Analysis and functional implications of phosphorylation of neuronal voltage-gated potassium channels

Abstract

Phosphorylation is the most common and abundant posttranslational modification to eukaryotic proteins, regulating a plethora of dynamic cellular processes. Here, we review and discuss recent advances in our knowledge of the breadth and importance of reversible phosphorylation in regulating the expression, localization and function of mammalian neuronal voltage-gated potassium (Kv) channels, key regulators of neuronal function. We highlight the role of modern mass spectrometric techniques and phosphospecific antibodies that reveal the extent and nature of phosphorylation at specific sites in Kv channels. We also emphasize the role of reversible phosphorylation in dynamically regulating diverse aspects of Kv channel biology. Finally, we discuss as important future directions the determination of the mechanistic basis for how altering phosphorylation state affects Kv channel expression, localization and function, the nature of macromolecular signaling complexes containing Kv channels and enzymes regulating their phosphorylation state, and the specific role of Kv channel phosphorylation in regulating neuronal function during physiological and pathophysiological events.

Introduction

Voltage-gated potassium or Kv channels are integral membrane proteins that allow for the selective flux of K⁺ ions across membranes [38]. These multisubunit polytopic integral proteins mediate K⁺ fluxes depending on their level of expression, their conduction properties, their activation, deactivation and inactivation characteristics, and the electrochemical gradient of specific ions across the cell membrane. Typically, opening of plasma membrane Kv channels leads to efflux of K⁺, as cells generally maintain a large chemical gradient of intracellular K⁺ [18]. This efflux generally leads to membrane hyperpolarization, given the large and negative equilibrium potential for K⁺. Plasma membrane Kv channels may also contribute to setting a cell’s resting potential, and in many cells in the body exert effects on numerous and diverse processes, such as cell division and differentiation, transcription, metabolism and cytoskeletal and membrane dynamics, though their effects on Ca²⁺ entry via plasma membrane voltage-gated Ca²⁺ channels [18]. In neurons, and in cardiac and skeletal muscle fibers, plasma membrane Kv channels modulate membrane excitability that triggers specialized functions such as secretion and contraction. Kv channel activity can either suppress the induction of such depolarizing excitatory events, or restore the cell’s resting membrane potential following a depolarizing event. The expression level of Kv channel proteins in the plasma membrane is regulated by diverse mechanisms including intracellular biosynthetic trafficking culminating in plasma membrane insertion, targeting of Kv channels to and their clustering at discrete subcellular sites within the plasma membrane, and regulated endocytosis followed by degradation or reinsertion. The activity of Kv channels in the plasma membrane is directly controlled by membrane potential, pH, redox potential, and binding of extracellular and intracellular ligands. The activity of Kv channels can also be modulated indirectly via signal transduction pathways leading to modifications of Kv channel intracellular domains, either through non-covalent binding of intracellular second messengers or interacting proteins, or through covalent posttranslational modifications mediated by a diverse repertoire of cytoplasmic modifying enzymes (reviewed in [27]).

Mammalian genomes contain on average ≈40 genes encoding the primary or α subunits of Kv channels, which are the transmembrane subunits that mediate conduction of K⁺ across membranes [38]. Kv channel α subunits have six transmembrane segments S1-S6, the first four (S1-S4) forming the voltage sensor, and the last two (S5-S6) forming the pore [38]. Kv channels can also contain transmembrane and/or cytoplasmic auxiliary subunits, which in themselves cannot form functional channels but that can impact the function of co-assembled α subunits. A wide variety of Kv channels can be formed by the combinatorial co-assembly of α and auxiliary subunits to generate a diversity of multisubunit Kv channel proteins with diverse structures and functions. The resultant Kv channels can also exhibit distinct sensitivities to modulation by intracellular second messengers, interacting proteins and covalent modification.

