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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.
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.
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.
Voltage-gated potassium channels (Kv) are the largest group of ion channels. Kv are involved in controlling the resting potential and action potential duration in the heart and brain. Additionally, these proteins participate in cell cycle progression as well as in several other important features in mammalian cell physiology, such as activation, differentiation, apoptosis, and cell volume control. Therefore, Kv remarkably participate in the cell function by balancing responses. The implication of Kv in physiological and pathophysiological cell growth is the subject of study, as Kv are proposed as therapeutic targets for tumor regression. Though it is widely accepted that Kv channels control proliferation by allowing cell cycle progression, their role is controversial. Kv expression is altered in many cancers, and their participation, as well as their use as tumor markers, is worthy of effort. There is an ever-growing list of Kv that remodel during tumorigenesis. This review focuses on the actual knowledge of Kv channel expression and their relationship with neoplastic proliferation. In this work, we provide an update of what is currently known about these proteins, thereby paving the way for a more precise understanding of the participation of Kv during cancer development.
Temperature-sensitive transient receptor potential (TRP) ion channels are members of the large tetrameric cation channels superfamily but are considered to be uniquely sensitive to heat, which has been presumed to be due to the existence of an unidentified temperature-sensing domain. Here we report that the homologous voltage-gated potassium (Kv) channels also exhibit high temperature sensitivity comparable to that of TRPV1, which is detectable under specific conditions when the voltage sensor is functionally decoupled from the activation gate through either intrinsic mechanisms or mutations. Interestingly, mutations could tune Shaker channel to be either heat-activated or heat-deactivated. Therefore, high temperature sensitivity is intrinsic to both TRP and Kv channels. Our findings suggest important physiological roles of heat-induced variation in Kv channel activities. Mechanistically our findings indicate that temperature-sensing TRP channels may not contain a specialized heat-sensor domain; instead, non-obligatory allosteric gating permits the intrinsic heat sensitivity to drive channel activation, allowing temperature-sensitive TRP channels to function as polymodal nociceptors.
Studies of the structure-function relationship in proteins for which no 3D structure is available are often based on inspection of multiple sequence alignments. Many functionally important residues of proteins can be identified because they are conserved during evolution. However, residues that vary can also be critically important if their variation is responsible for diversity of protein function and improved phenotypes. If too few sequences are studied, the support for hypotheses on the role of a given residue will be weak, but analysis of large multiple alignments is too complex for simple inspection. When a large body of sequence and functional data are available for a protein family, mature data mining tools, such as machine learning, can be applied to extract information more easily, sensitively and reliably. We have undertaken such an analysis of voltage-gated potassium channels, a transmembrane protein family whose members play indispensable roles in electrically excitable cells.
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.
The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca(2+)/calmodulin and modulation of voltage-dependent gating by extracellular Mg(2+). Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning.
Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is a major signaling molecule implicated in the regulation of various ion transporters and channels. Here we show that PIP(2) and intracellular MgATP control the activity of the KCNQ1/KCNE1 potassium channel complex. In excised patch-clamp recordings, the KCNQ1/KCNE1 current decreased spontaneously with time. This rundown was markedly slowed by cytosolic application of PIP(2) and fully prevented by application of PIP(2) plus MgATP. PIP(2)-dependent rundown was accompanied by acceleration in the current deactivation kinetics, whereas the MgATP-dependent rundown was not. Cytosolic application of PIP(2) slowed deactivation kinetics and also shifted the voltage dependency of the channel activation toward negative potentials. Complex changes in the current characteristics induced by membrane PIP(2) was fully restituted by a model originally elaborated for ATP-regulated two transmembrane-domain potassium channels. The model is consistent with stabilization by PIP(2) of KCNQ1/KCNE1 channels in the open state. Our data suggest a striking functional homology between a six transmembrane-domain voltage-gated channel and a two transmembrane-domain ATP-gated channel.
