Determinants of voltage-gated potassium channel surface expression and localization in Mammalian neurons

Neurons strictly regulate expression of a wide variety of voltage-dependent ion channels in their surface membranes to achieve precise yet dynamic control of intrinsic membrane excitability. Neurons also exhibit extreme morphological complexity that underlies diverse aspects of their function. Most ion channels are preferentially targeted to either the axonal or somatodendritic compartments, where they become further localized to discrete membrane subdomains. This restricted accumulation of ion channels enables local control of membrane signaling events in specific microdomains of a given compartment. Voltage-dependent K+ (Kv) channels act as potent modulators of diverse excitatory events such as action potentials, excitatory synaptic potentials, and Ca2+ influx. Kv channels exhibit diverse patterns of cellular expression, and distinct subtype-specific localization, in mammalian central neurons. Here we review the mechanisms regulating the abundance and distribution of Kv channels in mammalian neurons and discuss how dynamic regulation of these events impacts neuronal signaling.     

Modulation by Clamping: Kv4 and KChIP Interactions

The rapidly inactivating (A-type) potassium channels regulate membrane excitability that defines the fundamental mechanism of neuronal functions such as pain signaling. Cytosolic Kv channel-interacting proteins KChIPs that belong to neuronal calcium sensor (NCS) family of calcium binding EF-hand proteins co-assemble with Kv4 (Shal) α subunits to form a native complex that encodes major components of neuronal somatodendritic A-type K⁺ current, I_SA, in neurons and transient outward current, I_TO, in cardiac myocytes. The specific binding of auxiliary KChIPs to the Kv4 N-terminus results in modulation of gating properties, surface expression and subunit assembly of Kv4 channels. Here, I attempt to emphasize the interaction between KChIPs and Kv4 based on recent progress made in understanding the structure complex in which a single KChIP1 molecule laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner. Greater insights into molecular mechanism between KChIPs and Kv4 interaction may provide therapeutic potentials of designing compounds aimed at disrupting the protein–protein interaction for treatment of membrane excitability-related disorders. Special issue article in honor of Dr. Ji-Sheng Han.     

Expression of Kv1.3 potassium channels regulates density of cortical interneurons

The Kv1.3 protein is a member of the large family of voltage‐dependent K⁺ subunits (Kv channels), which assemble to form tetrameric membrane‐spanning channels that provide a selective pore for the conductance of K⁺ across the cell membrane. Kv1.3 differs from most other Kv channels in that deletion of Kv1.3 gene produces very striking changes in development and structure of the olfactory bulb, where Kv1.3 is expressed at high levels, resulting in a lower threshold for detection of odors, an increased number of synaptic glomeruli and alterations in the levels of a variety of neuronal signaling molecules. Because Kv1.3 is also expressed in the cerebral cortex, we have now examined the effects of deletion of the Kv1.3 gene on the expression of interneuron populations of the cerebral cortex. Using unbiased stereology we found an increase in the number of parvalbumin (PV) cells in whole cerebral cortex of Kv1.3^?/? mice relative to that in wild‐type mice, and a decrease in the number of calbindin (CB), calretinin (CR), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), and somatostatin (SOM) interneurons. These changes are accompanied by a decrease in the cortical volume such that the cell density of PV interneurons is significantly increased and that of SOM neurons is decreased in Kv1.3^?/? animals. Our studies suggest that, as in the olfactory bulb, Kv1.3 plays a unique role in neuronal differentiation and/or survival of interneuron populations and that expression of Kv1.3 is required for normal cortical function. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73:841–855, 2013     

Excitability of oxytocin neurons in paraventricular nucleus is regulated by voltage-gated potassium channels Kv4.2 and Kv4.3

Oxytocin is produced by neurons in the paraventricular nucleus (PVN) and the supraoptic nucleus in the hypothalamus. Various ion channels are considered to regulate the excitability of oxytocin neurons and its secretion. A-type currents of voltage-gated potassium channels (Kv channels), generated by Kv4.2/4.3 channels, are known to be involved in the regulation of neuron excitability. However, it is unclear whether the Kv4.2/4.3 channels participate in the regulation of excitability in PVN oxytocin neurons. Here, we investigated the contribution of the Kv4.2/4.3 channels to PVN oxytocin neuron excitability. By using transgenic rat brain slices with the oxytocin-monomeric red fluorescent protein 1 fusion transgene, we examined the excitability of oxytocin neurons by electrophysiological technique. In some oxytocin neurons, the application of Kv4.2/4.3 channel blocker increased firing frequency and membrane potential with extended action potential half-width. Our present study indicates the contribution of Kv4.2/4.3 channels to PVN oxytocin neuron excitability regulation.

