We report here several unusual features of inactivation of the rat Kv2.1 delayed rectifier potassium channel, expressed in Xenopus oocytes. The voltage dependence of inactivation was U-shaped, with maximum inactivation near 0 mV. During a maintained depolarization, development of inactivation was slow and only weakly voltage dependent (tau = 4 s at 0 mV; tau = 7 s at +80 mV). However, recovery from inactivation was strongly voltage dependent (e-fold for 20 mV) and could be rapid (tau = 0.27 s at -140 mV). Kv2.1 showed cumulative inactivation, where inactivation built up during a train of brief depolarizations. A single maintained depolarization produced more steady-state inactivation than a train of pulses, but there could actually be more inactivation with the repeated pulses during the first few seconds. We term this phenomenon "excessive cumulative inactivation." These results can be explained by an allosteric model, in which inactivation is favored by activation of voltage sensors, but the open state of the channel is resistant to inactivation.

In this study, we explored the possibility that two-pore domain potassium (K2P) channels are sufficient to support action potential (AP) generation in the absence of conventional voltage-gated potassium (KV) channels. Hodgkin-Huxley parameters were used to mimic the presence of voltage-gated sodium (NaV) channels in HEK-293 cells. Recombinant expression of either TREK-1 or TASK-3 channels was then used to generate a hyperpolarised resting membrane potential (RMP) leading to the characteristic non-linear current-voltage relationship expected of a K2P-mediated conductance. During conductance simulation experiments, both TASK-3 and TREK-1 channels were able to repolarise the membrane once AP threshold was reached, and at physiologically relevant current densities, this K2P-mediated conductance supported sustained AP firing. Moreover, the magnitude of the conductance correlated with the speed of the AP rise in a manner predicted from our computational studies. We discuss the physiological impact of axonal K2P channels and speculate on the possible clinical relevance of K2P channel modulation when considering the actions of general and local anaesthetics.

Kv7.1 (KCNQ1) channels are regulators of several physiological processes including vasodilatation, repolarization of cardiomyocytes, and control of secretory processes. A number of Kv7.1 pore mutants are sensitive to extracellular potassium. We hypothesized that extracellular potassium also modulates wild-type Kv7.1 channels. The Kv7.1 currents were measured in Xenopus laevis oocytes at different concentrations of extracellular potassium (1-50 mM). As extracellular potassium was elevated, Kv7.1 currents were reduced significantly more than expected from theoretical calculations based on the Goldman-Hodgkin-Katz flux equation. Potassium inhibited the steady-state current with an IC(50) of 6.0 ± 0.2 mM. Analysis of tail-currents showed that potassium increased the fraction of channels in the inactivated state. Similarly, the recovery from inactivation was slowed by potassium, suggesting that extracellular potassium stabilizes an inactivated state in Kv7.1 channels. The effect of extracellular potassium was absent in noninactivating Kv7.1/KCNE1 and Kv7.1/KCNE3 channels, further supporting a stabilized inactivated state as the underlying mechanism. Interestingly, coexpression of Kv7.1 with KCNE2 did not attenuate the inhibition by potassium. In a number of other Kv channels, including Kv1.5, Kv4.3, and Kv7.2-5 channels, currents were only minimally reduced by an increase in extracellular potassium as expected. These results show that extracellular potassium modulates Kv7.1 channels and suggests that physiological changes in potassium concentrations may directly control the function of Kv7.1 channels. This may represent a novel regulatory mechanism of excitability and of potassium transport in tissues expressing Kv7.1 channels.

Potassium channels selectively conduct K(+), primarily to the exclusion of Na(+), despite the fact that both ions can bind within the selectivity filter. Here we perform crystallographic titration and single-channel electrophysiology to examine the competition of Na(+) and K(+) binding within the filter of two NaK channel mutants; one is the potassium-selective NaK2K mutant and the other is the non-selective NaK2CNG, a CNG channel pore mimic. With high-resolution structures of these engineered NaK channel constructs, we explicitly describe the changes in K(+) occupancy within the filter upon Na(+) competition by anomalous diffraction. Our results demonstrate that the non-selective NaK2CNG still retains a K(+)-selective site at equilibrium, whereas the NaK2K channel filter maintains two high-affinity K(+) sites. A double-barrier mechanism is proposed to explain K(+) channel selectivity at low K(+) concentrations.

