The molecular chaperone hsp70 interacts with the cytosolic II-III loop of the Cav2.3 E-type voltage-gated Ca2+ channel.

Multiple types of voltage-activated Ca2+ channels (T, L, N, P, Q, R type) coexist in excitable cells and participate in synaptic differentiation, secretion, transmitter release, and neuronal plasticity. Ca2+ ions entering cells trigger these events through their interaction with the ion channel itself or through Ca2+ binding to target proteins initiating signalling cascades at cytosolic loops of the ion conducting subunit (Cava1). These loops interact with target proteins in a Ca2+-dependent or independent manner. In Cav2.3-containing channels the cytosolic linker between domains II and III confers a novel Ca2+ sensitivity to E-type Ca2+ channels including phorbol ester sensitive signalling via protein kinase C (PKC) in Cav2.3 transfected HEK-293 cells. To understand Ca2+ and phorbol ester mediated activation of Cav2.3 Ca2+ channels, protein interaction partners of the II-III loop were identified. FLAG-tagged II-III - loop of human Cav2.3 was over-expressed in HEK 293 cells, and the molecular chaperone hsp70, which is known to interact with PKC, was identified as a novel functional interaction partner. Immunopurified II-III loop-protein of neuronal and endocrine Cav2.3 splice variants stimulate autophosphorylation of PKCa, leading to the suggestion that hsp70--binding to the II-III loop--may act as an adaptor for Ca2+ dependent targeting of PKC to E-type Ca2+ channels.     

The C2A domain of synaptotagmin alters the kinetics of voltage-gated Ca2+ channels Ca(v)1.2 (Lc-type) and Ca(v)2.3 (R-type)

Biochemical and genetic studies implicate synaptotagmin (Syt 1) as a Ca2+ sensor for neuronal and neuroendocrine neurosecretion. Calcium binding to Syt 1 occurs through two cytoplasmic repeats termed the C2A and C2B domains. In addition, the C2A domain of Syt 1 has calcium-independent properties required for neurotransmitter release. For example, mutation of a polylysine motif (residues 189-192) reverses the inhibitory effect of injected recombinant Syt 1 C2A fragment on neurotransmitter release from PC12 cells. Here we examined the requirement of the C2A polylysine motif for Syt 1 interaction with the cardiac Cav1.2 (L-type) and the neuronal Cav2.3 (R-type) voltage-gated Ca2+ channels, two channels required for neurotransmission. We find that the C2A polylysine motif presents a critical interaction surface with Cav1.2 and Cav2.3 since truncated Syt 1 containing a mutated motif (Syt 1*1-264) was ineffective at modifying the channel kinetics. Mutating the polylysine motif also abolished C2A binding to Lc753-893, the cytosolic interacting domain of Syt 1 at Cav1.2 1 subunit. Syt 1 and Syt 1* harboring the mutation at the KKKK motif modified channel activation, while Syt 1* only partially reversed the syntaxin 1A effects on channel activity. This mutation would interfere with the assembly of Syt 1/channel/syntaxin into an exocytotic unit. The functional interaction of the C2A polylysine domain with Cav1.2 and Cav2.3 is consistent with tethering of the secretory vesicle to the Ca2+ channel. It indicates that calcium-independent properties of Syt 1 regulate voltage-gated Ca2+ channels and contribute to the molecular events underlying transmitter release.     

Molecular and biophysical basis of glutamate and trace metal modulation of voltage-gated Ca(v)2.3 calcium channels

Here, we describe a new mechanism by which glutamate (Glu) and trace metals reciprocally modulate activity of the Ca(v)2.3 channel by profoundly shifting its voltage-dependent gating. We show that zinc and copper, at physiologically relevant concentrations, occupy an extracellular binding site on the surface of Ca(v)2.3 and hold the threshold for activation of these channels in a depolarized voltage range. Abolishing this binding by chelation or the substitution of key amino acid residues in IS1-IS2 (H111) and IS2-IS3 (H179 and H183) loops potentiates Ca(v)2.3 by shifting the voltage dependence of activation toward more negative membrane potentials. We demonstrate that copper regulates the voltage dependence of Ca(v)2.3 by affecting gating charge movements. Thus, in the presence of copper, gating charges transition into the "ON" position slower, delaying activation and reducing the voltage sensitivity of the channel. Overall, our results suggest a new mechanism by which Glu and trace metals transiently modulate voltage-dependent gating of Ca(v)2.3, potentially affecting synaptic transmission and plasticity in the brain.     