Protein phosphorylation is the most common covalent posttranslational modification in signal transduction [57]. Phosphorylation, which is reversible and dynamic, affects virtually all cellular processes, including metabolism, growth, division, differentiation, motility, gene expression, translation, intracellular and intercellular communication [74]. It is estimated that 30% of all cellular proteins are targets of phosphorylation [5]. Phosphorylation consists of the transfer of the γ-phosphate group of ATP to the hydroxyl group on the side chains of serine, threonine or tyrosine residues of target proteins in a motif dependent context. This phosphoryl transfer reaction is enzymatically mediated by protein kinases or PKs, whereas enzymatic hydrolytic removal of phosphate from proteins is mediated protein phosphatases or PPs. Protein kinases constitute a major family of human genes, encoding ≈ 500 different PKs, of which ≈ 400 are specific for Ser/Thr, and ≈ 100 for Tyr) [74]. There exist ≈ 150 human PP genes (≈40 specific for Ser/Thr, and ≈100 for Tyr) [49]. The concerted activity of the PK and PP repertoire of cells determines the relative levels of target proteins found in their phosphorylated and dephosphorylated states. There is abundant and increasing evidence that a wide variety of Kv channels serve as direct targets of covalent modification by diverse PKs and PPs, whose activity is regulated by diverse signaling pathways, resulting in dynamic and reversible changes in Kv channel expression, localization, and function [56].

Neuronal plasticity through changes in Kv channel expression, localization and function

Since Kv channel activity allows the passage of K⁺ across the plasma membrane, a necessary requisite for this process is its presence within this cellular compartment. Therefore, the number of Kv channels of a specific type present in the plasma membrane will be critical in determining the impact of a given Kv channel on neuronal function. The subunit composition of Kv channels is achieved through combinatorial assembly of component subunits in the endoplasmic reticulum. As subunit assembly is cotranslational [8, 66], post-translational events such as phosphorylation are not thought to play a role in the assembly process. Moreover, no evidence has been provided for phosphorylation-dependent changes in the subunit composition of existing channels once they are assembled. Changes in the number of Kv channels present in the plasma membrane, through effects on biosynthetic intracellular trafficking, and through retrieval/reinsertion of existing Kv channels, represents a potential target for phosphorylation-dependent modulation of neuronal function (Fig. 1).

Figure 1. Mechanisms of Kv channel modulation by phosphorylation in neurons.

Reversible changes in phosphorylation of dendritic and axonal Kv channels can dynamically modulate their expression, localization and activity different diverse mechanisms, such as a) regulating intracellular trafficking (e.g. Kv1.2, Kv4.2), b) distribution within the plasma membrane (e.g. Kv2.1), and/or c) direct modulation of voltage-dependent gating (e.g. Kv2.1, Kv3.1, Kv7.2, Kv4.2). Kv channels primarily expressed in somatodendritic regions are in red, and those in axons in green.

In addition to providing a mechanism to regulate the overall levels of Kv channel expression, changes in phosphorylation state are thought to underlie changes in Kv channel localization at distinct sites within the plasma membrane (Fig. 1). Neurons are polarized cells with a cell body or soma, axons and dendrites, and because Kv channels are largely synthesized in cell bodies, protein transport within the cell to specific locations in the soma, axon and dendrites is a highly regulated process. Once targeted to a specific location, mechanisms must exist to anchor the plasma membrane Kv channels at discrete sites [33]. Accurate Kv channel compartmentalization to specific plasma membrane domains underlies neuronal function; dynamic changes in such localization can confer functional plasticity. Among different cell types in the body, the localization of Kv channels at discrete plasma membrane locations can have the largest impact on function, due to the large size and morphological complexity of many mammalian neurons. As such, neuronal function can be substantially impacted by changes in the restricted subcellular localization of Kv channels at or near chemical synapses, the axon initial segment, nodes of Ranvier, and other important neuronal functional domains [75].

Functional plasticity can also arise from modulating the activity of plasma membrane Kv channels through changes in their biophysical proprieties, such that the probability of a Kv channel being open or closed at a given membrane potential can be dynamically regulated (Fig. 1). As excitable molecules, Kv channels can respond to changes in membrane potential with conformational changes that lead to the opening or closing of the channel pore [71]. Dynamic changes in the gating of existing plasma membrane channel populations is another way in which phosphorylation can regulate the activity of neuronal Kv channels, by affecting the probability they will be open or closed at a given membrane potential.

There are numerous examples of neuronal signaling events that lead to dynamic and reversible changes in Kv channel expression, localization and function, with a resultant impact on neuronal function. As detailed below, these signaling events include fast synaptic transmission, including those that lead to synaptic plasticity in the form of long term potentiation and depression, neuromodulation through signaling pathways linked to GPCRs and other modalities, neuronal maturation and differentiation as regulated by growth factors, and events that trigger pathophysiological changes in neuronal form and function. The underlying assumption is that changes in the phosphorylation state of the Kv channel protein itself mediate these dynamic changes, however, only recently has direct experimental evidence been provided supporting a role for direct covalent modification of Kv channel subunits by phosphorylation. Here, we discuss some recent examples in which phosphorylation of Kv channel at specific sites has been defined through mass spectrometric-based proteomic and/or molecular biological approaches, application of phosphospecific antibodies has revealed regulation of phosphorylation at these sites in mammalian neurons, and changes in phosphorylation at these sites has been shown to underlie changes in Kv channel expression, localization and function in mammalian neurons.

