Novel splice variants of rat CaV2.1 that lack much of the synaptic protein interaction site are expressed in neuroendocrine cells

Voltage-gated Ca(2+) channels are responsible for the activation of the Ca(2+) influx that triggers exocytotic secretion. The synaptic protein interaction (synprint) site found in the II-III loop of Ca(V)2.1 and Ca(V)2.2 mediates a physical association with synaptic proteins that may be crucial for fast neurotransmission and axonal targeting. We report here the use of nested PCR to identify two novel splice variants of rat Ca(V)2.1 that lack much of the synprint site. Furthermore, we compare immunofluorescence data derived from antibodies directed against sequences in the Ca(V)2.1 synprint site and carboxyl terminus to show that channel variants lacking a portion of the synprint site are expressed in two types of neuroendocrine cells. Immunofluorescence data also suggest that such variants are properly targeted to neuroendocrine terminals. When expressed in a mammalian cell line, both splice variants yielded Ca(2+) currents, but the variant containing the larger of the two deletions displayed a reduced current density and a marked shift in the voltage dependence of inactivation. These results have important implications for Ca(V)2.1 function and for the mechanisms of Ca(V)2.1 targeting in neurons and neuroendocrine cells.     

Possible role during exocytosis of a Ca(2+)-activated channel in neurohypophysial granules.

Ion channels from bovine neurohypophysial granules were incorporated into artificial lipid bilayers. The larger amplitude channel is permeable to cations and exhibits multiple conductances. The channel opens only in the presence of free Ca2+, but is inhibited by relatively high Ca2+ concentrations. Release of vasopressin from permeabilized neurohypophysial terminals also shows a similar biphasic dependence on Ca2+. Release is selectively inhibited by low concentrations of the long-chain alcohol octanol, but not by high concentrations of ethanol, as is the neurosecretory granule Ca(2+)-activated cation channel. Furthermore, Ca(2+)-evoked release and channel activity are both inhibited by the long-chain tetraethylammonium analogs decamethonium and decyl-triethyl ammonium bromide. The close correlation between channel and release properties lead us to conclude that the Ca(2+)-activated channel is involved in peptide secretion.     

T-type Ca(2+) channels mediate neurotransmitter release in retinal bipolar cells

Transmitter release in neurons is thought to be mediated exclusively by high-voltage-activated (HVA) Ca(2+) channels. However, we now report that, in retinal bipolar cells, low-voltage-activated (LVA) Ca(2+) channels also mediate neurotransmitter release. Bipolar cells are specialized neurons that release neurotransmitter in response to graded depolarizations. Here we show that these cells express T-type Ca(2+) channel subunits and functional LVA Ca(2+) currents sensitive to mibefradil. Activation of these currents results in Ca(2+) influx into presynaptic terminals and exocytosis, which we detected as a capacitance increase in isolated terminals and the appearance of reciprocal currents in retinal slices. The involvement of T-type Ca(2+) channels in bipolar cell transmitter release may contribute to retinal information processing.     

Expression and functional characterization of the cardiac muscle ryanodine receptor Ca(2+) release channel in Chinese hamster ovary cells.

To study the function and regulation of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel, we expressed the RyR2 proteins in a Chinese hamster ovary (CHO) cell line, and assayed its function by single channel current recording and confocal imaging of intracellular Ca(2+) ([Ca(2+)](i)). The 16-kb cDNA encoding the full-length RyR2 was introduced into CHO cells using lipofectAmine and electroporation methods. Incorporation of microsomal membrane vesicles isolated from these transfected cells into lipid bilayer membrane resulted in single Ca(2+) release channel activities similar to those of the native Ca(2+) release channels from rabbit cardiac muscle SR membranes, both in terms of gating kinetics, conductance, and ryanodine modification. The expressed RyR2 channels were found to exhibit more frequent transitions to subconductance states than the native RyR2 channels and RyR1 expressed in CHO cells. Caffeine, an exogenous activator of RyR, induced release of [Ca(2+)](i) from these cells. Confocal imaging of cells expressing RyR2 did not detect spontaneous or caffeine-induced local Ca(2+) release events (i.e., "Ca(2+) sparks") typically seen in cardiac muscle. Our data show that the RyR2 expressed in CHO cells forms functional Ca(2+) release channels. Furthermore, the lack of localized Ca(2+) release events in these cells suggests that Ca(2+) sparks observed in cardiac muscle may involve cooperative gating of a group of Ca(2+) release channels and/or their interaction with muscle-specific proteins.     

