Durga P. Mohapatra

Regulation of ion channel localization and phosphorylation by neuronal activity

Voltage-dependent Kv2.1 K+ channels, which mediate delayed rectifier Kv currents (IK), are expressed in large clusters on the somata and dendrites of principal pyramidal neurons, where they regulate neuronal excitability. Here we report activity-dependent changes in the localization and biophysical properties of Kv2.1. In the kainate model of continuous seizures in rat, we find a loss of Kv2.1 clustering in pyramidal neurons in vivo. Biochemical analysis of Kv2.1 in the brains of these rats shows a marked dephosphorylation of Kv2.1. In cultured rat hippocampal pyramidal neurons, glutamate stimulation rapidly causes dephosphorylation of Kv2.1, translocation of Kv2.1 from clusters to a more uniform localization, and a shift in the voltage-dependent activation of IK. An influx of Ca2+ leading to calcineurin activation is both necessary and sufficient for these effects. Our finding that neuronal activity modifies the phosphorylation state, localization and function of Kv2.1 suggests an important link between excitatory neurotransmission and the intrinsic excitability of pyramidal neurons.

Localization and targeting of voltage-dependent ion channels in mammalian central neurons.

The intrinsic electrical properties and the synaptic input-output relationships of neurons are governed by the action of voltage-dependent ion channels. The localization of specific populations of ion channels with distinct functional properties at discrete sites in neurons dramatically impacts excitability and synaptic transmission. Molecular cloning studies have revealed a large family of genes encoding voltage-dependent ion channel principal and auxiliary subunits, most of which are expressed in mammalian central neurons. Much recent effort has focused on determining which of these subunits coassemble into native neuronal channel complexes, and the cellular and subcellular distributions of these complexes, as a crucial step in understanding the contribution of these channels to specific aspects of neuronal function. Here we review progress made on recent studies aimed to determine the cellular and subcellular distribution of specific ion channel subunits in mammalian brain neurons using in situ hybridization and immunohistochemistry. We also discuss the repertoire of ion channel subunits in specific neuronal compartments and implications for neuronal physiology. Finally, we discuss the emerging mechanisms for determining the discrete subcellular distributions observed for many neuronal ion channels.

Desensitization of Capsaicin-activated Currents in the Vanilloid Receptor TRPV1 Is Decreased by the Cyclic AMP-dependent Protein Kinase Pathway

Proinflammatory prostaglandin E2 is known to sensitize sensory neurons to noxious stimuli. This sensitization is mediated by the cAMP-dependent protein kinase (PKA) signal pathway. The capsaicin receptor TRPV1, a non-selective cation channel of sensory neurons involved in the sensation of inflammatory pain, is a target of PKA-mediated phosphorylation. Our goal was to investigate the influence of PKA on Ca2+-dependent desensitization of capsaicin-activated currents. By using site-directed mutagenesis, we created point mutations at PKA consensus sites and studied wild-type and mutant channels transiently expressed in HEK293t cells under whole-cell voltage clamp. We found that forskolin, a stimulator of adenylate cyclase, decreased desensitization of TRPV1. The selective PKA inhibitor H89 inhibited this effect. Mimicking phosphorylation at PKA consensus sites by replacing Ser-6, Ser-116, Thr-144, Thr-370, Ser-502, Ser-774, or Ser-820 with aspartate resulted in five mutations (S116D, T144D, T370D, S774D, and S820D) that exhibited decreased desensitization as well. However, disrupting phosphorylation by replacing respective sites with alanine resulted in four mutations (S6A, T144A, T370A, and S820A) with desensitization properties resembling those of the aspartate mutations. Significant changes in relative permeabilities for Ca2+ over Na+ or in capsaicin sensitivity could not explain changes in desensitization properties of mutant channels. In mutations S116A, S116D, T370A, and T370D, pretreatment of cells with forskolin did not reduce desensitization as compared with wild-type and other mutant channels. We conclude that Ser-116 and possibly Thr-370 are the most important residues involved in the mechanism of PKA-dependent reduction of desensitization of capsaicin-activated currents.

Regulation of Ca2+-dependent Desensitization in the Vanilloid Receptor TRPV1 by Calcineurin and cAMP-dependent Protein Kinase