Phosphorylation-dependent regulation of expression: Kv1.2

Kv1.2 is a component subunit of low-threshold, rapidly activating and non- or slowly activating Kv channels and is found at high levels in presynaptic terminals, in axon initial segments, and at juxtaparanodal regions of nodes of Ranvier [75]. Kv1.2 is extensively phosphorylated in mammalian brain (Fig. 2). A seminal study showed that muscarinic stimulation causes enhanced phosphorylation of tyrosine residues in Kv1.2 and suppression of Kv1.2 current [20]. Mutagenesis studies revealed that phosphorylation of Kv1.2 Y415 and Y417 causes disruption of Kv1.2 interaction with the cytoskeletal protein cortactin, and loss of interaction with cortactin diminishes the total Kv1.2 current in heterologous expression systems [16].

Figure 2. Kv1.2 and Kv2.1 are highly phosphorylated in rat brain.

Phosphorylation in Kv1.2 and Kv2.1 channels causes altered electrophoretic mobility on SDS-PAGE. Phosphorylation is revealed by differences in electrophoretic migration of these proteins in samples of rat brain membranes (RBM) analyzed by immunoblot without (-) or with (+) prior alkaline phosphatase (AP) treatment of the RBM.

Kv1.2 residues S434, S440, and S441 were also identified as in vivo phosphorylation sites by mass spectrometry analysis of Kv1.2 immunopurified from rat, mouse and human brain, and S449 in recombinant Kv1.2 expressed in HEK293 and COS cells [79]. To address which cellular population of Kv1.2 carries phosphorylation at these sites, recombinant Kv1.2 expressed in heterologous cells was subjected to Stable Isotope Labeling In Cell culture or SILAC [51]. Kv1.2 is an N-linked glycoprotein that is initially synthesized with a high mannose oligosaccharide chain that is later processed in the Golgi apparatus to a complex chain [67]. Analysis of Kv1.2 populations carrying different sugar chains from SILAC labeled cells revealed that phosphorylation at S440/S441 was specific to Kv1.2 with processed oligosaccharide chain, corresponding primarily to the cell surface population of Kv1.2 [36]. This result was supported in experiments employing a phosphospecific S440/S441-directed antibody that yielded specific reaction to Kv1.2 with a complex oligosaccharide chain on immunoblots, and selective immunocytochemical staining of cell surface Kv1.2 [79]. Site-directed mutagenesis studies showed that Ser-to-Ala mutations at either S440 or S441 diminished Kv1.2 surface expression levels, and increased the levels of intracellular Kv1.2, resulting in decreased ionic current [79]. Moreover, these mutations decreased the population of Kv1.2 carrying a processed oligosaccharide chain, and increased the population with a high mannose chain, suggesting that these mutations affected the biosynthetic delivery of Kv1.2 to the plasma membrane, as opposed to acting through enhanced endocytosis of cell surface channels, as is the case with tyrosine phosphorylation of Kv1.2. Eliminating phosphorylation at S449 (i.e., with the S449A mutation) led to decreased phosphorylation at S440/S441, and effects on trafficking similar to those seen in the S440A and S441A mutants. This suggests crosstalk between these phosphosites, in which S449 phosphorylation could be a prerequisite for S440 and S441 phosphorylation. More recent studies have identified S440 [7] and S449 [7, 23] as substrates for PKA, suggesting that signaling pathways leading to PKA activation could increase expression of Kv1.2 through effects on biosynthetic trafficking. Importantly, incorporation of Kv1.2 subunits confers phosphorylation-dependent trafficking to heteromeric Kv1 channel complexes [79]. As such phosphorylation of Kv1.2 may play a general role in providing phosphorylation-dependent regulation to the diverse repertoire of Kv1.2-containing heteromeric Kv1 channel complexes that predominate in mammalian brain [6, 59, 63]. In contrast to numerous studies showing phosphorylation-dependent changes in Kv1.2 expression levels, no studies have yet linked Kv1.2 phosphorylation to dynamic changes in subcellular localization or gating of Kv1.2 channels.