Subtype-specific expression of group III metabotropic glutamate receptors and Ca2+ channels in single nerve terminals

The release properties of glutamatergic nerve terminals are influenced by a number of factors, including the subtype of voltage-dependent calcium channel and the presence of presynaptic autoreceptors. Group III metabotropic glutamate receptors (mGluRs) mediate feedback inhibition of glutamate release by inhibiting Ca(2+) channel activity. By imaging Ca(2+) in preparations of cerebrocortical nerve terminals, we show that voltage-dependent Ca(2+) channels are distributed in a heterogeneous manner in individual nerve terminals. Presynaptic terminals contained only N-type (47.5%; conotoxin GVIA-sensitive), P/Q-type (3.9%; agatoxin IVA-sensitive), or both N- and P/Q-type (42.6%) Ca(2+) channels, although the remainder of the terminals (6.1%) were insensitive to these two toxins. In this preparation, two mGluRs with high and low affinity for l(+)-2-amino-4-phosphonobutyrate were identified by immunocytochemistry as mGluR4 and mGluR7, respectively. These receptors were responsible for 22.2 and 24.1% reduction of glutamate release, and they reduced the Ca(2+) response in 24.4 and 30.3% of the nerve terminals, respectively. Interestingly, mGluR4 was largely (73.7%) located in nerve terminals expressing both N- and P/Q-type Ca(2+) channels, whereas mGluR7 was predominantly (69.9%) located in N-type Ca(2+) channel-expressing terminals. This specific coexpression of different group III mGluRs and Ca(2+) channels may endow synaptic terminals with distinct release properties and reveals the existence of a high degree of presynaptic heterogeneity.     

Protein kinase C activation inhibits Cav1.3 calcium channel at NH2-terminal serine 81 phosphorylation site

The Ca(v)1.3 (alpha(1D)) variant of L-type Ca(2+) channels plays a vital role in the function of neuroendocrine and cardiovascular systems. In this article, we report on the molecular and functional basis of alpha(1D) Ca(2+) channel modulation by protein kinase C (PKC). Specifically, we show that the serine 81 (S81) phosphorylation site at the NH(2)-terminal region plays a critical role in alpha(1D) Ca(2+) channel modulation by PKC. The introduction of a negatively charged residue at position 81, by converting serine to aspartate, mimicked the PKC phosphorylation effect on alpha(1D) Ca(2+) channel. The modulation of alpha(1D) Ca(2+) channel by PKC was prevented by dialyzing cells with a 35-amino acid peptide mimicking the alpha(1D) NH(2)-terminal region comprising S81. In addition, the data revealed that only betaII- and epsilonPKC isozymes are implicated in this regulation. These novel findings have significant implications in the pathophysiology of alpha(1D) Ca(2+) channel and in the development of PKC isozyme-targeted therapeutics.     

Association of neuronal calcium channels with modular adaptor proteins.

Presynaptic voltage-gated calcium (Ca(2+)) channels mediate Ca(2+) influx into the presynaptic terminal that triggers synaptic vesicle fusion and neurotransmitter release. The immediate proximity of Ca(2+) channels to the synaptic vesicle release apparatus is critical for rapid and efficient synaptic transmission. In a series of biochemical experiments, we demonstrate a specific association of the cytosolic carboxyl terminus of the N-type Ca(2+) channel pore-forming alpha(1B) subunit with the modular adaptor proteins Mint1 and CASK. The carboxyl termini of alpha(1B) bind to the first PDZ domain of Mint1 (Mint1-1). The proline-rich region present in the carboxyl termini of alpha(1B) binds to the SH3 domain of CASK. Mint1-1 is specific for the E/D-X-W-C/S-COOH consensus, which defines a novel class of PDZ domains (class III). The Mint1-1 PDZ domain-binding motif is present only in the "long" carboxyl-terminal splice variants of N-type (alpha(1B)) and P/Q-type (alpha(1A)) Ca(2+) channels, but not in R-type (alpha(1E)) or L-type (alpha(1C)) Ca(2+) channels. Our results directly link presynaptic Ca(2+) channels to a macromolecular complex formed by modular adaptor proteins at synaptic junction and advance our understanding of coupling between cell adhesion and synaptic vesicle exocytosis.     

