Dodo Chikvashvili

Phosphorylation of a K+ channel alpha subunit modulates the inactivation conferred by a beta subunit. Involvement of cytoskeleton.

Voltage-gated K+ channels isolated from mammalian brain are composed of α and β subunits. Interaction between coexpressed Kv1.1 (α) and Kvβ1.1 (β) subunits confers rapid inactivation on the delayed rectifier-type current that is observed when α subunits are expressed alone. Integrating electrophysiological and biochemical analyses, we show that the inactivation of the αβ current is not complete even when α is saturated with β, and the αβ current has an inherent sustained component, indistinguishable from a pure α current. We further show that basal and protein kinase A-induced phosphorylations at Ser-446 of the α protein increase the extent, but not the rate, of inactivation of the αβ channel, without affecting the association between α and β. In addition, the extent of inactivation is increased by agents that lead to microfilament depolymerization. The effects of phosphorylation and of microfilament depolymerization are not additive. Taken together, we suggest that phosphorylation, via a mechanism that involves the interaction of the αβ channel with microfilaments, enhances the extent of inactivation of the channel. Furthermore, phosphorylation at Ser-446 also increases current amplitudes of the αβ channel as was shown before for the α channel. Thus, phosphorylation enhances in concert inactivation and current amplitudes, thereby leading to a substantial increase in A-type activity.

Direct Interaction of Target SNAREs with the Kv2.1 Channel MODAL REGULATION OF CHANNEL ACTIVATION AND INACTIVATION GATING

Previously we suggested that interaction between voltage-gated K+ channels and protein components of the exocytotic machinery regulated transmitter release. This study concerns the interaction between the Kv2.1 channel, the prevalent delayed rectifier K+ channel in neuroendocrine and endocrine cells, and syntaxin 1A and SNAP-25. We recently showed in islet β-cells that the Kv2.1 K+ current is modulated by syntaxin 1A and SNAP-25. Here we demonstrate, using co-immunoprecipitation and immunocytochemistry analyses, the existence of a physical interaction in neuroendocrine cells between Kv2.1 and syntaxin 1A. Furthermore, using concomitant co-immunoprecipitation from plasma membranes and two-electrode voltage clamp analyses in Xenopus oocytes combined with in vitro binding analysis, we characterized the effects of these interactions on the Kv2.1 channel gating pertaining to the assembly/disassembly of the syntaxin 1A/SNAP-25 (target (t)-SNARE) complex. Syntaxin 1A alone binds strongly to Kv2.1 and shifts both activation and inactivation to hyperpolarized potentials. SNAP-25 alone binds weakly to Kv2.1 and probably has no effect by itself. Expression of SNAP-25 together with syntaxin 1A results in the formation of t-SNARE complexes, with consequent elimination of the effects of syntaxin 1A alone on both activation and inactivation. Moreover, inactivation is shifted to the opposite direction, toward depolarized potentials, and its extent and rate are attenuated. Based on these results we suggest that exocytosis in neuroendocrine cells is tuned by the dynamic coupling of the Kv2.1 channel gating to the assembly status of the t-SNARE complex.

Fast inactivation of a brain K+ channel composed of Kv1.1 and Kvβ1.1 subunits modulated by G protein βγ subunits

Modulation of A‐type voltage‐gated K+ channels can produce plastic changes in neuronal signaling. It was shown that the delayed‐rectifier Kv1.1 channel can be converted to A‐type upon association with Kvβ1.1 subunits; the conversion is only partial and is modulated by phosphorylation and microfilaments. Here we show that, in Xenopus oocytes, expression of Gβ1γ2 subunits concomitantly with the channel (composed of Kv1.1 and Kvβ1.1 subunits), but not after the channels expression in the plasma membrane, increases the extent of conversion to A‐type. Conversely, scavenging endogenous Gβγ by co‐expression of the C‐terminal fragment of the β‐adrenergic receptor kinase reduces the extent of conversion to A‐type. The effect of Gβγ co‐expression is occluded by treatment with dihydrocytochalasin B, a microfilament‐disrupting agent shown previously by us to enhance the extent of conversion to A‐type, and by overexpression of Kvβ1.1. Gβ1γ2 subunits interact directly with GST fusion fragments of Kv1.1 and Kvβ1.1. Co‐expression of Gβ1γ2 causes co‐immunoprecipitation with Kv1.1 of more Kvβ1.1 subunits. Thus, we suggest that Gβ1γ2 directly affects the interaction between Kv1.1 and Kvβ1.1 during channel assembly which, in turn, disrupts the ability of the channel to interact with microfilaments, resulting in an increased extent of A‐type conversion.