Phosphorylation-dependent regulation of expression, localization and function: Kv2.1

Voltage-gated channels containing Kv2.1 α subunits are examples of Kv channels where dynamic changes in phosphorylation state have been shown to fundamentally alter expression, localization and function. Kv2.1 is expressed in the somatic and dendritic regions of most mammalian neurons [73, 75]. In many neurons, Kv channels containing Kv2.1 contribute the bulk of sustained outward K⁺ current [14, 48], and play a major role in regulating neuronal excitability during periods of high frequency firing [9, 43]. Kv2.1 is extensively phosphorylated (Fig. 2) in mammalian central neurons [41, 47, 65]. Seminal work by Choi and colleagues revealed that delayed rectifier current was enhanced in mouse cortical neurons during apoptosis, and that the K⁺ efflux associated with the increased channel Kv activity was required for induction of apoptosis [81]. Later studies revealed that Kv2.1-containing channels mediated these pro-apoptotic increases in delayed rectifier K⁺ current [52]. Increased phosphorylation of Kv2.1 on S800 via p38 MAP kinase activation led to enhanced K⁺ current levels [58] through enhanced SNARE-mediated membrane insertion [53]. A number of recent studies have suggested that a wide variety of pro-apoptotic stimuli trigger increased Kv2.1 currents in diverse types of mammalian neurons [22, 64, 80]. Determination of the molecular mechanism whereby enhanced phosphorylation of Kv2.1 at S800 leads to enhanced insertion of Kv2.1 in the plasma membrane would provide important information as to our basic understanding of regulated Kv channel expression, and may provide novel targets for reducing neuronal apoptosis triggered by a variety of pro-apoptotic stimuli. A recent paper offered an intriguing finding that suppression of apoptosis in human hepatoma cells upon infection with hepatitis C virus occurs through viral-induced inhibition of p38 MAPK. This prevents pro-apoptotic p38 MAPK-mediated Kv2.1 phosphorylation and the resultant pro-apoptotic increases in Kv2.1 expression [37]. The authors suggest that Kv2.1 represents a novel target for antiviral therapy.

Kv2.1 is found extensively phosphorylated (Fig. 2) in mammalian central neurons [47], where it is present in large somatodendritic clusters [3, 29, 73]. Kv2.1 is rapidly dephosphorylated in response to treatments that increase neuronal cytosolic Ca²⁺. This occurs in vivo in response to epileptic seizures [41] and hypoxia [21, 40]. A similar dephosphorylation occurs in cultured hippocampal neurons in vitro, in response to elevated cytosolic Ca²⁺ and activation of calcineurin. This can be triggered by glutamate stimulation acting through ionotropic receptors [41, 46], hypoxia leading to either Ca²⁺ release from neuronal mitochondria [40] or astrocyte-mediated glutamate spillover [42], and/or carbachol stimulation of muscarinic receptors leading to IP₃-mediated Ca²⁺ release from intracellular stores [44]. These changes in Kv2.1 phosphorylation state are associated with a dramatic change in localization in neurons in vivo and in neurons in culture [39-41, 46]. Treatments that trigger Kv2.1 dephosphorylation lead to dispersion of channel clusters (Fig. 3), leading to a more uniform somatodendritic expression of Kv2.1 but no marked changes in overall plasma membrane expression levels [41]. The role of the dynamic dispersal of Kv2.1 clusters is not yet clear, but may involve lowering the density of plasma membrane Kv2.1 channels at specific clustering sites, which are associated with intracellular Ca2+ signaling domains, astrocyte end feet [10], and in spinal motor neurons, cholinergic synapses [45].

Figure 3. Excitatory stimulation causes changes in Kv2.1 localization in rat hippocampal neurons.

Glutamate treatment (10 μM, 15 min) of cultured hippocampal neurons induces dispersion of clusters of Kv2.1 (green). Neurons were double stained with anti-MAP2 antibody (red) as a neuronal somatodendritic marker.