Inwardly rectifying potassium (Kir) channels are expressed in many cell types and contribute to a wide range of physiological processes. Particularly, Kir4.1 channels are involved in the astroglial spatial potassium buffering. In this work, we examined the effects of the cationic amphiphilic drug quinacrine on Kir4.1 channels heterologously expressed in HEK293 cells, employing the patch clamp technique. Quinacrine inhibited the currents of Kir4.1 channels in a concentration and voltage dependent manner. In inside-out patches, quinacrine inhibited Kir4.1 channels with an IC50 value of 1.8±0.3μM and with extremely slow blocking and unblocking kinetics. Molecular modeling combined with mutagenesis studies suggested that quinacrine blocks Kir4.1 by plugging the central cavity of the channels, stabilized by the residues E158 and T128. Overall, this study shows that quinacrine blocks Kir4.1 channels, which would be expected to impact the potassium transport in several tissues.

Calcitriol (an active metabolite of vitamin D) modulates the expression of hundreds of human genes by activation of the vitamin D nuclear receptor (VDR). However, VDR-mediated transcriptional modulation does not fully explain various phenotypic effects of calcitriol. Recently a fast non-genomic response to vitamin D has been described, and it seems that mitochondria are one of the targets of calcitriol. These non-classical calcitriol targets open up a new area of research with potential clinical applications. The goal of our study was to ascertain whether calcitriol can modulate mitochondrial function through regulation of the potassium channels present in the inner mitochondrial membrane.

Kv1.2 is a prominent potassium channel subtype in the nervous system and serves as an important structural template for investigation of ion channel function. However, Kv1.2 voltage-dependence exhibits dramatic cell-to-cell variability due to a gating mode shift that is regulated by an unknown mechanism. We report that this variable behavior is regulated by the extracellular redox environment. Exposure to reducing agents promotes a shift in gating properties towards an 'inhibited' gating mode that resists opening, and causes channels to exhibit pronounced use-dependent activation during trains of repetitive depolarizations. This sensitivity to extracellular redox potential is absent in other Kv1 channels, but is apparent in heteromeric channels containing Kv1.2 subunits, and overlaps with the reported physiological range of extracellular redox couples. Mutagenesis of candidate cysteine residues fails to abolish redox sensitivity. Therefore, we suggest that an extrinsic, redox-sensitive binding partner imparts these properties.

Besides their role in the generation of action potentials, voltage-gated potassium channels are implicated in cellular processes ranging from cell division to cell death. The K(+) channel regulator protein (KCNRG), identified as a putative tumor suppressor, reduces K(+) currents through human K(+) channels hKv1.1 and hKv1.4 expressed in Xenopus oocytes. Current attenuation requires the presence of the N-terminal T1 Domain and immunoprecipitation experiments suggest association of KCNRG with the N-terminus of the channel. Our data indicates that KCNRG is an ER-associated protein, which we propose regulates Kv1 family channel proteins by retaining a fraction of channels in endomembranes.

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare, and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K(+)) channels to infect cells. Time of addition assays using K(+) channel modulating agents demonstrated that K(+) channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K(+) channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, two-pore domain K(+) channels (K2P) were identified as the K(+) channel family mediating BUNV K(+) channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

Purkinje neuron dysfunction is associated with cerebellar ataxia. In a mouse model of spinocerebellar ataxia type 1 (SCA1), reduced potassium channel function contributes to altered membrane excitability resulting in impaired Purkinje neuron spiking. We sought to determine the relationship between altered membrane excitability and motor dysfunction in SCA1 mice.

The computational ability of CNS neurons depends critically on the specific localization of ion channels in the somatodendritic and axonal membranes. Neuronal dendrites receive synaptic inputs at numerous spines and integrate them in time and space. The integration of synaptic potentials is regulated by voltage-gated potassium (Kv) channels, such as Kv4.2, which are specifically localized in the dendritic membrane. The synaptic potentials eventually depolarize the membrane of the axon initial segment, thereby activating voltage-gated sodium channels to generate action potentials. Specific Kv channels localized in the axon initial segment, such as Kv1 and Kv7 channels, determine the shape and the rate of action potentials. Kv1 and Kv7 channels present at or near nodes of Ranvier and in presynaptic terminals also influence the propagation of action potentials and neurotransmitter release. The physiological significance of proper Kv channel localization is emphasized by the fact that defects in the trafficking of Kv channels are observed in several neurological disorders including epilepsy. In this review, we will summarize the current understanding of the mechanisms of Kv channel trafficking and discuss how they contribute to the establishment and maintenance of the specific localization of Kv channels in neurons.