Abbreviation: PVN, paraventricular nucleus; Oxt-mRFP1, Oxt-monometric red fluorescent protein 1; PaTx-1, Phrixotoxin-1; TEA, Tetraethylammonium Chloride; TTX, tetrodotoxin; aCSF, artificial cerebrospinal fluid;PBS, phosphate buffered saline 3v, third ventricle.     

Molecular diversity and function of voltage-gated (Kv) potassium channels in epithelial cells

Voltage-gated K+ channels belonging to Kv1-9 subfamilies are widely expressed in excitable cells where they play an essential role in membrane hyperpolarization during an action potential and in the propagation of action potentials along the plasma membrane. Early patch clamp studies on epithelial cells revealed the presence of K+ currents with biophysical and pharmacologic properties characteristic of Kv channels expressed in excitable cells. More recently, molecular approaches including PCR and the availability of more selective antibodies directed against Kv alpha and auxiliary subunits, have demonstrated that epithelial cells from various organ systems, express a remarkable diversity Kv channel subunits. Unlike neurons and myocytes however, epithelial cells do not typically generate action potentials or exhibit dynamic changes in membrane potential necessary for activation of Kv alpha subunits. Moreover, the fact that many Kv channels expressed in epithelial cells exhibit inactivation suggest that their activities are relatively transient, making it difficult to ascribe a functional role for these channels in transepithelial electrolyte or nutrient transport. Other proposed functions have included (i) cell migration and wound healing, (ii) cell proliferation and cancer, (iii) apoptosis and (iv) O2 sensing. Certain Kv channels, particularly Kv1 and Kv2 subfamily members, have been shown to be involved in the proliferation of prostate, colon, lung and breast carcinomas. In some instances, a significant increase in Kv channel expression has been correlated with tumorogenesis suggesting the possibility of using these proteins as markers for transformation and perhaps reducing the rate of tumor growth by selectively inhibiting their functional activity.     

Differential modulation of cardiac potassium channels by Grb adaptor proteins

Scaffolding growth factor receptor-bound (Grb) adaptor proteins are components of macromolecular signaling complexes at the plasma membrane and thus are putative regulators of ion channel activity. The present study aimed to define the impact of Grb adaptor proteins on the function of cardiac K⁺ channels. To this end channel proteins were coinjected with the adaptor proteins in Xenopus oocytes and channel activity analyzed with two-electrode voltage-clamp. It is shown that coexpression of Grb adaptor proteins can reduce current amplitudes of coexpressed channels. Grb7 and 10 significantly inhibited functional currents generated by hERG, Kv1.5 and Kv4.3 channels. Only Grb10 significantly inhibited KCNQ1/KCNE1 K⁺ channels, and only Grb7 reduced Kir2.3 activity, whereas neither Grb protein significantly affected the closely related Kir2.1 and Kir2.2 channels. The present observations for the first time provide evidence for a selective and modulatory role of Grb adaptor proteins in the functional expression of cardiac K⁺ channels.     

The Sensorless Pore Module of Voltage-gated K+ Channel Family 7 Embodies the Target Site for the Anticonvulsant Retigabine

KCNQ (voltage-gated K⁺ channel family 7 (Kv7)) channels control cellular excitability and underlie the K⁺ current sensitive to muscarinic receptor signaling (the M current) in sympathetic neurons. Here we show that the novel anti-epileptic drug retigabine (RTG) modulates channel function of pore-only modules (PMs) of the human Kv7.2 and Kv7.3 homomeric channels and of Kv7.2/3 heteromeric channels by prolonging the residence time in the open state. In addition, the Kv7 channel PMs are shown to recapitulate the single-channel permeation and pharmacological specificity characteristics of the corresponding full-length proteins in their native cellular context. A mutation (W265L) in the reconstituted Kv7.3 PM renders the channel insensitive to RTG and favors the conductive conformation of the PM, in agreement to what is observed when the Kv7.3 mutant is heterologously expressed. On the basis of the new findings and homology models of the closed and open conformations of the Kv7.3 PM, we propose a structural mechanism for the gating of the Kv7.3 PM and for the site of action of RTG as a Kv7.2/Kv7.3 K⁺ current activator. The results validate the modular design of human Kv channels and highlight the PM as a high-fidelity target for drug screening of Kv channels.     