Kv2.1 phosphorylation state also regulates the voltage dependence and kinetics of activation. Intracellular dialysis of heterologous cells expressing recombinant Kv2.1 with alkaline phosphatase (AP) causes a hyperpolarizing shift in the voltage-dependent activation, and an increase in activation kinetics at the activation midpoint voltage of the Kv2.1 current [44, 47, 54]. The effects of AP treatment are largely eliminated in a C-terminal truncation mutant of Kv2.1, suggesting that the phosphorylation-dependent regulation of voltage dependent activation was due to phosphorylation in this region [47]. When transferred to other Kv channels that did not normally respond to these treatments, the Kv2.1 C-terminus could confer sensitivity to both AP- and carbachol-induced modulation of voltage-dependent gating [44]. However, the sites mediating this modulation remained elusive for some time, in part due to almost 100 potential phospho-acceptor residues in the Kv2.1 C-terminus. Application of an approach employing immunopurification of endogenous Kv2.1 in brain, and recombinant Kv2.1 expressed in heterologous cells, followed by tandem mass spectrometric identification of sites covalently modified with phosphate in vivo led to identification of 16 serine and threonine phosphorylation sites, 15 of which were located in the C-terminus [54]. To specifically identify residues regulated by treatments that modulate Kv2.1 localization and function, samples from control cultures, and cultures stimulated to activate calcineurin, were subjected to SILAC labeling, allowing for quantitative mass spectrometric identification of eight sites regulated by calcineurin [54]. Individually mutating these sites to alanine partially mimicked the effects of stimuli that induced dephosphorylation in yielding hyperpolarizing shifts in the voltage-dependence of activation. Mutations at more than one site gave larger magnitude effects, suggesting that multisite phosphorylation could yield graded regulation of Kv2.1 function [54]. Importantly, these alanine mutants were also partially refractory to subsequent dephosphorylation upon calcineurin activation or AP treatment [54]. These analyses also allowed for generation of phosphospecific antibodies against a subset of those sites that exhibited calcineurin-mediated dephosphorylation and affected Kv2.1 gating. Experimental application of these antibodies revealed that individual phosphorylation sites on Kv2.1 exhibit distinct regulation [39]. One site (S603) exhibited an exceptionally sensitive and bidirectional regulation with changes in neuronal activity, suggesting that diverse stimuli that affect neuronal activity could regulate Kv2.1 gating. Activity-dependent dephosphorylation of Kv2.1 leads to an increased probability that channels will be open at a given membrane potential, resulting in homeostatic suppression of neuronal excitability [43]. That the numerous phosphorylation sites on Kv2.1 subunits within the channel tetramer could be independently regulated suggests a wide spectrum of channel localization and function can be obtained through regulation of Kv2.1 phosphorylation. The large number of phosphorylation sites present on Kv2.1 subunits, the fact that Kv2.1 channels exists as a tetramers of these subunits, and that in other tetrameric ion channels phosphorylation sites can be dominant (i.e., phosphorylation of only one subunit confers the phosphorylated phenotype to the entire tetrameric complex) or recessive, such that 3–4 subunits must be phosphorylated to influence function [72], suggests that regulation of Kv2.1 expression, localization and function through multisite phosphorylation is complex.

Phosphorylation-dependent regulation of function: Kv3.1

The Kv3.1 channel is highly expressed in numerous fast-spiking neurons in mammalian brain [75], and is prominently found in the auditory system in the anterior ventral cochlear nucleus (AVCN) and the medial nucleus of the trapezoid body (MNTB) neurons. Similar to Kv2.1, the voltage dependence of Kv3.1 activation is modulated by phosphorylation state, in that AP treatment causes a hyperpolarizing shift in the voltage-dependent activation of Kv3.1 currents in MNTB neurons [34]. Treatment with CK2 inhibitors mimicked the effects of AP on the voltage-dependent activation of the Kv3.1 channel in auditory neurons [34]. PKC phosphorylates the S503 residue located in the C-terminus of Kv3.1. This phosphorylation causes suppression in Kv3.1 current, due to a decreased P_o. Studies utilizing phosphospecific antibodies specific for Kv3.1 phosphorylation at S503 in auditory neurons show that this site is phosphorylated in quiet environments, and dephosphorylation occurs at this site correlates with high frequency auditory stimulation, producing an increased Kv3.1 current and enhanced acoustic perception in vivo [69].

