Dafna Singer-Lahat

Calcium channel beta subunit heterogeneity: functional expression of cloned cDNA from heart, aorta and brain.

Complementary DNAs encoding three novel and distinct beta subunits (CaB2a, CaB2b and CaB3) of the high voltage activated (L‐type) calcium channel have been isolated from rabbit heart. Their deduced amino acid sequence is homologous to the beta subunit originally cloned from skeletal muscle (CaB1). CaB2a and CaB2b are splicing products of a common primary transcript (CaB2). Northern analysis and specific amplification of CaB2 and CaB3 specific cDNAs by polymerase chain reactions showed that CaB2 is predominantly expressed in heart, aorta and brain, whereas CaB3 is most abundant in brain but also present in aorta, trachea, lung, heart and skeletal muscle. A partial DNA sequence complementary to a third variant of the CaB2 gene, subtype CaB2c, has also been cloned from rabbit brain. Coexpression of CaB2a, CaB2b and CaB3 with alpha 1heart enhances not only the expression in the oocyte of the channel directed by the cardiac alpha 1 subunit alone, but also effects its macroscopic characteristics such as drug sensitivity and kinetics. These results together with the known alpha 1 subunit heterogeneity, suggest that different types of calcium currents may depend on channel subunit composition.

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.

Modulation of cardiac Ca2+ channels in Xenopus oocytes by protein kinase C.

L‐Type calcium channel was expressed in Xenopus laevis oocytes injected with RNAs coding for different cardiac Cu2+ channel subunits, or with total heart RNA. The effects of activation of protein kinase C (PKC) by the phorbol ester PMA (4β‐phorbol 12‐myristate 13‐acetate) were studied. Currents through channels composed of the main (α1) subunit alone were initially increased and then decreased by PMA. A similar biphasic modulation was observed when the α1 subunit was expressed in combination with α2/δ, β and/or γ subunits, and when the channels were expressed following injection of total rat heart RNA. No effects on the voltage dependence of activation were observed. The effects of PMA were blocked by staurosporine, a protein kinase inhibitor. β subunit moderated the enhancement caused by PMA. We conclude that both enhancement and inhibition of cardiac L‐type Ca2+ currents by PKC are mediated via an effect on the α1 subunit, while the β subunit may play a mild modulatory role.

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.

Voltage-gated Potassium Channel as a Facilitator of Exocytosis

Voltage‐gated ion channels are well characterized for their function in excitability signals. Accumulating studies, however, have established an ion‐independent function for the major classes of ion channels in cellular signaling. During the last few years we established a novel role for Kv2.1, a voltage‐gated potassium (Kv) channel, classically known for its role of repolarizing the membrane potential, in facilitation of exocytosis. Kv2.1 induces facilitation of depolarization‐induced release through its direct interaction with syntaxin, a protein component of the exocytotic machinery, independently of the potassium ion flow through the channels pore. Here, we review our recent studies, further characterize the phenomena (using chromaffin cells and carbon fiber amperometry), and suggest plausible mechanisms that can underlie this facilitation of release.

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.