage-independent Kir channels has been extensively studied, the mechanism of how PIP2 potentiates the activity of voltage-dependent ion channels remains uknown. In this work, we study PIP2 activation of a voltage-dependent potassium channel (Kv7.1). Kv7.1 channels have a canonical Kv structure with a central pore-gate domain (PGD) surrounded by four peripheral voltage-sensing domains (VSDs). Kv channel activation is thought to involve two general steps. First, membrane depolarization is sensed by gating charges in the VSDs resulting in conformational changes within the VSDs from the resting state to the activated state. After all four VSDs have been activated, the second general step in Kv activation involves a concerted motion during which the PGD opens to allow ion permeation. In this study we ask if PIP2 regulates activation of Kv7.1 by potentiating the early VSD conformational changes, the concerted opening of the PGD, or both. Using the voltage-clamp fluorometry (VCF) technique to assay local conformation changes in the VSDs and the PGD simultaneously, we are able to show that although depletion of PIP2 eliminates the ionic current, the early conformational changes in the VSD do not require PIP2. This result indicates that the later conformational changes that couple VSD activation to the opening of the PGD are eliminated by PIP2 depletion. We continue this work by dissecting the molecular details of why PIP2 is required for opening of the PGD in response to VSD activation. These results may provide insights on the common principle of how Kv channels are modulated by PIP2.

Polyunsaturated Fatty Acids Modify the Gating of Kv Channels

Polyunsaturated fatty acids (PUFAs) have been reported to exhibit antiarrhythmic properties, which are attributed to their capability to modulate ion channels. This PUFAs ability has been reported to be due to their effects on the gating properties of ion channels. In the present review, we will focus on the role of PUFAs on the gating of two Kv channels, Kv1.5 and Kv11.1. Kv1.5 channels are blocked by n−3 PUFAs of marine [docosahexaenoic acid (DHA) and eicosapentaenoic acid] and plant origin (alpha-linolenic acid, ALA) at physiological concentrations. The blockade of Kv1.5 channels by PUFAs steeply increased in the range of membrane potentials coinciding with those of Kv1.5 channel activation, suggesting that PUFAs-channel binding may derive a significant fraction of its voltage sensitivity through the coupling to channel gating. A similar shift in the activation voltage was noted for the effects of n–6 arachidonic acid (AA) and DHA on Kv1.1, Kv1.2, and Kv11.1 channels. PUFAs-Kv1.5 channel interaction is time-dependent, producing a fast decay of the current upon depolarization. Thus, Kv1.5 channel opening is a prerequisite for the PUFA-channel interaction. Similar to the Kv1.5 channels, the blockade of Kv11.1 channels by AA and DHA steeply increased in the range of membrane potentials that coincided with the range of Kv11.1 channel activation, suggesting that the PUFAs-Kv channel interactions are also coupled to channel gating. Furthermore, AA regulates the inactivation process in other Kv channels, introducing a fast voltage-dependent inactivation in non-inactivating Kv channels. These results have been explained within the framework that AA closes voltage-dependent potassium channels by inducing conformational changes in the selectivity filter, suggesting that Kv channel gating is lipid dependent.

Voltage Sensor Inactivation in Potassium Channels

In voltage-gated potassium (Kv) channels membrane depolarization causes movement of a voltage sensor domain. This conformational change of the protein is transmitted to the pore domain and eventually leads to pore opening. However, the voltage sensor domain may interact with two distinct gates in the pore domain: the activation gate (A-gate), involving the cytoplasmic S6 bundle crossing, and the pore gate (P-gate), located externally in the selectivity filter. How the voltage sensor moves and how tightly it interacts with these two gates on its way to adopt a relaxed conformation when the membrane is depolarized may critically determine the mode of Kv channel inactivation. In certain Kv channels, voltage sensor movement leads to a tight interaction with the P-gate, which may cause conformational changes that render the selectivity filter non-conductive (“P/C-type inactivation”). Other Kv channels may preferably undergo inactivation from pre-open closed-states during voltage sensor movement, because the voltage sensor temporarily uncouples from the A-gate. For this behavior, known as “preferential” closed-state inactivation, we introduce the term “A/C-type inactivation”. Mechanistically, P/C- and A/C-type inactivation represent two forms of “voltage sensor inactivation.”