Concerted Trafficking Regulation of Kv2.1 and KATP Channels by Leptin in Pancreatic β-Cells

In pancreatic β-cells, voltage-gated potassium 2.1 (Kv2.1) channels are the dominant delayed rectifier potassium channels responsible for action potential repolarization. Here, we report that leptin, a hormone secreted by adipocytes known to inhibit insulin secretion, causes a transient increase in surface expression of Kv2.1 channels in rodent and human β-cells. The effect of leptin on Kv2.1 surface expression is mediated by the AMP-activated protein kinase (AMPK). Activation of AMPK mimics whereas inhibition of AMPK occludes the effect of leptin. Inhibition of Ca²⁺/calmodulin-dependent protein kinase kinase β, a known upstream kinase of AMPK, also blocks the effect of leptin. In addition, the cAMP-dependent protein kinase (PKA) is involved in Kv2.1 channel trafficking regulation. Inhibition of PKA prevents leptin or AMPK activators from increasing Kv2.1 channel density, whereas stimulation of PKA is sufficient to promote Kv2.1 channel surface expression. The increased Kv2.1 surface expression by leptin is dependent on actin depolymerization, and pharmacologically induced actin depolymerization is sufficient to enhance Kv2.1 surface expression. The signaling and cellular mechanisms underlying Kv2.1 channel trafficking regulation by leptin mirror those reported recently for ATP-sensitive potassium (K_ATP) channels, which are critical for coupling glucose stimulation with membrane depolarization. We show that the leptin-induced increase in surface K_ATP channels results in more hyperpolarized membrane potentials than control cells at stimulating glucose concentrations, and the increase in Kv2.1 channels leads to a more rapid repolarization of membrane potential in cells firing action potentials. This study supports a model in which leptin exerts concerted trafficking regulation of K_ATP and Kv2.1 channels to coordinately inhibit insulin secretion.     

Mass spectrometry-based phosphoproteomics reveals multisite phosphorylation on mammalian brain voltage-gated sodium and potassium channels

Voltage-gated sodium and potassium channels underlie electrical activity of neurons, and are dynamically regulated by diverse cell signaling pathways that ultimately exert their effects by altering the phosphorylation state of channel subunits. Recent mass spectrometric-based studies have led to a new appreciation of the extent and nature of phosphorylation of these ion channels in mammalian brain. This has allowed for new insights into how neurons dynamically regulate the localization, activity and expression through multisite ion channel phosphorylation.     

The Kv4.2 mediates excitatory activity-dependent regulation of neuronal excitability in rat cortical neurons

Neuronal excitability can cooperate with synaptic transmission to control the information storage. This regulation of neuronal plasticity can be affected by alterations in neuronal inputs and accomplished by modulation of voltage-dependent ion channels. In this study, we report that enhanced excitatory input negatively regulated neuronal excitability. Enhanced excitatory input by glutamate, electric field stimulation or high K⁺ increased transient outward K⁺ current, whereas did not affect the delayed rectifier K⁺ current in rat cultured cortical neurons. Both the voltage-dependent K⁺ channel 4.2 and 4.3 subunits contributed to the increase. The increase in the K⁺ current density by Kv4.2 was ascribed to its cytoplasmic membrane translocation, which was mediated by NMDA type of glutamate receptor. Furthermore, enhanced excitatory input inhibited neuronal excitability. Taken together, our results suggest that excitatory neurotransmission affects neuronal excitability via the regulation of the K⁺ channel membrane translocation.     

Defective trafficking of Kv2.1 channels in MPTP‐induced nigrostriatal degeneration

Intracellular protein trafficking is tightly regulated, and improper trafficking might be the fundamental provocateur for human diseases including neurodegeneration. In neurons, protein trafficking to and from the plasma membrane affects synaptic plasticity. Voltage‐gated potassium channel 2.1 (Kv2.1) is a predominant delayed rectifier potassium (K⁺) current, and electrical activity patterns of dopamine (DA) neurons within the substantia nigra are generated and modulated by the orchestrated function of different ion channels. The pathological hallmark of Parkinson's disease (PD) is the progressive loss of these DA neurons, resulting in the degeneration of striatal dopaminergic terminals. However, whether trafficking of Kv2.1 channels contributes to PD remains unclear. In this study, we demonstrated that MPTP/MPP⁺ increases the surface expression of the Kv2.1 channel and causes nigrostriatal degeneration by using a subchronic MPTP mouse model. The inhibition of the Kv2.1 channel by using a specific blocker, guangxitoxin‐1E, protected nigrostriatal projections against MPTP/MPP⁺ insult and thus facilitated the recovery of motor coordination. These findings highlight the importance of trafficking of Kv2.1 channels in the pathogenesis of PD.

