Ilana Lotan

Activation of Protein Kinase C Alters Voltage Dependence of a Na+ Channel

Phorbol esters and purified protein kinase C (PKC) have been shown to down-modulate the voltage-dependent Na+ channels expressed in Xenopus oocytes injected with chick brain RNA. We used the two-electrode voltage-clamp technique to demonstrate that a Na+ channel expressed in oocytes injected with RNA coding for the alpha subunit of the channel alone (VA200, a variant of rat brain type IIA) is also inhibited by PKC activation. The inhibition of Na+ currents, expressed in oocytes injected with either alpha subunit RNA (rat) or total brain RNA (chick), is voltage-dependent, being stronger at negative potentials. It appears to result mainly from a shift in the activation curve to the right and possibly a decrease in the steepness of the voltage dependence of activation. There is little effect on the inactivation process and maximal Na+ conductance. Thus, PKC modulates the Na+ channel by a mechanism involving changes in voltage-dependent properties of its main, channel-forming alpha subunit.

A potential site of functional modulation by protein kinase A in the cardiac Ca2+ channel α 1C subunit

The well‐characterized enhancement of the cardiac Ca2+ L‐type current by protein kinase A (PKA) is not observed when the corresponding channel is expressed in Xenopus oocytes, possibly because it is fully phosphorylated in the basal state. However, the activity of the expressed channel is reduced by PKA inhibitors. Using this paradigm as an assay to search for PKA sites relevant to channel modulation, we have found that mutation of serine 1928 of the α 1C subunit to alanine abolishes the modulation of the expressed channel by PKA inhibitors. This effect was independent of the presence of the β subunit. Phosphorylation of serine 1928 of α 1C may mediate the modulatory effect of PKA on the cardiac voltage‐dependent Ca2+ channel.

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.

Evidence for the existence of a cardiac specific isoform of the α1 subunit of the voltage dependent calcium channel

Biochemical, pharmacological and electrophysiological evidence implies the existence of tissue specific isoforms of the L‐type VDCC. The α1 and α2 subunits of the skeletal muscle calcium channel have been previously cloned and their amino acid sequence deduced. Here we report the isolation and sequencing of a partial cDNA that encodes a heart specific isoform of the α1 subunit. The amino acid sequence deduced from this part cDNA clone shows 64.7% similarity with the skeletal muscle α1 subunit. Northern analysis reveals 2 hybridizing bands, 8.5 and 13 kb, in contrast to one 6.5 kb band in the skeletal muscle. Selective inhibition of mRNA expression in Xenopus oocytes by complementary oligodeoxynucleotides derived from the heart clone provides further evidence that the cDNA corresponds to an essential component of the VDCC. These data further support the existence of tissue‐specific isoforms of the L‐type VDCC.

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.

Modulation of vertebrate brain Na+ and K+ channels by subtypes of protein kinase C

Effects of purified subtypes I, II and III of protein kinase C (PKC) on voltage‐dependent transient K+ (A) and Na+ channels were studied in Xenopus oocytes injected with chick brain RNA. The experiments were performed in the constant presence of 10 nM β‐phorbol 12‐myristate‐13‐acetate (PMA). Intracellular injection of subtype I (τ) reduced the A‐current (I A), with no effect on Na+ current (I Na). PKC subtype II (β1, + β2) and III (α) reduced both currents. PKC did not affect the response to kainate. Inactivated (heated) or unactivated (injected in the absence of PMA) enzyme and vehicle alone had no effect. Our results strongly suggest that I Na, and I A in vertebrate neurons are modulated by PKC; all PKC subtypes exert a similar effect on the A‐channel while only subtypes II and III modulate the Na+ channel.

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 of the skeletal muscle sodium channel α-subunit by the β1 -subunit

Co‐expression of cloned sodium channel β1 ‐subunit with the rat skeletal muscle‐subunit (αμI) accelerated the macroscopic current decay, enhanced the current amplitude, shifted the steady state inactivation curve to more negative potentials and decreased the time required for complete recovery from inactivation. Sodium channels expressed from skeletal muscle mRNA showed a similar behaviour to that observed from , indicating that β1 restores ‘physiological’ behaviour. Northern blot analysis revealed that the Na+ channel β1‐subunit is present in high abundance (about 0.1%) in rat heart, brain and skeletal muscle, and the hybridization with untranslated region of the ‘brain’ β1 cDNA to skeletal muscle and heart mRNA indicated that the diffferent Na + channel α‐subunits in brain, skeletal muscle and heart may share a common β1 ‐subunit.