CB1 receptors diminish both Ca(2+) influx and glutamate release through two different mechanisms active in distinct populations of cerebrocortical nerve terminals

We have investigated the mechanisms by which activation of cannabinoid receptors reduces glutamate release from cerebrocortical nerve terminals. Glutamate release evoked by depolarization of nerve terminals with high KCl (30 mmol/L) involves N and P/Q type Ca(2+)channel activation. However, this release of glutamate is independent of Na(+) or K(+) channel activation as it was unaffected by blockers of these channels (tetrodotoxin -TTX- or tetraethylammonium TEA). Under these conditions in which only Ca(2+) channels contribute to pre-synaptic activity, the activation of cannabinoid receptors with WIN55,212-2 moderately reduced glutamate release (26.4 +/- 1.2%) by a mechanism that in this in vitro model is resistant to TTX and consistent with the inhibition of Ca(2+) channels. However, when nerve terminals are stimulated with low KCl concentrations (5-10 mmol/L) glutamate release is affected by both Ca(2+) antagonists and also by TTX and TEA, indicating the participation of Na(+) and K(+) channel firing in addition to Ca(2+) channel activation. Interestingly, stimulation of nerve terminals with low KCl concentrations uncovered a mechanism that further inhibited glutamate release (81.78 +/- 4.9%) and that was fully reversed by TEA. This additional mechanism is TTX-sensitive and consistent with the activation of K(+) channels. Furthermore, Ca(2+) imaging of single boutons demonstrated that the two pre-synaptic mechanisms by which cannabinoid receptors reduce glutamate release operate in distinct populations of nerve terminals.     

Biochemical and biophysical evidence for gamma 2 subunit association with neuronal voltage-activated Ca2+ channels.

A novel gene (Cacng2; gamma(2)) encoding a protein similar to the voltage-activated Ca(2+) channel gamma(1) subunit was identified as the defective gene in the epileptic and ataxic mouse, stargazer. In this study, we analyzed the association of this novel neuronal gamma(2) subunit with Ca(2+) channels of rabbit brain, and the function of the gamma(2) subunit in recombinant neuronal Ca(2+) channels expressed in Xenopus oocytes. Our results showed that the gamma(2) subunit and a closely related protein (called gamma(3)) co-sedimented and co-immunoprecipitated with neuronal Ca(2+) channel subunits in vivo. Electrophysiological analyses showed that gamma(2) co-expression caused a significant decrease in the current amplitude of both alpha(1B)(alpha(1)2.2)-class (36.8%) and alpha(1A)(alpha(1)2.1)-class (39.7%) Ca(2+) channels (alpha(1)beta(3)alpha(2)delta). Interestingly, the inhibitory effects of the gamma(2) subunit on current amplitude were dependent on the co-expression of the alpha(2)delta subunit. In addition, co-expression of gamma(2) or gamma(1) also significantly decelerates the activation kinetics of alpha(1B)-class Ca(2+) channels. Taken together, these results suggest that the gamma(2) subunit is an important constituent of the neuronal Ca(2+) channel complex and that it down-regulates neuronal Ca(2+) channel activity. Furthermore, the gamma(2) subunit likely contributes to the fine-tuning of neuronal Ca(2+) channels by counterbalancing the effects of the alpha(2)delta subunit.     

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.     

The involvement of voltage-operated calcium channels in somato-dendritic oxytocin release

Magnocellular neurons of the supraoptic nucleus (SON) secrete oxytocin and vasopressin from axon terminals in the neurohypophysis, but they also release large amounts of peptide from their somata and dendrites, and this can be regulated both by activity-dependent Ca(2+) influx and by mobilization of intracellular Ca(2+). This somato-dendritic release can also be primed by agents that mobilise intracellular Ca(2+), meaning that the extent to which it is activity-dependent, is physiologically labile. We investigated the role of different Ca(2+) channels in somato-dendritic release; blocking N-type channels reduced depolarisation-induced oxytocin release from SONs in vitro from adult and post-natal day 8 (PND-8) rats, blocking L-type only had effect in PND-8 rats, while blocking other channel types had no significant effect. When oxytocin release was primed by prior exposure to thapsigargin, both N- and L-type channel blockers reduced release, while P/Q and R-type blockers were ineffective. Using confocal microscopy, we found immunoreactivity for Ca(v)1.2 and 1.3 channel subunits (which both form L-type channels), 2.1 (P/Q type), 2.2 (N-type) and 2.3 (R-type) in the somata and dendrites of both oxytocin and vasopressin neurons, and the intensity of the immunofluorescence signal for different subunits differed between PND-8, adult and lactating rats. Using patch-clamp electrophysiology, the N-type Ca(2+) current density increased after thapsigargin treatment, but did not alter the voltage sensitivity of the channel. These results suggest that the expression, location or availability of N-type Ca(2+) channels is altered when required for high rates of somato-dendritic peptide release.     