Disturbances in glucose-tolerance, insulin-release, and stress-induced hyperglycemia upon disruption of the Ca(v)2.3 (alpha 1E) subunit of voltage-gated Ca(2+) channels

Multiple types of voltage-activated Ca(2+) channels (T, L, N, P, Q, R type) coordinate Ca(2+)-dependent processes in neurons and neuroendocrine cells. Expressional and functional data have suggested a role for Ca(v)2.3 Ca(2+) channels in endocrine processes. To verify its role in vivo, Ca(v)2.3(-/-) mutant mice were generated, thus deficient in alpha 1E/R-type Ca(2+) channel. Intraperitoneal injection of D-glucose showed that glucose tolerance was markedly reduced, and insulin release into plasma was impaired in Ca(v)2.3-deficient mice. In isolated islets of Langerhans from these animals, no glucose-induced insulin release was detected. Further, in stressed Ca(v)2.3-deficient mice, the rate of glucose release into the blood was only 29% of that observed for wild-type animals. Thus, the deletion of Ca(v)2.3 causes deficits not only in insulin release but also in stress-induced hyperglycemia. The complex phenotype of Ca(v)2.3-deficient mice has dual components related to endocrine and neurological defects. The present findings provide direct evidence of a functional role for the Ca(v)2.3 subunit in hormone secretion and glucose homeostasis.     

Mice lacking Ca(v)2.3 (alpha1E) calcium channel exhibit hyperglycemia.

To investigate the functional role of Ca(v)2.3 channel in glucose homeostasis, we performed in vivo glucose tolerance and insulin tolerance tests together with stress-induced glucose release tests using mice deficient in Ca(v)2.3 channel (Ca(v)2.3-/-). The Ca(v)2.3-/- mice were significantly heavier than wild-type mice. In glucose tolerance and insulin tolerance tests, Ca(v)2.3-/- mice showed a significantly higher blood glucose level compared to wild-type mice. However, stress-induced blood glucose changes in Ca(v)2.3-/- mice were similar to those in wild-type mice. These results suggest that Ca(v)2.3 channel plays a role in glucose homeostasis by reducing insulin sensitivity and that Ca(v)2.3-/- mice exhibit symptoms resembling non-insulin-dependent diabetes mellitus.     

Syntaxin 1A modulates the voltage-gated L-type calcium channel (Ca(v)1.2) in a cooperative manner

Syntaxin 1A (Sx1A) modifies the activity of voltage-gated Ca2+ channels acting via the cytosolic and the two vicinal cysteines (271 and 272) at the transmembrane domain. Here we show that Sx1A modulates the Lc-type Ca2+ channel, Cav1.2, in a cooperative manner, and we explore whether channel clustering or the Sx1A homodimer is responsible for this activity. Sx1A formed homodimers but, when mutated at the two vicinal transmembrane domain cysteines, was unable to either dimerize or modify the channel activity suggesting disulfide bond formation. Moreover, applying global molecular dynamic search established a theoretical prospect of generating a disulfide bond between two Sx1A transmembrane helices. Nevertheless, Sx1A activity was not correlated with Sx1A homodimer. Application of a vicinal thiol reagent, phenylarsine oxide, abolished Sx1A action indicating the accessibility of Cys-271,272 thiols. Sx1A inhibition of channel activity was restored by phenylarsine oxide antidote, 2,3-dimercaptopropanol, consistent with thiol interaction of Sx1A. In addition, the supralinear mode of channel inhibition was correlated to the monomeric form of Sx1A and was apparent only when the three channel subunits alpha11.2/alpha2delta1/beta2a were present. This functional demonstration of cooperativity suggests that the three-subunit channel responds as a cluster, and Sx1A monomers associate with a dimer (or more) of a three-subunit Ca2+ channel. Consistent with channel cluster linked to Sx1A, a conformational change driven by membrane depolarization and Ca2+ entry would rapidly be transduced to the exocytotic machinery. As shown herein, the supralinear relationship between Sx1A and the voltage-gated Ca2+ channel within the cluster could convey the cooperativity that distinguishes the process of neurotransmitter release.     