Phosphorylation-dependent regulation of expression, localization and function: Kv4.2

Kv4.2 is a component subunit of dendritic transient A-type K⁺ current in neurons, and is critically involved in dynamic regulation of dendritic excitability and plasticity [24]. A number of studies have demonstrated complex phosphorylation-dependent regulation of Kv4.2-based transient A-type K⁺ current activity in response to elevated excitatory synaptic activity [35]. In vitro phosphorylation of recombinant fragments of Kv4.2 revealed PKA [2], Erk [1], and CamKII [76] -mediated phosphorylation of Kv4.2. Phosphorylation by CamKII in vitro occurs at S438 and S459, and CamKII activation enhances Kv4.2 cell surface expression in heterologous cells and in neurons [76]. Erk-mediated phosphorylation of Kv4.2 leads to decreased Kv4.2 currents [61]. Phosphospecific antibodies specific for Kv4.2 triply phosphorylated by Erk at T602, T607 and S616 revealed increased Erk-mediated Kv4.2 phosphorylation in response to status epilepticus in rats [32]. PKA-mediated phosphorylation at S552 is enhanced by coexpression with auxiliary KChIP and DPPX subunits, and is associated with cell surface but not intracellular Kv4.2 in heterologous cells and in neurons [62, 68]. Enhanced surface expression of Kv4.2 induced by interaction with cytoplasmic KChIP auxiliary subunits requires phosphorylation at S552 [30]. Phosphorylation at S552 also plays a prominent role in regulating Kv4.2 expression in neuronal dendrites [25] through PKA phosphorylation [15]. Pharmacological activation of PKA leads to Kv4.2 internalization from dendritic spines, whereas PKA inhibition blocks AMPA receptor-mediated internalization. This process is inhibited by the S552A mutation, suggesting that activity-dependent internalization of Kv4.2 is dependent on PKA phosphorylation on S552 [15]. As such, regulation of plasma membrane levels of Kv4.2 by phosphorylation at S552 is complex and multimodal, in that phosphorylation during biosynthetic trafficking serves to promote increased expression, whereas phosphorylation of plasma membrane Kv4.2 leads to their internalization. Moreover, Kv4.2 is associated with A-kinase anchored proteins or AKAPs, which may allow for efficient coupling between PKA activation and Kv4.2 modulation [30]. Phosphospecific antibodies specific for Kv4.2 phosphorylated at S552 revealed no obvious changes in phosphorylation in response to status epilepticus, in animal subjects where changes in Erk phosphorylation of Kv4.2 were observed [32]. Mass spectrometric analyses identified four phosphorylation sites (S548, S552, S572 and S575) of which only S552 had been revealed in previous in vitro phosphorylation studies [62]. The role of these other in vivo sites in Kv4.2 expression, localization and function has not been investigated.

Activation of ERK leads to a change in the voltage-dependent-dependent activation of neuronal Kv4.2 channels, causing a rightward shift in the activation curve and the decreased rate of recovery from inactivation [82]. ERK directly phosphorylates Kv4.2 at T602, T607, and S616 [61]. Site directed mutagenesis revealed that this effect is mediated primarily by phosphorylation at T607 [61]. Kv4.2 phosphorylation by PKA at T38 and S552 residues, and S552 phosphorylation upon PKA pharmacological activation has been shown in hippocampal slices [2]. PKA-dependent phosphorylation on this residue causes depolarizing shift in activation curve of Kv4.2, but only in presence of KChIP auxiliary subunits [60]. The activity-dependent suppression of Kv4.2 channels through Erk and PKA phosphorylation has been proposed to underlay certain forms of neuronal plasticity, including LTP [35].

Phosphorylation-dependent regulation of function: Kv7.2 and Kv7.3

Kv7/KCNQ channels are the molecular correlates of the M-type current [77]. These channels localize in axon initial segments and nodes of Ranvier throughout the nervous system and modulate neuronal excitability, bursting and neurotransmitter release under G_q/11 activity modulation [17]. Kv7.2 and Kv7.3 subunits are associated with PKs such as PKC through AKAPs [19]. PKC activation through phorbol 12-myristate 13-acetate (PMA) treatment causes a hyperpolarizing shift in the activation curve for Kv7.2 expressed in Xenopus oocytes. Conversely, AP treatment of recombinant Kv7.2 expressed in HEK293 cells causes the opposite effect [50]. In contrast, Src tyrosine kinase associates with Kv7 channels and diminishes the probability of opening of Kv7.2/Kv7.3 heteromultimers in heterologous cells, and overexpression of Src causes decreased M-current amplitude in sympathetic neurons [12]. This effect seems to be dependent on Kv7.3 phosphorylation at Y67 and Y349 [28]. In addition, T217 (in Kv7.2) and T246 (in Kv7.3), which localize in the S4-S5 loop, have been identified as phosphorylation sites by tandem mass spectrometry analysis of recombinant Kv7.2/Kv7.3 channels. Mutation of these sites to aspartate/glutamate residues eliminates channel activity, suggesting a potential role of phosphorylation at these sites in regulating Kv7.2/Kv7.3 activity [70]. Given their high level of expression at axon initial segments and at nodes of Ranvier, dynamic regulation of the function of Kv7.2 and Kv7.3 could impact neuronal excitability.