The vanilloid receptor TRPV1 is a polymodal nonselective cation channel of nociceptive sensory neurons involved in the perception of inflammatory pain. TRPV1 exhibits desensitization in a Ca2+-dependent manner upon repeated activation by capsaicin or protons. The cAMP-dependent protein kinase (PKA) decreases desensitization of TRPV1 by directly phosphorylating the channel presumably at sites Ser116 and Thr370. In the present study we investigated the influence of protein phosphatase 2B (calcineurin) on Ca2+-dependent desensitization of capsaicin- and proton-activated currents. By using site-directed mutagenesis, we generated point mutations at PKA and protein kinase C consensus sites and studied wild type (WT) and mutant channels transiently expressed in HEK293t or HeLa cells under whole cell voltage clamp. We found that intracellular application of the cyclosporin A·cyclophilin A complex (CsA·CyP), a specific inhibitor of calcineurin, significantly decreased desensitization of capsaicin- or proton-activated TRPV1-WT currents. This effect was similar to that obtained by extracellular application of forskolin (FSK), an indirect activator of PKA. Simultaneous applications of CsA·CyP and FSK in varying concentrations suggested that these substances acted independently from each other. In mutation T370A, application of CsA·CyP did not reduce desensitization of capsaicin-activated currents as compared with WT and to mutant channels S116A and T144A. In a double mutation at candidate protein kinase C phosphorylation sites, application of CsA·CyP or FSK decreased desensitization of capsaicin-activated currents similar to WT channels. We conclude that Ca2+-dependent desensitization of TRPV1 might be in part regulated through channel dephosphorylation by calcineurin and channel phosphorylation by PKA possibly involving Thr370 as a key amino acid residue.

Calcium- and Metabolic State-Dependent Modulation of the Voltage-Dependent Kv2.1 Channel Regulates Neuronal Excitability in Response to Ischemia

Ischemic stroke is often accompanied by neuronal hyperexcitability (i.e., seizures), which aggravates brain damage. Therefore, suppressing stroke-induced hyperexcitability and associated excitoxicity is a major focus of treatment for ischemic insults. Both ATP-dependent and Ca2+-activated K+ channels have been implicated in protective mechanisms to suppress ischemia-induced hyperexcitability. Here we provide evidence that the localization and function of Kv2.1, the major somatodendritic delayed rectifier voltage-dependent K+ channel in central neurons, is regulated by hypoxia/ischemia-induced changes in metabolic state and intracellular Ca2+ levels. Hypoxia/ischemia in rat brain induced a dramatic dephosphorylation of Kv2.1 and the translocation of surface Kv2.1 from clusters to a uniform localization. In cultured rat hippocampal neurons, chemical ischemia (CI) elicited a similar dephosphorylation and translocation of Kv2.1. These events were reversible and were mediated by Ca2+ release from intracellular stores and calcineurin-mediated Kv2.1 dephosphorylation. CI also induced a hyperpolarizing shift in the voltage-dependent activation of neuronal delayed rectifier currents (IK), leading to enhanced IK and suppressed neuronal excitability. The IK blocker tetraethylammonium reversed the ischemia-induced suppression of excitability and aggravated ischemic neuronal damage. Our results show that Kv2.1 can act as a novel Ca2+- and metabolic state-sensitive K+ channel and suggest that dynamic modulation of IK/Kv2.1 in response to hypoxia/ischemia suppresses neuronal excitability and could confer neuroprotection in response to brief ischemic insults.

The Kv2.1 C Terminus Can Autonomously Transfer Kv2.1-Like Phosphorylation-Dependent Localization, Voltage-Dependent Gating, and Muscarinic Modulation to Diverse Kv Channels

Modulation of K+ channels is widely used to dynamically regulate neuronal membrane excitability. The voltage-gated K+ channel Kv2.1 is an abundant delayed rectifier K+ (IK) channel expressed at high levels in many types of mammalian central neurons where it regulates diverse aspects of membrane excitability. Neuronal Kv2.1 is constitutively phosphorylated, localized in high-density somatodendritic clusters, and has a relatively depolarized voltage dependence of activation. Here, we show that the clustering and voltage-dependent gating of endogenous Kv2.1 in cultured rat hippocampal neurons are modulated by cholinergic stimulation, a common form of neuromodulation. The properties of neuronal Kv2.1 are recapitulated in recombinant Kv2.1 expressed in human embryonic kidney 293 (HEK293) cells, but not COS-1 cells, because of cell background-specific differences in Kv2.1 phosphorylation. As in neurons, Kv2.1 in HEK293 cells is dynamically regulated by cholinergic stimulation, which leads to Ca2+/calcineurin-dependent dephosphorylation of Kv2.1, dispersion of channel clusters, and hyperpolarizing shifts in the voltage-dependent gating properties of the channel. Immunocytochemical, biochemical, and biophysical analyses of chimeric Kv channels show that the Kv2.1 cytoplasmic C-terminal domain can act as an autonomous domain sufficient to transfer Kv2.1-like clustering, voltage-dependent activation, and cholinergic modulation to diverse Kv channels. These findings provide novel mechanistic insights into cholinergic modulation of ion channels and regulation of the localization and voltage-dependent gating properties of the abundant neuronal Kv2.1 channel by cholinergic and other neuromodulatory stimuli.

