N- and P/Q-type Ca2+ channels in adrenal chromaffin cells

Ca2+ is the most ubiquitous second messenger found in all cells. Alterations in [Ca2+]i contribute to a wide variety of cellular responses including neurotransmitter release, muscle contraction, synaptogenesis and gene expression. Voltage-dependent Ca2+ channels, found in all excitable cells (Hille 1992), mediate the entry of Ca2+ into cells following depolarization. Ca2+ channels are composed of a large pore-forming subunit, called the alpha1 subunit, and several accessory subunits. Ten different alpha1 subunit genes have been identified and classified into three families, Ca(v1-3) (Dunlap et al. 1995, Catterall 2000). Each alpha1 gene produces a unique Ca2+ channel. Although chromaffin cells express several different types of Ca2+ channels, this review will focus on the Cav(2.1) and Cav(2.2) channels, also known as P/Q- and N-type respectively (Nowycky et al. 1985, Llinas et al. 1989b, Wheeler et al. 1994). These channels exhibit physiological and pharmacological properties similar to their neuronal counterparts. N-, P/Q and to a lesser extent R-type Ca2+ channels are known to regulate neurotransmitter release (Hirning et al. 1988, Horne & Kemp 1991, Uchitel et al. 1992, Luebke et al. 1993, Takahashi & Momiyama 1993, Turner et al. 1993, Regehr & Mintz 1994, Wheeler et al. 1994, Wu & Saggau 1994, Waterman 1996, Wright & Angus 1996, Reid et al. 1997). N- and P/Q-type Ca2+ channels are abundant in nerve terminals where they colocalize with synaptic vesicles. Similarly, these channels play a role in neurotransmitter release in chromaffin cells (Garcia et al. 2006). N- and P/Q-type channels are subject to many forms of regulation (Ikeda & Dunlap 1999). This review pays particular attention to the regulation of N- and P/Q-type channels by heterotrimeric G-proteins, interaction with SNARE proteins, and channel inactivation in the context of stimulus-secretion coupling in adrenal chromaffin cells.     

BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells.

BK channel activation by brief depolarizations requires Ca2+ influx through L- and Q-type Ca2+ channels in rat chromaffin cells. Ca2+- and voltage-dependent BK-type K+ channels contribute to action potential repolarization in rat adrenal chromaffin cells. Here we characterize the Ca2+ currents expressed in these cells and identify the Ca2+ channel subtypes that gate the activation of BK channels during Ca2+ influx. Selective Ca2+ channel antagonists indicate the presence of at least four types of high-voltage-gated Ca2+ channels: L-, N-, P, and Q type. Mean amplitudes of the L-, N-, P-, and Q-type Ca2+ currents were 33, 21, 12, and 24% of the total Ca2+ current, respectively. Five-millisecond Ca2+ influx steps to 0 mV were employed to assay the contribution of Ca2+ influx through these Ca2+ channels to the activation of BK current. Blockade of L-type Ca2+ channels by 5 microM nifedipine or Q-type Ca2+ channels by 2 microM Aga IVA reduced BK current activation by 77 and 42%, respectively. In contrast, blockade of N-type Ca2+ channels by brief applications of 1-2 microM CnTC MVIIC or P-type Ca2+ channels by 50-100 nM Aga IVA reduced BK current activation by only 11 and 12%, respectively. Selective blockade of L- and Q-type Ca2+ channels also eliminated activation of BK current during action potentials, whereas almost no effects were seen by the selective blockade of N- or P-type Ca2+ channels. Finally, the L-type Ca2+ channel agonist Bay K 8644 promoted activation of BK current by brief Ca2+ influx steps by more than twofold. These data show that, despite the presence of at least four types of Ca2+ channels in rat chromaffin cells, BK channel activation in rat chromaffin cells is predominantly coupled to Ca2+ influx through L- and Q-type Ca2+ channels.     