The C-terminus of human Ca(v)2.3 voltage-gated calcium channel interacts with alternatively spliced calmodulin-2 expressed in two human cell lines

Ca(v)2.3 containing voltage-activated Ca(2+) channels are expressed in excitable cells and trigger neurotransmitter and peptide-hormone release. Their expression remote from the fast release sites leads to the accumulation of presynaptic Ca(2+) which can both, facilitate and inhibit the influx of Ca(2+) ions through Ca(v)2.3. The facilitated Ca(2+) influx was recently related to hippocampal postsynaptic facilitation and long term potentiation. To analyze Ca(2+) mediated modulation of cellular processes more in detail, protein partners of the carboxy terminal tail of Ca(v)2.3 were identified by yeast-2-hybrid screening, leading in two human cell lines to the detection of a novel, extended and rarely occurring splice variant of calmodulin-2 (CaM-2), called CaM-2-extended (CaM-2-ext). CaM-2-ext interacts biochemically with the C-terminus of Ca(v)2.3 similar to the classical CaM-2 as shown by co-immunoprecipitation. Functionally, only CaM-2-ext reduces whole cell inward currents significantly. The insertion of the novel 46 nts long exon and the consecutive expression of CaM-2-ext must be dependent on a new upstream translation initiation site which is only rarely used in the tested human cell lines. The structure of the N-terminal extension is predicted to be more hydrophobic than the remaining CaM-2-ext protein, suggesting that it may help to dock it to the lipophilic membrane surrounding.     

Identification of the calcium channel alpha 1E (Ca(v)2.3) isoform expressed in atrial myocytes

The consensus octapeptide repeat motif of the barley seed storage protein C hordein, Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln, forms the epitope of two anti-prolamin monoclonal antibodies (Mabs), IFRN 0061 and 0614. The Mabs were found to exhibit unusual temperature-dependent binding characteristics, recognising C hordein and a peptide corresponding to the consensus repeat at 5 degrees C but not at 37 degrees C, as determined by enzyme-linked immunosorbent assay (ELISA). The K(d) of IFRN 0614 for the consensus peptide was found to be 1.2x10(12) mol(-1) at 12 degrees C, but no constant could be calculated at 37 degrees C due to a lack of binding. Similar ELISA binding characteristics were observed with an anti-C hordein polyclonal antiserum and a Mab raised to the consensus peptide. Circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy showed that the protein and the consensus peptide exist in a temperature-dependent equilibrium of poly-L-proline II type structures and beta-turn conformations. Whilst thermodynamic and kinetic effects may reduce antibody binding at higher temperatures, they cannot account for the complete loss of Mab recognition at higher temperatures. It seems likely that the Mabs preferentially recognise the Pro-Gln-Gln-Pro-Phe-Pro-Gln-Gln motif when presented in a conformation which may correspond to the poly-L-proline II type conformation which dominates the CD and FTIR spectra at 4-12 degrees C.     

Identification of a region of RyR1 that participates in allosteric coupling with the alpha(1S) (Ca(V)1.1) II-III loop.

In skeletal muscle, excitation-contraction (EC) coupling and retrograde signaling are thought to result from direct interactions between the ryanodine receptor (RyR1) and the alpha(1) subunit of the dihydropyridine receptor (alpha(1S)). Previous work has shown that the s53 region of alpha(1S) (residues 720-765 in the II-III loop) and regions R10 (1635-2636) and R9 (2659-3720) of RyR1 are involved in this signaling. Using the yeast two-hybrid system, we here report an interaction between s53 and the sR16 region of RyR1 (1837-2168, within R10), whereas no interaction was seen using upstream residues of the alpha(1S) II-III loop (s31, 666-709). The specificity of the s53-sR16 interaction was tested by using fragments of the cardiac RyR (RyR2) and DHPR (alpha(1C)) that correspond to sR16 and s53, respectively. No interaction was observed for sR16 x c53 (alpha(1C) 850-897), but weak interaction was occasionally observed for s53 x cR16 (RyR2 1817-2142). To test the functional significance of the s53 x sR16 interaction, we expressed in dyspedic myotubes a chimeric RyR (chimeraR16) in which sR16 was substituted for the corresponding region of RyR2. ChimeraR16 was found to mediate weak skeletal-type EC coupling. To test the necessity of sR16 sequence for coupling, we used "chimeraR16-rev," in which sR16 and a small upstream region of RyR1 were replaced by RyR2 sequence. ChimeraR16-rev did not differ from RyR1 in its ability to mediate EC coupling. Thus, interaction between residues 720-765 of alpha(1S) and residues 1837-2168 of RyR1 appears to contribute to but is not essential for EC coupling in skeletal muscle.     