Glycerotoxin from Glycera convoluta stimulates neurosecretion by up-regulating N-type Ca2+ channel activity

We report here the purification of glycerotoxin from the venom of Glycera convoluta, a novel 320 kDa protein capable of reversibly stimulating spontaneous and evoked neurotransmitter release at the frog neuromuscular junction. However, glycerotoxin is ineffective at the murine neuromuscular junction, which displays a different subtype of voltage- dependent Ca(2+) channels. By sequential and selective inhibition of various types of Ca(2+) channels, we found that glycerotoxin was acting via Ca(v)2.2 (N-type). In neuroendocrine cells, it elicits a robust, albeit transient, influx of Ca(2+) sensitive to the Ca(v)2.2 blockers omega-conotoxin GVIA and MVIIA. Moreover, glycerotoxin triggers a Ca(2+) transient in human embryonic kidney (HEK) cells over-expressing Ca(v)2.2 but not Ca(v)2.1 (P/Q-type). Whole-cell patch-clamp analysis of Ca(v)2.2 expressing HEK cells revealed an up-regulation of Ca(2+) currents due to a leftward shift of the activation peak upon glycerotoxin addition. A direct interaction between Ca(v)2.2 and this neurotoxin was revealed by co-immunoprecipitation experiments. Therefore, glycerotoxin is a unique addition to the arsenal of tools available to unravel the mechanism controlling Ca(2+)-regulated exocytosis via the specific activation of Ca(v)2.2.     

Ca(2+) changes induced by different presynaptic nicotinic receptors in separate populations of individual striatal nerve terminals

Presynaptic nicotinic acetylcholine receptors likely play a modulatory role in the nerve terminal. Using laser-scanning confocal microscopy, we have characterized physiological responses obtained on activation of presynaptic nicotinic receptors by measuring calcium changes in individual nerve terminals (synaptosomes) isolated from the rat corpus striatum. Nicotine (500 nM) induced Ca(2+) changes in a subset (10-25%) of synaptosomes. The Ca(2+) responses were dependent on extracellular Ca(2+) and desensitized very slowly (several minutes) on prolonged exposure to agonist. The nicotine-induced Ca(2+) responses were dose-dependent and were completely blocked by dihydro-beta-erythroidine (5 microM), differentially affected by mecamylamine (10 microM) and alpha-conotoxin MII (100 nM), and not affected by alpha-bungarotoxin (500 nM). Immunocytochemical studies using well-characterized monoclonal antibodies revealed the presence of the alpha4 and alpha3/alpha5 nicotinic subunits. The nicotine-induced responses were unaffected by prior depolarization or by a mixture of Ca(2+) channel toxins including omega-conotoxin MVIIC (500 nM), omega-conotoxin GVIA (500 nM) and agatoxin TK (200 nM). Our results indicate that nicotinic receptors present on striatal nerve terminals induce Ca(2+) entry largely without involving voltage-gated Ca(2+) channels, most likely by direct permeation via the receptor channel itself. In addition, at least two subpopulations of presynaptic nicotinic receptors reside on separate terminals in the striatum, suggesting distinct modulatory roles.     