K+ Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin

Kv channels inhibit release indirectly by hyperpolarizing membrane potential, but the significance of Kv channel interaction with the secretory apparatus is not known. The Kv2.1 channel is commonly expressed in the soma and dendrites of neurons, where it could influence the release of neuropeptides and neurotrophins, and in neuroendocrine cells, where it could influence hormone release. Here we show that Kv2.1 channels increase dense-core vesicle (DCV)-mediated release after elevation of cytoplasmic Ca2+. This facilitation occurs even after disruption of pore function and cannot be explained by changes in membrane potential and cytoplasmic Ca2+. However, triggering release increases channel binding to syntaxin, a secretory apparatus protein. Disrupting this interaction with competing peptides or by deleting the syntaxin association domain of the channel at the C terminus blocks facilitation of release. Thus, direct association of Kv2.1 with syntaxin promotes exocytosis. The dual functioning of the Kv channel to influence release, through its pore to hyperpolarize the membrane potential and through its C-terminal association with syntaxin to directly facilitate release, reinforces the requirements for repetitive firing for exocytosis of DCVs in neuroendocrine cells and in dendrites.

Modulation by protein kinase C activation of rat brain delayed-rectifier K+ channel expressed in Xenopus oocytes

The modulation by protein kinase C (PKC) of the RCK1 K+ channel was investigated in Xenopus oocytes by integration of two‐electrode voltage clamp, site‐directed mutagenesis and SDS‐PAGE analysis techniques. Upon application of β‐phorbol 12‐myristate 13‐acetate (PMA) the current was inhibited by 50–90%. No changes in the voltage sensitivity of the channel, changes in membrane surface area or selective elimination of RCK1 protein from the plasma membrane could be detected. The inhibition was mimicked by 1‐oleoyl‐2‐acetylrac‐glycerol (OAG) but not by αPMA, and was blocked by staurosporine and calphostin C. Upon deletion of most of the N‐terminus a preceding enhancement of about 40% of the current was prominent in response to PKC activation. Its physiological significance is discussed. The N‐terminus deletion eliminated 50% of the inhibition. However, phosphorylation of none of the ten classical PKC phosphorylation sites on the channel molecule could account, by itself or in combination with others, for the inhibition. Thus, our results show that PKC activation can modulate the channel conductance in a bimodal fashion. The N‐terminus is involved in the inhibition, however, not via its direct phosphorylation.

Selective Interaction of Syntaxin 1A with KCNQ2: Possible Implications for Specific Modulation of Presynaptic Activity

KCNQ2/KCNQ3 channels are the molecular correlates of the neuronal M-channels, which play a major role in the control of neuronal excitability. Notably, they differ from homomeric KCNQ2 channels in their distribution pattern within neurons, with unique expression of KCNQ2 in axons and nerve terminals. Here, combined reciprocal coimmunoprecipitation and two-electrode voltage clamp analyses in Xenopus oocytes revealed a strong association of syntaxin 1A, a major component of the exocytotic SNARE complex, with KCNQ2 homomeric channels resulting in a ∼2-fold reduction in macroscopic conductance and ∼2-fold slower activation kinetics. Remarkably, the interaction of KCNQ2/Q3 heteromeric channels with syntaxin 1A was significantly weaker and KCNQ3 homomeric channels were practically resistant to syntaxin 1A. Analysis of different KCNQ2 and KCNQ3 chimeras and deletion mutants combined with in-vitro binding analysis pinpointed a crucial C-terminal syntaxin 1A-association domain in KCNQ2. Pull-down and coimmunoprecipitation analyses in hippocampal and cortical synaptosomes demonstrated a physical interaction of brain KCNQ2 with syntaxin 1A, and confocal immunofluorescence microscopy showed high colocalization of KCNQ2 and syntaxin 1A at presynaptic varicosities. The selective interaction of syntaxin 1A with KCNQ2, combined with a numerical simulation of syntaxin 1As impact in a firing-neuron model, suggest that syntaxin 1As interaction is targeted at regulating KCNQ2 channels to fine-tune presynaptic transmitter release, without interfering with the function of KCNQ2/3 channels in neuronal firing frequency adaptation.

Direct Interaction of Endogenous Kv Channels with Syntaxin Enhances Exocytosis by Neuroendocrine Cells

K+ efflux through voltage-gated K+ (Kv) channels can attenuate the release of neurotransmitters, neuropeptides and hormones by hyperpolarizing the membrane potential and attenuating Ca2+ influx. Notably, direct interaction between Kv2.1 channels overexpressed in PC12 cells and syntaxin has recently been shown to facilitate dense core vesicle (DCV)-mediated release. Here, we focus on endogenous Kv2.1 channels and show that disruption of their interaction with native syntaxin after ATP-dependent priming of the vesicles by Kv2.1 syntaxin–binding peptides inhibits Ca2+ -triggered exocytosis of DCVs from cracked PC12 cells in a specific and dose-dependent manner. The inhibition cannot simply be explained by the impairment of the interaction of syntaxin with its SNARE cognates. Thus, direct association between endogenous Kv2.1 and syntaxin enhances exocytosis and in combination with the Kv2.1 inhibitory effect to hyperpolarize the membrane potential, could contribute to the known activity dependence of DCV release in neuroendocrine cells and in dendrites where Kv2.1 commonly expresses and influences release.