An activity-dependent increased role for L-type calcium channels in exocytosis is regulated by adrenergic signaling in chromaffin cells

Chromaffin cells of the adrenal medulla represent a primary output of the sympathetic nervous system. Their electrical stimulation evokes the fusion of large dense core granules with the cell membrane and the exocytic release of multiple transmitter molecules into the circulation. There the transmitters contribute to the regulation of basic metabolism of the organism. Under physiological activity, granule fusion and transmitter release are limited by activity-dependent Ca(2+) influx, entering through multiple isoforms of voltage-gated calcium channels. In this study we utilize perforated-patch voltage-clamp recordings and depolarize mouse chromaffin cells in situ with action potential-like waveforms to mimic physiological firing. We measure calcium influx through specific isoforms and measure cell capacitance as an index of granule fusion. Combining these approaches we calculate specific stimulus-secretion efficiencies for L-type, N-type, P/Q-type and R-type calcium channels under varied physiological activity levels. Current influx through all channel subtypes exhibited an activity-dependent depression. As expected P/Q-type channels, while responsible for modest Ca(2+) influx, are tightly coupled to catecholamine secretion under all conditions. We further find that stimulation designed to match sympathetic input under the acute stress response recruits L-type channels to a state of enhanced stimulus-secretion efficiency. N- and R-type channels do not undergo activity-dependent recruitment and remain loosely coupled to the secretion. Thus, only L-type channels exhibit activity-dependent changes in their stimulus-secretion function under physiological stimulation. Lastly, we show that treatment with the beta-adrenergic agonist, isoproterenol, specifically blocks the increase in the stimulus-secretion function of L-type channels. Thus, increased cell firing specifically enhances stimulus-secretion coupling of L-type Ca(2+) channels in chromaffin cells in situ. This mechanism is regulated by an adrenergic signaling pathway.     

Voltage-gated calcium channels in adult rat inferior colliculus neurons

The inferior colliculus (IC) plays a key role in the processing of auditory information and is thought to be an important site for genesis of wild running seizures that evolve into tonic-clonic seizures. IC neurons are known to have Ca(2+) channels but neither their types nor their pharmacological properties have been as yet characterized. Here, we report on biophysical and pharmacological properties of Ca(2+) channel currents in acutely dissociated neurons of adult rat IC, using electrophysiological and molecular techniques. Ca(2+) channels were activated by depolarizing pulses from a holding potential of -90 mV in 10 mV increments using 5 mM barium (Ba(2+)) as the charge carrier. Both low (T-type, VA) and high (HVA) threshold Ca(2+) channel currents that could be blocked by 50 microM cadmium, were recorded. Pharmacological dissection of HVA currents showed that nifedipine (10 microM, L-type channel blocker), omega-conotoxin GVIA (1 microM, N-type channel blocker), and omega-agatoxin TK (30 nM, P-type channel blocker) partially suppressed the current by 21%, 29% and 22%, respectively. Since at higher concentration (200 nM) omega-agatoxin TK also blocks Q-type channels, the data suggest that Q-type Ca(2+) channels carry approximately 16% of HVA current. The fraction of current (approximately 12%) resistant to the above blockers, which was blocked by 30 microM nickel and inactivated with tau of 15-50 ms, was considered as R-type Ca(2+) channel current. Consistent with the pharmacological evidences, Western blot analysis using selective Ca(2+) channel antibodies showed that IC neurons express Ca(2+) channel alpha(1A), alpha(1B), alpha(1C), alpha(1D), and alpha(1E) subunits. We conclude that IC neurons express functionally all members of HVA Ca(2+) channels, but only a subset of these neurons appear to have developed functional LVA channels.     

Synapse-to-synapse variation of calcium channel subtype contributions in large mossy fiber terminals of mouse hippocampus