S-nitrosation controls gating and conductance of the alpha 1 subunit of class C L-type Ca(2+) channels

Modulation of smooth muscle, L-type Ca(2+) channels (class C, Ca(V)1.2b) by thionitrite S-nitrosoglutathione (GSNO) was investigated in the human embryonic kidney 293 expression system at the level of whole-cell and single-channel currents. Extracellular administration of GSNO (2 mM) rapidly reduced whole-cell Ba(2+) currents through channels derived either by expression of alpha1C-b or by coexpression of alpha1C-b plus beta2a and alpha2-delta. The non-thiol nitric oxide (NO) donors 2,2-diethyl-1-nitroso-oxhydrazin (2 mM) and 3-morpholinosydnonimine-hydrochloride (2 mM), which elevated cellular cGMP levels to a similar extent as GSNO, failed to affect Ba(2+) currents significantly. Intracellular administration of copper ions, which promote decomposition of the thionitrite, antagonized its inhibitory effect, and loading of cells with high concentrations of dithiothreitol (2 mM) prevented the effect of GSNO on alpha1C-b channels. Intracellular loading of cells with oxidized glutathione (2 mM) affected neither alpha1C-b channel function nor their modulation by GSNO. Analysis of single-channel behavior revealed that GSNO inhibited Ca(2+) channels mainly by reducing open probability. The development of GSNO-induced inhibition was associated with the transient occurrence of a reduced conductance state of the channel. Our results demonstrate that GSNO modulates the alpha1 subunit of smooth muscle L-type Ca(2+) channels by an intracellular mechanism that is independent of NO release and stimulation of guanylyl cyclase. We suggest S-nitrosation of intracellularly located sulfhydryl groups as an important determinant of Ca(2+) channel gating and conductance.     

Calcium microdomains in regulated exocytosis

Katz and co-workers showed that Ca(2+) triggers exocytosis. The existence of sub-micrometer domains of greater than 100 microM [Ca(2+)](i) was postulated on theoretical grounds. Using a modified, low-affinity aequorin, Llinas et al. were the first to demonstrate the existence of Ca(2+) 'microdomains' in squid presynaptic terminals. Over the past several years, it has become clear that individual Ca(2+) nano- and microdomains forming around the mouth of voltage-gated Ca(2+) channels ascertain the tight coupling of fast synaptic vesicle release to membrane depolarization by action potentials. Recent work has established different geometric arrangements of vesicles and Ca(2+) channels at different central synapses and pointed out the role of Ca(2+) syntillas - localized, store operated Ca(2+) signals - in facilitation and spontaneous release. The coupling between Ca(2+) increase and evoked exocytosis is more sluggish in peripheral terminals and neuroendocrine cells, where channels are less clustered and Ca(2+) comes from different sources, including Ca(2+) influx via the plasma membrane and the mobilization of Ca(2+) from intracellular stores. Finally, also non- (electrically) excitable cells display highly localized Ca(2+) signaling domains. We discuss in particular the organization of structural microdomains of Bergmann glia, specialized astrocytes of the cerebellum that have only recently been considered as secretory cells. Glial microdomains are the spatial substrate for functionally segregated Ca(2+) signals upon metabotropic activation. Our review emphasizes the large diversity of different geometric arrangements of vesicles and Ca(2+) sources, leading to a wide spectrum of Ca(2+) signals triggering release.     

Differential expression of high voltage-activated Ca2+ channel types in the rostral reticular thalamic nucleus of the absence epileptic WAG/Rij rat

In the WAG/Rij rat, a model for human absence epilepsy, spike-wave discharges (SWD) and absence epileptic behavior develop after the age of 3 months. The rostral part of the reticular thalamic nucleus (rRTN) is involved in SWD. Ca(2+) channels play a central role in the initiation and maintenance of burst firing activity of thalamic cells. We hypothesize that a changed expression of alpha(1)-subunits of one or more high voltage-activated Ca(2+) channel types in the rRTN underlies the development of SWD. To test this hypothesis we compared 3- and 6-month-old WAG/Rij rats with nonepileptic, age-matched control rats. By immunocytochemistry, the expressions of alpha(1)1.3-, alpha(1)2.1-, alpha(1)2.2-, and alpha(1)2.3-subunits were shown in both strains, demonstrating the presence of Ca(v)1.3, Ca(v)2.1, Ca(v)2.2, and Ca(v)2.3 channels, respectively. Quantification of channel expression indicates that the development of SWD in WAG/Rij rats is concomitant with an increased expression of Ca(v)2.1 channels in the rRTN. These channels are mainly presynaptic, as revealed by double immunofluorescence involving the presynapse marker syntaxin. The mechanism by which this increase could be related to the occurrence of SWD has been discussed.     

A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.