Rat proximal and distal colon are net K+ secretory and net K+ absorptive epithelia, respectively. Chronic dietary K+ loading increases net K+ secretion in the proximal colon and transforms net K+ absorption to net K+ secretion in the distal colon, but changes in apical K+ channel expression are unclear. We evaluated expression/activity of apical K+ (BK) channels in surface colonocytes in proximal and distal colon of control and K+-loaded animals using patch-clamp recording, immunohistochemistry, and Western blot analyses. In controls, BK channels were more abundant in surface colonocytes from K+ secretory proximal colon (39% of patches) than in those from K+-absorptive distal colon (12% of patches). Immunostaining demonstrated more pronounced BK channel α-subunit protein expression in surface cells and cells in the upper 25% of crypts in proximal colon, compared with distal colon. Dietary K+ loading had no clear-cut effects on the abundance, immunolocalization, or expression of BK channels in proximal colon. By contrast, in distal colon, K+ loading 1) increased BK channel abundance in patches from 12 to 41%; 2) increased density of immunostaining in surface cells, which extended along the upper 50% of crypts; and 3) increased expression of BK channel α-subunit protein when assessed by Western blotting (P < 0.001). Thus apical BK channels are normally more abundant in K+ secretory proximal colon than in K+ absorptive distal colon, and apical BK channel expression in distal (but not proximal) colon is greatly stimulated as part of the enhanced K+ secretory response to dietary K+ loading.

Expressed in Xenopus oocytes, KvLQT1 channel subunits yield a small, rapidly activating, voltage- dependent potassium conductance. When coexpressed with the minK gene product, a slowly activating and much larger potassium current results. Using fluctuation analysis and single-channel recordings, we have studied the currents formed by human KvLQT1 subunits alone and in conjunction with human or rat minK subunits. With low external K+, the single-channel conductances of these three channel types are estimated to be 0.7, 4.5, and 6.5 pS, respectively, based on noise analysis at 20 kHz bandwidth of currents at +50 mV. Power spectra computed over the range 0.1 Hz-20 kHz show a weak frequency dependence, consistent with current interruptions occurring on a broad range of time scales. The broad spectrum causes the apparent single-channel current value to depend on the bandwidth of the recording, and is mirrored in very "flickery" single-channel events of the channels from coexpressed KvLQT1 and human minK subunits. The increase in macroscopic current due to the presence of the minK subunit is accounted for by the increased apparent single-channel conductance it confers on the expressed channels. The rat minK subunit also confers the property that the outward single-channel current is increased by external potassium ions.

The inactivation gating of hERG channels is important for the channel function and drug-channel interaction. Whereas hERG channels are highly selective for K+, we have found that inactivated hERG channels allow Na+ to permeate in the absence of K+. This provides a new way to directly monitor and investigate hERG inactivation. By using whole cell patch clamp method with an internal solution containing 135 mM Na+ and an external solution containing 135 mM NMG+, we recorded a robust Na+ current through hERG channels expressed in HEK 293 cells. Kinetic analyses of the hERG Na+ and K+ currents indicate that the channel experiences at least two states during the inactivation process, an initial fast, less stable state followed by a slow, more stable state. The Na+ current reflects Na+ ions permeating through the fast inactivated state but not through the slow inactivated state or open state. Thus the hERG Na+ current displayed a slow inactivation as the channels travel from the less stable, fast inactivated state into the more stable, slow inactivated state. Removal of fast inactivation by the S631A mutation abolished the Na+ current. Moreover, acceleration of fast inactivation by mutations T623A, F627Y, and S641A did not affect the hERG Na+ current, but greatly diminished the hERG K+ current. We also found that external Na+ potently blocked the hERG outward Na+ current with an IC50 of 3.5 mM. Mutations in the channel pore and S6 regions, such as S624A, F627Y, and S641A, abolished the inhibitory effects of external Na+ on the hERG Na+ current. Na+ permeation and blockade of hERG channels provide novel ways to extend our understanding of the hERG gating mechanisms.

Voltage-gated potassium channels KCNQ2-5 generate the M-current, which controls neuronal excitability. KCNQ2-5 subunits each harbor a high-affinity anticonvulsant drug-binding pocket containing an essential tryptophan (W265 in human KCNQ3) conserved for >500 million years, yet lacking a known physiological function. Here, phylogenetic analysis, electrostatic potential mapping, in silico docking, electrophysiology, and radioligand binding assays reveal that the anticonvulsant binding pocket evolved to accommodate endogenous neurotransmitters including γ-aminobutyric acid (GABA), which directly activates KCNQ5 and KCNQ3 via W265. GABA, and endogenous metabolites β-hydroxybutyric acid (BHB) and γ-amino-β-hydroxybutyric acid (GABOB), competitively and differentially shift the voltage dependence of KCNQ3 activation. Our results uncover a novel paradigm: direct neurotransmitter activation of voltage-gated ion channels, enabling chemosensing of the neurotransmitter/metabolite landscape to regulate channel activity and cellular excitability.