Cholesterol, an essential lipid component of cellular plasma membranes, regulates fluidity, mechanical integrity, raft structure and may specifically interact with membrane proteins. Numerous effects on ion channels by cholesterol, including changes in current amplitude, voltage dependence and gating kinetics, have been reported. We have previously described such changes in the voltage-gated potassium channel Kv1.3 of lymphocytes by cholesterol and its analog 7-dehydrocholesterol (7DHC). In voltage-gated channels membrane depolarization induces movement of the voltage sensor domains (VSD), which is transmitted by a coupling mechanism to the pore domain (PD) to open the channel. Here, we investigated whether cholesterol effects were mediated by the VSD to the pore or the PD was the direct target. Specificity was tested by comparing Kv1.3 and Kv10.1 channels having different VSD-PD coupling mechanisms. Current recordings were performed with two-electrode voltage-clamp fluorometry, where movement of the VSDs was monitored by attaching fluorophores to external cysteine residues introduced in the channel sequence. Loading the membrane with cholesterol or 7DHC using methyl-β-cyclodextrin induced changes in the steady-state and kinetic parameters of the ionic currents while leaving fluorescence parameters mostly unaffected in both channels. Non-stationary noise analysis revealed that reduction of single channel conductance rather than that of open probability caused the observed current decrease. Furthermore, confocal laser scanning and stimulated emission depletion microscopy demonstrated significant changes in the distribution of these ion channels in response to sterol loading. Our results indicate that sterol-induced effects on ion channel gating directly target the pore and do not act via the VSD.
The uteri, spontaneously active or Ca(2+) (6 mM) induced, were allowed to equilibrate, and to inhibit voltage-gated potassium (K(V)) channels 1 mM 4-amino pyridine (4-AP) was applied for 15 min before adding H(2)O(2). H(2)O(2) was added cumulatively: 2 μM, 20 μM, 200 μM, 400 μM, and 3 mM. Average time for H(2)O(2) concentrations (2, 20, 200, and 400) μM to reach its full effect was 15 min. H(2)O(2) 3 mM had a prolonged effect and therefore was left to act for 30 min. Two-way ANOVA showed significant differences in time dependency between spontaneous and Ca(2+)-induced rat uteri after applying 3 mM H(2)O(2) (type of contraction, P = 0.0280), but not 400 μM H(2)O(2) (P = 0.9271). Our results indicate that H(2)O(2) oxidises channel intracellular thiol groups and activates the channel, inducing relaxation. Cell antioxidative defence system quickly activates glutathione peroxidase (GSHPx) defence mechanism but not catalase (CAT) defence mechanism. Intracellular redox mechanisms repair the oxidised sites and again establish deactivation of K(V) channels, recuperating contractility. In conclusion, our results demonstrate that K(V) channels can be altered in a time-dependent manner by reversible redox-dependent intracellular alterations.
The Amyloid Precursor Protein (APP) is involved in the regulation of multiple cellular functions via protein-protein interactions and has been most studied with respect to Alzheimer's disease (AD). Abnormal processing of the single transmembrane-spanning C99 fragment of APP contributes to the formation of amyloid plaques, which are causally related to AD. Pathological C99 accumulation is thought to associate with early cognitive defects in AD. Here, unexpectedly, sequence analysis revealed that C99 exhibits 24% sequence identity with the KCNE1 voltage-gated potassium (Kv) channel β subunit, comparable to the identity between KCNE1 and KCNE2-5 (21-30%). This suggested the possibility of C99 regulating Kv channels.
Voltage-gated potassium channels are integral membrane proteins selectively permeable for potassium ions and activated upon change of membrane potential. Voltage-gated potassium channels of the Kv1.3 type were discovered both in plasma membrane and in inner mitochondrial membrane (mito Kv1.3 channels). For some time Kv1.3 channels located both in plasma membrane and in mitochondria are considered as a potentially new molecular target in several pathologies including some cancer disorders. Lipophilic nontoxic organic inhibitors of Kv1.3 channels may potentially find a clinical application to support therapy of some cancer diseases such as breast, pancreas and lung cancer, melanoma or chronic lymphocytic leukaemia (B-CLL). Inhibition of T lymphocyte Kv1.3 channels may be also important in treatment of chronic and acute respiratory diseases including severe pulmonary complication in corona virus disease Covid 19, however further studies are needed to confirm this supposition. Statins are small-molecule organic compounds, which are lipophilic and are widely used in treatment of hypercholesterolemia and atherosclerosis. Electrophysiological studies performed in our laboratory showed that statins: pravastatin, mevastatin and simvastatin are effective inhibitors of Kv1.3 channels in cancer cells of human T cell line Jurkat. We showed that application of the statins in the concentration range from 1.5 μM to 50 μM inhibited the channels in a concentration-dependent manner. The inhibitory effect was the most potent in case of simvastatin and the least potent in case of pravastatin. The inhibition was partially irreversible in case of simvastatin and fully reversible in case of pravastatin and mevastatin. It was accompanied by a significant acceleration of the current inactivation rate without any significant change of the activation rate. Mechanism of the inhibition is probably complex, including a direct interaction with the channel protein and perturbation of lipid bilayer structure, leading to stabilization of the inactivated state of the channels.