Nitric oxide signalling augments neuronal voltage-gated L-type (Ca(v)1) and P/q-type (Ca(v)2.1) channels in the mouse medial nucleus of the trapezoid body

Nitric Oxide (NO) is a diffusible second messenger that modulates ion channels, intrinsic excitability and mediates synaptic plasticity. In light of its activity-dependent generation in the principal neurons of the medial nucleus of the trapezoid body (MNTB), we have investigated its potential modulatory effects on native voltage-gated calcium channels (Ca(V)) within this nucleus. Whole-cell patch recordings were made from brain slices from P13-15 CBA mice. Slices were incubated with the inhibitor of neuronal nitric oxide synthase (nNOS) 7-nitroindazole (10 μM) and pharmacological blockers used to isolate Ca(2+) current subtypes. Unpaired observations in the presence and absence of the NO-donors sodium nitroprusside (SNP, 100 μM) or Diethyl-ammonium-nonoate (DEA, 100 μM) were made to elucidate NO-dependent modulation of the expressed Ca(V) subtypes. A differential effect of NO on the calcium channel subtypes was observed: Ca(V)1 and Ca(V)2.1 (L+R- and P/Q+R-type) conductances were potentiated, whereas N+R-type (Ca(V)2.2) and R-type (Ca(V)2.3) current amplitudes were unaffected. L+R-type currents increased from 0.36 ± 0.04 nA to 0.64 ± 0.11 nA and P/Q+R-type from 0.55 ± 0.09 nA to 0.94 ± 0.05 nA, thereby changing the balance and relative contribution of each subtype to the whole cell calcium current. In addition, N+R-type half-activation voltage was left shifted following NO exposure. NO-dependent modulation of P/Q+R and N+R-type, but not L+R-type, channels was removed by inhibition of soluble guanylyl cyclase (sGC) activity. This data demonstrates a differential effect of NO signalling on voltage-gated calcium entry, by distinct NO-dependent pathways.     

Altered cocaine effects in mice lacking Ca(v)2.3 (alpha(1E)) calcium channel

Much evidence indicates that calcium channel plays a role in cocaine-induced behavioral responses. We assessed the contributions of Ca(v)2.3 (alpha(1E)) calcium channel to cocaine effects using Ca(v)2.3 knockout mice (Ca(v)2.3-/-). Acute administration of cocaine enhanced the locomotor activity in wild-type mice (Ca(v)2.3+/+), but failed to produce any response in Ca(v)2.3-/- mice. Repeated exposure to cocaine induced the behavioral sensitization and conditioned place preference in both genotypes. Pretreatment with a D1-receptor antagonist, SCH23390, blocked the cocaine-induced place preference in Ca(v)2.3+/+ mice; however, it had no significant effect in Ca(v)2.3-/- mice. Microdialysis and RT-PCR analysis revealed that the levels of extracellular dopamine and dopamine D1 and D2 receptor mRNAs were not altered in Ca(v)2.3-/- mice. These data indicate that Ca(v)2.3 channel contributes to the locomotor-stimulating effect of cocaine, and the deletion of Ca(v)2.3 channel reveals the presence of a novel pathway leading to cocaine rewarding which is insensitive to D1 receptor antagonist.     