Potassium channels form physical complexes with solute transporters in vivo, yet little is known about their range of possible signaling modalities and the underlying mechanisms. The KCNQ2/3 potassium channel, which generates neuronal M-current, is voltage-gated and its activity is also stimulated by binding of various small molecules. KCNQ2/3 forms reciprocally regulating complexes with sodium-coupled myo-inositol transporters (SMITs) in mammalian neurons. Here, we report that the neurotransmitter γ-aminobutyric acid (GABA) and other small molecules directly regulate myo-inositol transport in rat dorsal root ganglia, and by human SMIT1-KCNQ2/3 complexes in vitro, by inducing a distinct KCNQ2/3 pore conformation. Reciprocally, SMIT1 tunes KCNQ2/3 sensing of GABA and related metabolites. Ion permeation and mutagenesis studies suggest that SMIT1 and GABA similarly alter KCNQ2/3 pore conformation but via different KCNQ subunits and molecular mechanisms. KCNQ channels therefore act as chemosensors to enable co-assembled myo-inositol transporters to respond to diverse stimuli including neurotransmitters, metabolites and drugs.

Potassium channels have been discovered in the inner mitochondrial membrane of various cells. These channels can regulate the mitochondrial membrane potential, the matrix volume, respiration and reactive species generation. Therefore, it is believed that their activation is cytoprotective in various tissues. In our study, the single-channel activity of a large-conductance calcium-activated potassium channel (mitoBKCa) was measured by the patch-clamp technique on mitoplasts derived from mitochondria isolated from human glioma U-87 MG cells. Here, we show for the first time that mechanical stimulation of mitoBKCa channels results in an increased probability of channel opening. However, the mechanosensitivity of mitoBKCa channels was variable with some channels exhibiting no mechanosensitivity. We detected the expression of mechanosensitive BKCa-STREX exon in U-87 MG cells and hypotesize, based on previous studies demonstrating the presence of multiple BKCa splice variants that variable mechanosensitivity of mitoBKCa could be the result of the presence of diverse BKCa isoforms in mitochondria of U-87 MG cells. Our findings indicate the possible involvement of the mitoBKCa channel in mitochondria activities in which changes in membrane tension and shape play a crucial role, such as fusion/fission and cristae remodeling.

Voltage-gated potassium ion channels (Kv) play an important role in a variety of cellular processes, including the functioning of excitable cells, regulation of apoptosis, cell growth and differentiation, the release of neurotransmitters and hormones, maintenance of cardiac activity, etc. Failure in the functioning of Kv channels leads to severe genetic disorders and the development of tumors, including malignant ones. Understanding the mechanisms underlying Kv channels functioning is a key factor in determining the cause of the diseases associated with mutations in the channels, and in the search for new drugs. The mechanism of activation of the channels is a topic of ongoing debate, and a consensus on the issue has not yet been reached. This review discusses the key stages in studying the mechanisms of functioning of Kv channels and describes the basic models of their activation known to date.

Although bipolar disorder (BP) is one of the most heritable psychiatric conditions, susceptibility genes for the disorder have yet to be conclusively identified. It is likely that variants in multiple genes across multiple pathways contribute to the genotype-phenotype relationship in the affected population. Recent evidence from genome-wide association studies implicates an entire class of genes related to the structure and regulation of ion channels, suggesting that the etiology of BP may arise from channelopathies. In this review, we examine the evidence for this hypothesis, with a focus on the potential role of voltage-gated potassium channels. We consider evidence from genetic and expression studies, and discuss the potential underlying biology. We consider animal models and treatment implications of the involvement of potassium ion channelopathy in BP. Finally, we explore intriguing parallels between BP and epilepsy, the signature channelopathy of the central nervous system.

Drugs that inhibit the cardiac rapid delayed rectifier potassium ion current (IKr) channel can be proarrhythmic and their clinical use has been associated with sudden unexpected death (SUD). Since SUD is about 20 times more common among people with epilepsy than in the general population, and some data indicate that drug treatment may contribute, we tested the hypothesis that the classic antiepileptic drugs phenytoin (PHT), carbamazepine (CBZ), and phenobarbital (PB) have a potential to block IKr. The whole cell patch-clamp recording technique was used to study the effects on IKr channels expressed by the human ether-a-go-go related gene (HERG) stably expressed in Human Embryo Kidney (HEK) 293 cells. Tail currents, which are purely related to HERG, were blocked with an IC50 (the concentration when 50% inhibition was obtained compared to control values) of 240 microM for PHT and 3 mM for PB. A 20% inhibition of tail currents was obtained at CBZ concentrations of 250 and 500 microM. Collective data show that drugs with the same margins (ratio HERG IC50/unbound therapeutic concentration), as PHT and PB, may have arrhythmogenic potential, especially when used in predisposed patients and in the case of drug-drug interactions. SUD in epilepsy is generally a seizure-related phenomenon. However, our data suggest that PHT and PB may play a contributing role, perhaps by making some patients more vulnerable to the cardiovascular depression induced by seizures.