The influence of membrane cholesterol content on a variety of ion channel conductances in numerous cell models has been shown, but studies exploring its role in auditory hair cell physiology are scarce. Recent evidence shows that cholesterol depletion affects outer hair cell electromotility and the voltage-gated potassium currents underlying tall hair cell development, but the effects of cholesterol on the major ionic currents governing auditory hair cell excitability are unknown. We investigated the effects of a cholesterol-depleting agent (methyl beta cyclodextrin, MβCD) on ion channels necessary for the early stages of sound processing. Large-conductance BK-type potassium channels underlie temporal processing and open in a voltage- and calcium-dependent manner. Voltage-gated calcium channels (VGCCs) are responsible for calcium-dependent exocytosis and synaptic transmission to the auditory nerve. Our results demonstrate that cholesterol depletion reduced peak steady-state calcium-sensitive (BK-type) potassium current by 50% in chick cochlear hair cells. In contrast, MβCD treatment increased peak inward calcium current (~30%), ruling out loss of calcium channel expression or function as a cause of reduced calcium-sensitive outward current. Changes in maximal conductance indicated a direct impact of cholesterol on channel number or unitary conductance. Immunoblotting following sucrose-gradient ultracentrifugation revealed BK expression in cholesterol-enriched microdomains. Both direct impacts of cholesterol on channel biophysics, as well as channel localization in the membrane, may contribute to the influence of cholesterol on hair cell physiology. Our results reveal a new role for cholesterol in the regulation of auditory calcium and calcium-activated potassium channels and add to the growing evidence that cholesterol is a key determinant in auditory physiology.
Kv7 (KCNQ) voltage-gated potassium channels are critical regulators of neuronal excitability and are candidate targets for development of antiseizure medications. Drug discovery efforts have identified small molecules that modulate channel function and reveal mechanistic insights into Kv7 channel physiological roles. While Kv7 channel activators have therapeutic benefits, inhibitors are useful for understanding channel function and mechanistic validation of candidate drugs. In this study, we reveal the mechanism of a Kv7.2/Kv7.3 inhibitor, ML252. We used docking and electrophysiology to identify critical residues involved in ML252 sensitivity. Most notably, Kv7.2[W236F] or Kv7.3[W265F] mutations strongly attenuate ML252 sensitivity. This tryptophan residue in the pore is also required for sensitivity to certain activators, including retigabine and ML213. We used automated planar patch clamp electrophysiology to assess competitive interactions between ML252 and different Kv7 activator subtypes. A pore-targeted activator (ML213) weakens the inhibitory effects of ML252, whereas a distinct activator subtype (ICA-069673) that targets the voltage sensor does not prevent ML252 inhibition. Using transgenic zebrafish larvae expressing an optical reporter (CaMPARI) to measure neural activity in-vivo, we demonstrate that Kv7 inhibition by ML252 increases neuronal excitability. Consistent with in-vitro data, ML213 suppresses ML252 induced neuronal activity, while the voltage-sensor targeted activator ICA-069673 does not prevent ML252 actions. In summary, this study establishes a binding site and mechanism of action of ML252, classifying this poorly understood drug as a pore-targeted Kv7 channel inhibitor that binds to the same tryptophan residue as commonly used pore-targeted Kv7 activators. ML213 and ML252 likely have overlapping sites of interaction in the pore Kv7.2 and Kv7.3 channels, resulting in competitive interactions. In contrast, the VSD-targeted activator ICA-069673 does not prevent channel inhibition by ML252.