A tyrosine residue in TM6 of the Vanilloid Receptor TRPV1 involved in desensitization and calcium permeability of capsaicin-activated currents

The Vanilloid Receptor TRPV1 is a non-selective cation channel with a high relative Ca(2+) permeability. TRPV1 exhibits slow desensitization, a potential mechanism regulating adaptation of peripheral sensory neurons to noxious stimuli. The predicted folding pattern of TRPV1 resembles that of voltage-gated channels. Sequence alignment of segments 6 of TRPV1 and voltage-gated Na(+) channels reveals a conserved aromatic amino acid that in Na(+) channels is involved in fast inactivation and pharmacological block. We found that replacing this tyrosine Y671 by positively charged lysine (K) completely abrogated Ca(2+)-dependent desensitization. Y671K also exhibited significant reduction in Ca(2+) permeability that was not responsible for the lack in desensitization. Substitution of Y671 with negatively charged aspartate or uncharged alanine slightly altered desensitization but left Ca(2+) permeability unchanged. Substitution of Y671 with positively charged arginine produced a phenotype similar to Y671K. We propose that residue Y671 is critical for the high relative Ca(2+) permeability of TRPV1 and participates in the structural rearrangements of the channel protein leading to Ca(2+)-dependent desensitization.

SynDIG1: an activity-regulated AMPA receptor-interacting transmembrane protein that regulates excitatory synapse development

During development of the central nervous system, precise synaptic connections between presynaptic and postsynaptic neurons are formed. While significant progress has been made in our understanding of AMPA receptor trafficking during synaptic plasticity, less is known about the molecules that recruit AMPA receptors to nascent synapses during synaptogenesis. Here we identify a type II transmembrane protein (SynDIG1) that regulates AMPA receptor content at developing synapses in dissociated rat hippocampal neurons. SynDIG1 colocalizes with AMPA receptors at synapses and at extrasynaptic sites and associates with AMPA receptors in heterologous cells and brain. Altered levels of SynDIG1 in cultured neurons result in striking changes in excitatory synapse number and function. SynDIG1-mediated synapse development is dependent on association with AMPA receptors via its extracellular C terminus. Intriguingly, SynDIG1 content in dendritic spines is regulated by neuronal activity. Altogether, we define SynDIG1 as an activity-regulated transmembrane protein that regulates excitatory synapse development.

Bidirectional Activity-Dependent Regulation of Neuronal Ion Channel Phosphorylation

Activity-dependent dephosphorylation of neuronal Kv2.1 channels yields hyperpolarizing shifts in their voltage-dependent activation and homoeostatic suppression of neuronal excitability. We recently identified 16 phosphorylation sites that modulate Kv2.1 function. Here, we show that in mammalian neurons, compared with other regulated sites, such as serine (S)563, phosphorylation at S603 is supersensitive to calcineurin-mediated dephosphorylation in response to kainate-induced seizures in vivo, and brief glutamate stimulation of cultured hippocampal neurons. In vitro calcineurin digestion shows that supersensitivity of S603 dephosphorylation is an inherent property of Kv2.1. Conversely, suppression of neuronal activity by anesthetic in vivo causes hyperphosphorylation at S603 but not S563. Distinct regulation of individual phosphorylation sites allows for graded and bidirectional homeostatic regulation of Kv2.1 function. S603 phosphorylation represents a sensitive bidirectional biosensor of neuronal activity.

RGS6, a Modulator of Parasympathetic Activation in Heart

Rationale: Parasympathetic regulation of heart rate is mediated by acetylcholine binding to G protein–coupled muscarinic M2 receptors, which activate heterotrimeric Gi/o proteins to promote G protein–coupled inwardly rectifying K+ (GIRK) channel activation. Regulator of G protein signaling (RGS) proteins, which function to inactivate G proteins, are indispensable for normal parasympathetic control of the heart. However, it is unclear which of the more than 20 known RGS proteins function to negatively regulate and thereby ensure normal parasympathetic control of the heart. Objective: To examine the specific contribution of RGS6 as an essential regulator of parasympathetic signaling in heart. Methods and Results: We developed RGS6 knockout mice to determine the functional impact of loss of RGS6 on parasympathetic regulation of cardiac automaticity. RGS6 exhibited a uniquely robust expression in the heart, particularly in sinoatrial and atrioventricular nodal regions. Loss of RGS6 provoked dramatically exaggerated bradycardia in response to carbachol in mice and isolated perfused hearts and significantly enhanced the effect of carbachol on inhibition of spontaneous action potential firing in sinoatrial node cells. Consistent with a role of RGS6 in G protein inactivation, RGS6-deficient atrial myocytes exhibited a significant reduction in the time course of acetylcholine-activated potassium current (IKACh) activation and deactivation, as well as the extent of IKACh desensitization. Conclusions: RGS6 is a previously unrecognized, but essential, regulator of parasympathetic activation in heart, functioning to prevent parasympathetic override and severe bradycardia. These effects likely result from actions of RGS6 as a negative regulator of G protein activation of GIRK channels.