Functional diversity in neuronal voltage-gated calcium channels by alternative splicing of Ca(v)alpha1

Alternative splicing is a critical mechanism used extensively in the mammalian nervous system to increase the level of diversity that can be achieved by a set of genes. This review focuses on recent studies of voltage-gated calcium (Ca) channel Ca_vα₁ subunit splice isoforms in neurons. Voltage-gated Ca channels couple changes in neuronal activity to rapid changes in intracellular Ca levels that in turn regulate an astounding range of cellular processes. Only ten genes have been identified that encode Ca_vα₁ subunits, an insufficient number to account for the level of functional diversity among voltage-gated Ca channels. The consequences of regulated alternative splicing among the genes that comprise voltage-gated Ca channels permits specialization of channel function, optimizing Ca signaling in different regions of the brain and in different cellular compartments. Although the full extent of alternative splicing is not yet known for any of the major subtypes of voltage-gated Ca channels, it is already clear that it adds a rich layer of structural and functional diversity”.     

Dynamin- and Rab5-Dependent Endocytosis of a Ca(2+)-Activated K(+) Channel, KCa2.3

Regulation of the number of ion channels at the plasma membrane is a critical component of the physiological response. We recently demonstrated that the Ca(2+)-activated K(+) channel, KCa2.3 is rapidly endocytosed and enters a Rab35- and EPI64C-dependent recycling compartment. Herein, we addressed the early endocytic steps of KCa2.3 using a combination of fluorescence and biotinylation techniques. We demonstrate that KCa2.3 is localized to caveolin-rich domains of the plasma membrane using fluorescence co-localization, transmission electron microscopy and co-immunoprecipitation (co-IP). Further, in cells lacking caveolin-1, we observed an accumulation of KCa2.3 at the plasma membrane as well as a decreased rate of endocytosis, as assessed by biotinylation. We also demonstrate that KCa2.3 and dynamin II are co-localized following endocytosis as well as demonstrating they are associated by co-IP. Further, expression of K44A dynamin II resulted in a 2-fold increase in plasma membrane KCa2.3 as well as a 3-fold inhibition of endocytosis. Finally, we evaluated the role of Rab5 in the endocytosis of KCa2.3. We demonstrate that expression of a dominant active Rab5 (Q79L) results in the accumulation of newly endocytosed KCa2.3 on to the membrane of the Rab5-induced vacuoles. We confirmed this co-localization by co-IP; demonstrating that KCa2.3 and Rab5 are associated. As expected, if Rab5 is required for the endocytosis of KCa2.3, expression of a dominant negative Rab5 (S34N) resulted in an approximate 2-fold accumulation of KCa2.3 at the plasma membrane. This was confirmed by siRNA-mediated knockdown of Rab5. Expression of the dominant negative Rab5 also resulted in a decreased rate of KCa2.3 endocytosis. These results demonstrate that KCa2.3 is localized to a caveolin-rich domain within the plasma membrane and is endocytosed in a dynamin- and Rab5-dependent manner prior to entering the Rab35/EPI64C recycling compartment and returning to the plasma membrane.     

Role of alpha1 2.3 subunit I-II linker sites in the enhancement of Ca(v) 2.3 current by phorbol 12-myristate 13-acetate and acetyl-beta-methylcholine

Potentiation of Ca(v) 2.3 currents by phorbol 12-myristate 13-acetate (PMA) or acetyl-beta-methylcholine (MCh) may be due to protein kinase C (PKC)-mediated phosphorylation of the alpha1 2.3 subunit. Mutational analysis of potential PKC sites unique to the alpha1 2.3 subunit revealed several sites in the II-III linker that are specific to MCh (Kamatchi, G., Franke, R., Lynch, C., III, and Sando, J. (2004) J. Biol. Chem. 279, 4102-4109). To identify sites responsive to PMA, Ser/Thr --> Ala mutations were made in potential PKC sites homologous to the alpha1 2.3 and 2.2 subunits, both of which respond to PMA. Wild type alpha1 2.3 or mutants were expressed in Xenopus oocytes in combination with beta1b and alpha2/delta subunits and muscarinic M1 receptors. Inward current (I(Ba)) was recorded using Ba2+ as the charge carrier. Thr-365 of the I-II linker was identified as the primary site of PMA action, and this site also was required, along with the previously identified MCh-selective sites, for the MCh response. Ser-369 and Ser-1995 contributed to current enhancement only if Thr-365 also was available. Mutation of the essential sites to Asp increased the basal I(Ba) and caused a corresponding decrease in the PMA or MCh responses, consistent with possible regulation of these sites by phosphorylation. These results suggest that PMA and MCh both activate a pathway that can regulate the common PMA-sensitive sites in the I-II linker but that MCh also activates an additional pathway required for regulation of the MCh-unique sites, especially in the II-III linker.     