     

Membrane channels as integrators of G-protein-mediated signaling

A variety of extracellular stimuli regulate cellular responses via membrane receptors. A well-known group of seven-transmembrane domain-containing proteins referred to as G protein-coupled receptors, directly couple with the intracellular GTP-binding proteins (G proteins) across cell membranes and trigger various cellular responses by regulating the activity of several enzymes as well as ion channels. Many specific populations of ion channels are directly controlled by G proteins; however, indirect modulation of some channels by G protein-dependent phosphorylation events and lipid metabolism is also observed. G protein-mediated diverse modifications affect the ion channel activities and spatio-temporally regulate membrane potentials as well as of intracellular Ca^2 + concentrations in both excitatory and non-excitatory cells. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.     

High spatial density is associated with non-conducting Kv channels from two families

Ion channels are well known for their ability to regulate the cell membrane potential. However, many ion channels also have functions that do not involve ion conductance. Kv2 channels are one family of ion channels whose non-conducting functions are central to mammalian cell physiology. Kv2.1 and Kv2.2 channels form stable contact sites between the endoplasmic reticulum and plasma membrane via an interaction with endoplasmic reticulum resident proteins. To perform this structural role, Kv2 channels are expressed at extremely high densities on the plasma membranes of many cell types, including central pyramidal neurons, α-motoneurons, and smooth muscle cells. Research from our lab and others has shown that the majority of these plasma membrane Kv2.1 channels do not conduct potassium in response to depolarization. The mechanism of this channel silencing is unknown but is thought to be dependent on channel density in the membrane. Furthermore, the prevalence of a non-conducting population of Kv2.2 channels has not been directly tested. In this work we make improved measurements of the numbers of conducting and non-conducting Kv2.1 channels expressed in HEK293 cells and expand the investigation of non-conducting channels to three additional Kv α-subunits: Kv2.2, Kv1.4, and Kv1.5. By comparing the numbers of gating and conducting channels in individual HEK293 cells, we found that on average, only 50% of both Kv2.1 and Kv2.2 channels conducted potassium and, as previously suggested, that fraction decreased with increased channel density in the plasma membrane. At the highest spatial densities tested, which are comparable with those found at Kv2 clusters in situ, only 20% of Kv2.1 and Kv2.2 channels conducted potassium. We also show for the first time that Kv1.4 and Kv1.5 exhibit density-dependent silencing, suggesting that this phenomenon has an underlying mechanism that is shared by Kv channels from multiple families.     

Mossy fibre contact triggers the targeting of Kv4.2 potassium channels to dendrites and synapses in developing cerebellar granule neurons

Compartmentalization of neuronal function is achieved by highly localized clustering of ion channels in discrete subcellular membrane domains. Voltage-gated potassium (Kv) channels exhibit highly variable cellular and subcellular patterns of expression. Here, we describe novel activity-dependent synaptic targeting of Kv4.2, a dendritic Kv channel, in cerebellar granule cells (GCs). In vivo, Kv4.2 channels are highly expressed in cerebellar glomeruli, specializations of GC dendrites that form synapses with mossy fibres. In contrast, in cultured GCs, Kv4.2 was found localized, not to dendrites but to cell bodies. To investigate the role of synaptic contacts, we developed a co-culture system with cells from pontine grey nucleus, the origin of mossy fibres. In these co-cultures, synaptic structures formed, and Kv4.2 was now targeted to these synaptic sites in a manner dependent on synaptic activity. Activation of NMDA- and/or AMPA-type glutamate receptors was necessary for the targeting of Kv4.2 in co-cultures, and activation of these receptor systems in GC monocultures induced dendritic targeting of Kv4.2 in the absence of synapse formation. These results indicate that the proper targeting of Kv4.2 channels is dynamically regulated by synaptic activity. This activity-dependent regulation of Kv4.2 localization provides a crucial yet dynamic link between synaptic activity and dendritic excitability.     