The voltage-dependent potassium channel subunit Kv2.1 regulates insulin secretion from rodent and human islets independently of its electrical function.

Aims/hypothesisIt is thought that the voltage-dependent potassium channel subunit Kv2.1 (Kv2.1) regulates insulin secretion by controlling beta cell electrical excitability. However, this role of Kv2.1 in human insulin secretion has been questioned. Interestingly, Kv2.1 can also regulate exocytosis through direct interaction of its C-terminus with the soluble NSF attachment receptor (SNARE) protein, syntaxin 1A. We hypothesised that this interaction mediates insulin secretion independently of Kv2.1 electrical function.MethodsWild-type Kv2.1 or mutants lacking electrical function and syntaxin 1A binding were studied in rodent and human beta cells, and in INS-1 cells. Small intracellular fragments of the channel were used to disrupt native Kv2.1–syntaxin 1A complexes. Single-cell exocytosis and ion channel currents were monitored by patch-clamp electrophysiology. Interaction between Kv2.1, syntaxin 1A and other SNARE proteins was probed by immunoprecipitation. Whole-islet Ca2+-responses were monitored by ratiometric Fura red fluorescence and insulin secretion was measured.ResultsUpregulation of Kv2.1 directly augmented beta cell exocytosis. This happened independently of channel electrical function, but was dependent on the Kv2.1 C-terminal syntaxin 1A-binding domain. Intracellular fragments of the Kv2.1 C-terminus disrupted native Kv2.1–syntaxin 1A interaction and impaired glucose-stimulated insulin secretion. This was not due to altered ion channel activity or impaired Ca2+-responses to glucose, but to reduced SNARE complex formation and Ca2+-dependent exocytosis.Conclusions/interpretationDirect interaction between syntaxin 1A and the Kv2.1 C-terminus is required for efficient insulin exocytosis and glucose-stimulated insulin secretion. This demonstrates that native Kv2.1–syntaxin 1A interaction plays a key role in human insulin secretion, which is separate from the channel’s electrical function.

Non-conducting function of the Kv2.1 channel enables it to recruit vesicles for release in neuroendocrine and nerve cells

Regulation of exocytosis by voltage-gated K+ channels has classically been viewed as inhibition mediated by K+ fluxes. We recently identified a new role for Kv2.1 in facilitating vesicle release from neuroendocrine cells, which is independent of K+ flux. Here, we show that Kv2.1-induced facilitation of release is not restricted to neuroendocrine cells, but also occurs in the somatic-vesicle release from dorsal-root-ganglion neurons and is mediated by direct association of Kv2.1 with syntaxin. We further show in adrenal chromaffin cells that facilitation induced by both wild-type and non-conducting mutant Kv2.1 channels in response to long stimulation persists during successive stimulation, and can be attributed to an increased number of exocytotic events and not to changes in single-spike kinetics. Moreover, rigorous analysis of the pools of released vesicles reveals that Kv2.1 enhances the rate of vesicle recruitment during stimulation with high Ca2+, without affecting the size of the readily releasable vesicle pool. These findings place a voltage-gated K+ channel among the syntaxin-binding proteins that directly regulate pre-fusion steps in exocytosis.

VAMP2 interacts directly with the N terminus of Kv2.1 to enhance channel inactivation

Recently, we demonstrated that the Kv2.1 channel plays a role in regulated exocytosis of dense-core vesicles (DCVs) through direct interaction of its C terminus with syntaxin 1A, a plasma membrane soluble NSF attachment receptor (SNARE) component. We report here that Kv2.1 interacts with VAMP2, the vesicular SNARE partner that is also present at high concentration in neuronal plasma membrane. This is the first report of VAMP2 interaction with an ion channel. The interaction was demonstrated in brain membranes and characterized using electrophysiological and biochemical analyses in Xenopus oocytes combined with an in vitro binding analysis and protein modeling. Comparative study performed with wild-type and mutant Kv2.1, wild-type Kv1.5, and chimeric Kv1.5N/Kv2.1 channels revealed that VAMP2 enhanced the inactivation of Kv2.1, but not of Kv1.5, via direct interaction with the T1 domain of the N terminus of Kv2.1. Given the proposed role for surface VAMP2 in the regulation of the vesicle cycle and the important role for the sustained Kv2.1 current in the regulation of dendritic calcium entry during high-frequency stimulation, the interaction of VAMP2 with Kv2.1 N terminus may contribute, alongside with the interaction of syntaxin with Kv2.1 C terminus, to the activity dependence of DCV release.