Future perspectives

As detailed above, changes in Kv channel phosphorylation by physiological and pathophysiological events that alter the cellular tone of PK and PP activity can modulate Kv channel expression, localization and function to fundamentally alter neuronal function. A combination of molecular biological, pharmacological, biochemical, cell biological and biophysical approaches have yielded important insights into how such regulation is achieved through Kv channel phosphorylation. Recent application of approaches employing antibody-based immunopurification of brain Kv channels and tandem mass spectrometric identification of amino acid residues chemically modified with phosphate has recently emerged as a powerful approach to determine in vivo patterns of Kv channel phosphorylation. This approach has also proved effective for identifying in vivo phosphorylation sites on voltage- and calcium-activated BK [78] and voltage-gated Na⁺ [4] channels. A fundamental assumption of this approach is that many of the sites identified as in vivo phosphorylation sites on native Kv channels will be those important in regulating Kv channel expression, localization and function, and vice-versa. Generation of phosphospecific antibodies that specifically recognize Kv channels phosphorylated at identified sites is emerging as an important step to determine how these sites are regulated by physiological and pathophysiological stimuli that impact neuronal function.

The tremendous technological advances in mass spectrometric techniques over the last few years have allowed great advances in the study of Kv channel phosphorylation [7, 23, 54, 70, 78, 79]. The improvement in sensitivity and the development of quantitative methods such as SILAC have allowed the quantitative determination of phosphorylation sites in Kv channel proteins expressed in heterologous cells [54, 55, 79]. Future studies in cultured neurons, or studies using post vivo labeling techniques such as isobaric tags for relative and absolute quantitation (iTRAQ) will help to elucidate the nature and extent of altered Kv channel phosphorylation in different physiological (e.g. development, neuronal differentiation, aging) and pathophysiological (e.g. epilepsy, stroke, schizophrenia) processes affecting neuronal function.

Several examples of signaling complexes containing Kv channels, PKs, and/or PPs have been identified in various mammalian tissues [11, 13, 19, 60]. However, the basis of the dynamic modulation and localization of these complexes in neurons is not yet completely understood. Future research on the extent and nature of Kv channel involvement in such signaling complexes, and the impact of macromolecular assembly of Kv channels with other signaling proteins at specific sites in neurons, using optical techniques such as FRET and quantitative mass spectrometric approaches will help to understand the mechanistic processes underlying the dynamic modulation of Kv channels through phosphorylation.

The mechanistic basis for regulation of Kv channel expression, localization and function remains an important yet poorly understood research area. Few mammalian Kv channel structures are available, and those that are available (e.g. Kv1.2; [31]) are of Kv channels that lack the C-terminal domains that are the targets of most of the known phosphorylation-dependent modulatory events. Moreover, in general these structures are obtained from expression systems (bacterial, fungal, insect) that may be inherently unable to generate Kv channels with phosphorylation patterns similar to those of neuronal Kv channels. The detailed structural information needed to determine the mechanistic basis of how changes in Kv channel phosphorylation state can dramatically impact their expression, localization and function awaits determination, through X-ray crystallographic or other approaches, of Kv channel structures in different phosphorylation states.

In summary, phosphorylation is a common mechanism that regulates expression, localization and function of neuronal Kv channels. Numerous candidate phosphorylation sites have been identified using predictive algorithms and site directed mutagenesis approaches. More recently, mass spectrometric approaches have provided complementary data as to which sites are phosphorylated in vivo. Phosphospecific antibodies have allowed for insights into the extent and nature of modulation at specific sites on neuronal Kv channels in response to specific physiological and pathophysiological stimuli. However, in only a few cases have these different approaches been employed in concert to generate a more holistic picture of Kv channel phosphorylation. Moreover, little is known of the impact of altered Kv channel phosphorylation on neuronal function in a living animal, such information may come from knock in mouse technologies that have been used to gain in vivo insights for other phosphoproteins [26].