Updating In Vivo and In Vitro Phosphorylation and Methylation Sites of Voltage‐Gated Kv7.2 Potassium Channels

Voltage‐gated Kv7.2 potassium channels regulate neuronal excitability. The gating of these channels is tightly controlled by various mediators and neurotransmitters acting via G protein‐coupled receptors; the underlying signaling cascades involve phosphatidylinositol‐4,5‐bisphosphate (PIP₂), Ca²⁺/calmodulin, and phosphorylation. Recent studies found that the PIP₂ sensitivity of Kv7.2 channels is affected by two posttranslational modifications, phosphorylation and methylation, harboured within putative PIP₂‐binding domains. In this study, we updated phosphorylation and methylation sites in Kv7.2 either heterologously expressed in mammalian cells or as GST‐fusion proteins exposed to recombinant protein kinases by using LC–MS/MS. In vitro kinase assays revealed that CDK5, protein kinase C (PKC) alpha, PKA, p38 MAPK, CamKIIα, and GSK3β could mediate phosphorylation. Taken together, we provided a comprehensive map of phosphorylation and methylation in Kv7.2 within protein–protein and protein–lipid interaction domains. This may help to interpret the functional roles of individual PTM sites in Kv7.2 channels. All MS data are available via ProteomeXchange with the identifier PXD005567.     

Ethanol inhibits Kv7.2/7.3 channel open probability by reducing the PI(4,5)P2 sensitivity of Kv7.2 subunit

Ethanol often causes critical health problems by altering the neuro-nal activities of the central and peripheral nerve systems. One of the cellular targets of ethanol is the plasma membrane proteins including ion channels and receptors. Recently, we reported that ethanol elevates membrane excitability in sympathetic neurons by inhibiting Kv7.2/7.3 channels in a cell type-specific manner. Even though our studies revealed that the inhibitory effects of ethanol on the Kv7.2/7.3 channel was diminished by the increase of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI (4,5)P₂), the molecular mechanism of ethanol on Kv7.2/7.3 channel inhibition remains unclear. By investigating the kinetics of Kv7.2/7.3 current in high K⁺ solution, we found that ethanol inhibited Kv7.2/7.3 channels through a mechanism distinct from that of tetraethylammonium (TEA) which enters into the pore and blocks the gate of the channels. Using a non-stationary noise analysis (NSNA), we demonstrated that the inhibitory effect of ethanol is the result of reduction of open probability (P_O) of the Kv7.2/7.3 channel, but not of a single channel current (i) or channel number (N). Finally, ethanol selectively facilitated the kinetics of Kv7.2 current suppression by voltage-sensing phosphatase (VSP)-induced PI(4,5)P₂ depletion, while it slowed down Kv7.2 current recovery from the VSP-induced inhibition. Together our results suggest that ethanol regulates neuronal activity through the reduction of open probability and PI(4,5)P₂ sensitivity of Kv7.2/7.3 channels.     

Proteomic analysis highlights the molecular complexities of native Kv4 channel macromolecular complexes

Voltage-gated K(+) (Kv) channels are key determinants of membrane excitability in the nervous and cardiovascular systems, functioning to control resting membrane potentials, shape action potential waveforms and influence the responses to neurotransmitters and neurohormones. Consistent with this functional diversity, multiple types of Kv currents, with distinct biophysical properties and cellular/subcellular distributions, have been identified. Rapidly activating and inactivating Kv currents, typically referred to as I(A) (A-type) in neurons, for example, regulate repetitive firing rates, action potential back-propagation (into dendrites) and modulate synaptic responses. Currents with similar properties, referred to as I(to,f) (fast transient outward), expressed in cardiomyocytes, control the early phase of myocardial action potential repolarization. A number of studies have demonstrated critical roles for pore-forming (α) subunits of the Kv4 subfamily in the generation of native neuronal I(A) and cardiac I(to,f) channels. Studies in heterologous cells have also suggested important roles for a number of Kv channel accessory and regulatory proteins in the generation of functional I(A) and I(to,f) channels. Quantitative mass spectrometry-based proteomic analysis is increasingly recognized as a rapid and, importantly, unbiased, approach to identify the components of native macromolecular protein complexes. The recent application of proteomic approaches to identify the components of native neuronal (and cardiac) Kv4 channel complexes has revealed even greater complexity than anticipated. The continued emphasis on development of improved biochemical and analytical proteomic methods seems certain to accelerate progress and to provide important new insights into the molecular determinants of native ion channel protein complexes.