Lectins modulate the internalization of recombinant interferon-alpha A and the induction of 2',5'-oligo(A) synthetase

After binding to specific cell surface receptors, interferon-alpha (IFN-alpha) along with its receptor is internalized by the cells. However, the physiological significance of the internalization of IFN is not known. We have found that the lectin concanavalin A (ConA), which does not inhibit the binding of 125I-rIFN-alpha A, inhibits both the internalization of 125I-rIFN-alpha A and the rIFN-alpha A-induced increase in the levels of 2',5'-oligo(A) synthetase mRNA and enzymatic activity in the B lymphoblastoid cell line Daudi. The reduced level of IFN-induced 2',5'-oligo(A) synthetase in ConA-treated cells was due neither to direct inhibition of the enzymatic activity nor to generalized inhibition of protein or RNA synthesis. The dose-response curves were similar for the effect of ConA to inhibit 125I-rIFN-alpha A internalization and 2',5'-oligo(A) synthetase induction. The correlation between the ConA-mediated inhibition of both 125I-rIFN-alpha A internalization and 2',5'-oligo(A) synthetase induction suggests that internalization of rIFN-alpha A plays a role in the responses to rIFN-alpha A. However, since ConA inhibits protein mobility in the plasma membrane, it is possible that ConA is also preventing aggregation of IFN receptors or interactions between IFN receptors and signal transducing proteins in the plasma membrane that may be necessary for responses to IFN.     

Tyrosine kinases act directly on the alpha1 subunit to modulate Ca(v)2.2 calcium channels.

Voltage-operated calcium channels are modulated by tyrosine kinases in different cell types. In this study, I(Ba) was measured by the whole cell voltage-clamp technique in single COS-7 cells overexpressing the Ca(v)2.2 calcium channels encoding N-type currents. Bath application of genistein, a nonselective PTK inhibitor (50-300 microM), concentration-dependently inhibited calcium channel currents, whereas the inactive structural analogue, daidzein, was without effect over the same concentration range. Similarly, PP1, a src family-selective tyrosine kinase inhibitor, inhibited I(Ba) in a concentration-dependent manner (500 nM-5 microM) over a range of test potentials. Expression of the Ca(v)2.2alpha1 (alpha(1B)) subunit alone gave rise to functional channels, and genistein (100 microM) also inhibited currents elicited by the alpha(1B) subunit alone. These results indicate that tyrosine kinase inhibitors are likely to inhibit Ca(v)2.2 calcium channels via an action on the pore-forming alpha(1) subunit and suggest that an endogenous member of the Src family may play a physiological role in modulating these channels.     

Characterization of the gating brake in the I-II loop of Ca(v)3.2 T-type Ca(2+) channels

Mutations in the I-II loop of Ca(v)3.2 channels were discovered in patients with childhood absence epilepsy. All of these mutations increased the surface expression of the channel, whereas some mutations, and in particular C456S, altered the biophysical properties of channels. Deletions around C456S were found to produce channels that opened at even more negative potentials than control, suggesting the presence of a gating brake that normally prevents channel opening. The goal of the present study was to identify the minimal sequence of this brake and to provide insights into its structure. A peptide fragment of the I-II loop was purified from bacteria, and its structure was analyzed by circular dichroism. These results indicated that the peptide had a high alpha-helical content, as predicted from secondary structure algorithms. Based on homology modeling, we hypothesized that the proximal region of the I-II loop may form a helix-loop-helix structure. This model was tested by mutagenesis followed by electrophysiological measurement of channel gating. Mutations that disrupted the helices, or the loop region, had profound effects on channel gating, shifting both steady state activation and inactivation curves, as well as accelerating channel kinetics. Mutations designed to preserve the helical structure had more modest effects. Taken together, these studies showed that any mutations in the brake, including C456S, disrupted the structural integrity of the brake and its function to maintain these low voltage-activated channels closed at resting membrane potentials.