The initiation and propagation of the action potential (AP) along an axon allows neurons to convey information rapidly and across distant sites. Although AP properties have typically been characterized at the soma and proximal axon, knowledge of the propagation of APs toward distal axonal domains of mammalian CNS neurons remains limited. We used genetically encoded voltage indicators (GEVIs) to image APs with submillisecond temporal resolution simultaneously at different locations along the long axons of dissociated hippocampal neurons from rat embryos of either sex. We found that APs became sharper and showed remarkable fidelity as they traveled toward distal axons, even during a high-frequency train. Blocking voltage-gated potassium channels (Kv) with 4-AP resulted in an increase in AP width in all compartments, which was stronger at distal locations and exacerbated during AP trains. We conclude that the higher levels of Kv channel activity in distal axons serve to sustain AP fidelity, conveying a reliable digital signal to presynaptic boutons.SIGNIFICANCE STATEMENT The AP represents the electrical signal carried along axons toward distant presynaptic boutons where it culminates in the release of neurotransmitters. The nonlinearities involved in this process are such that small changes in AP shape can result in large changes in neurotransmitter release. Since axons are remarkably long structures, any distortions that APs suffer along the way have the potential to translate into a significant modulation of synaptic transmission, particularly in distal domains. To avoid these issues, distal axons have ensured that signals are kept remarkably constant and insensitive to modulation during a train, despite the long distances traveled. Here, we uncover the mechanisms that allow distal axonal domains to provide a reliable and faithful digital signal to presynaptic terminals.
Voltage-gated potassium channels (Kv) and cyclic nucleotide-binding domain-containing cation channels HCN, CNG, and KCNH are the evolutionarily related families of ion channels in animals. Their homologues were found in several lineages of eukaryotes and prokaryotes; however, the actual phylogenetic and structural diversity of these ion channels remains unclear. In this work, we present a taxonomically broad investigation of evolutionary relationships and structural diversity of Kv, HCN, CNG, and KCNH and their homologues in eukaryotes focusing on channels from different protistan groups. We demonstrate that both groups of channels consist of a more significant number of lineages than it was shown before, and these lineages can be grouped in two clusters termed Kv-like channels and CNBD-channels. Moreover, we, for the first time, report the unusual two-repeat tandem Kv-like channels and CNBD-channels in several eukaryotic groups, i.e. dinoflagellates, oomycetes, and chlorarachniophytes. Our findings reveal still underappreciated phylogenetic and structural diversity of eukaryotic ion channel lineages.
The hypothalamic paraventricular nucleus (PVN) is an important site in the regulation of the autonomic nervous system. Specifically, PVN neurons projecting to the rostral ventrolateral medulla (PVN-RVLM) play a regulatory role in the determination of the sympathetic outflow in the cardiovascular system. In the PVN-RVLM neurons, the estrogen receptor β is expressed. However, to date, the effects of estrogen on PVN-RVLM neurons have not been reported. The present study investigated estrogen-mediated modulation of two voltage-gated potassium channel (Kv) subunits, Kv4.2 and Kv4.3, that are expressed predominantly in PVN neurons and the functional current of Kv4.2 and Kv4.3, the transient outward potassium current (IA).
Voltage-gated potassium (Kv) channels enable potassium efflux and membrane repolarization in excitable tissues. Many Kv channels undergo a progressive loss of ion conductance in the presence of a prolonged voltage stimulus, termed slow inactivation, but the atomic determinants that regulate the kinetics of this process remain obscure. Using a combination of synthetic amino acid analogs and concatenated channel subunits we establish two H-bonds near the extracellular surface of the channel that endow Kv channels with a mechanism to time the entry into slow inactivation: an intra-subunit H-bond between Asp447 and Trp434 and an inter-subunit H-bond connecting Tyr445 to Thr439. Breaking of either interaction triggers slow inactivation by means of a local disruption in the selectivity filter, while severing the Tyr445-Thr439 H-bond is likely to communicate this conformational change to the adjacent subunit(s). DOI: http://dx.doi.org/10.7554/eLife.01289.001.
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