Both N- and P/Q-type voltage-dependent calcium channels are involved in fast transmitter release in the hippocampus, but are differentially regulated. Although variable contributions of voltage-dependent calcium channel subtypes to presynaptic Ca2+ influx have been suggested to give a neural network of great diversity, their presence has only been demonstrated in a culture system and has remained unclear in the brain. Here, the individual large mossy fiber presynaptic terminal was labeled with Ca2+/Sr2+-sensitive fluorescent dextrans in the hippocampal slice of the mouse. The fractional contribution of voltage-dependent calcium channel subtypes to presynaptic Ca2+/Sr2+ influx was directly measured by the sensitivity of Ca2+/Sr2+-dependent fluorescent increment to subtype-selective neurotoxins, omega-conotoxin GVIA (an N-type selective blocker), omega-agatoxin IVA (a P/Q-type selective blocker) and SNX-482 (an R-type selective blocker). Synapse-to-synapse comparison of large mossy fiber terminals revealed that the contributions of N- and R-type voltage-dependent calcium channels varied more widely than that of P/Q-type. Even two large mossy fiber presynaptic terminals neighboring on the same axon differed in the fractional contributions of N- and R-type voltage-dependent calcium channels. On the other hand, these terminals were similar in the fractional contributions of P/Q-type voltage-dependent calcium channels. These results provide direct evidence that individual large mossy fiber synapses are differential in the contribution of N- and R-type voltage-dependent calcium channel subtypes to presynaptic Ca2+/Sr2+ influx. We suggest that the synapse-to-synapse variation of presynaptic voltage-dependent calcium channel subtype contributions may be one of the mechanisms amplifying diversity of the hippocampal network.     

Action potential afterdepolarization mediated by a Ca2+-activated cation conductance in myenteric AH neurons

We investigated the nature of afterdepolarizing potentials in AH neurons from the guinea-pig duodenum using whole-cell patch-clamp recordings in intact myenteric ganglia. Afterdepolarizing potentials were minimally activated following action-potential firing under normal conditions, but after application of charybdotoxin (40 nM) or tetraethyl ammonium (TEA; 10-20 mM) to the bathing solution, prominent afterdepolarizing potentials followed action potentials. The whole-cell current underlying afterdepolarizing potentials (I(ADP)) in the presence of TEA (10-20 mM) reversed at -38 mV and was not voltage-dependent. Reduction of NaCl in the bathing (Krebs) solution to 58 mM shifted the reversal potential of the I(ADP) to -58 mV, suggesting that the current underlying the afterdepolarizing potential was carried by a mixture of cations. The relative contributions of Na(+) and K(+) to this current were estimated to be about 1:5. Substitution of external Na(+) with N-methyl D-glucamine blocked the current while replacement of internal Cl(-) with gluconate did not block the I(ADP). The I(ADP) was also inhibited when CsCl-filled patch pipettes were used. The I(ADP) was blocked or substantially decreased in amplitude in the presence of N-type Ca(2+) channel antagonists, omega-conotoxin GVIA and omega-conotoxin MVIIC, respectively, and was eliminated by external Cd(2+), indicating that it was dependent on Ca(2+) entry. The I(ADP) was also inhibited by ryanodine (10-20 microM), indicating that Ca(2+)-induced Ca(2+) release was involved in its activation. Niflumic acid consistently inhibited the I(ADP) with an IC(50) of 63 microM. Using antibodies against the pore-forming subunits of L-, N- and P/Q-type voltage-gated Ca(2+) channels, we have demonstrated that myenteric AH neurons express N- and P/Q, but not L-type voltage-gated Ca(2+) channels. We conclude that the ADP in myenteric AH neurons, in the presence of an L-type Ca(2+)-channel blocker, is generated by the opening of Ca(2+)-activated non-selective cation channels following action potential-mediated Ca(2+) entry mainly through N-type Ca(2+) channels. Ca(2+) release from ryanodine-sensitive stores triggered by Ca(2+) entry contributes significantly to the activation of this current.     

Blocker-resistant Ca2+ currents in rat CA1 hippocampal pyramidal neurons

Ca(2+) currents resistant to organic Ca(2+) channel antagonists are present in different types of central neurons. Here, we describe the properties of such currents in CA1 neurons acutely dissociated from rat hippocampus. Blocker-resistant Ca(2+) currents were isolated by combined application of N-, P/Q- and L-type Ca(2+) current antagonists (omega-conotoxin GVIA 2 microM; omega-conotoxin MVIIC 3 microM; omega-agatoxin IVA 200 nM; nifedipine 10 microM) and constituted approximately 21% of the total Ba(2+) current.The blocker-resistant current showed properties similar to R-type currents in other cell types, i.e. voltages of half-maximal inactivation and activation of -76 and -17 mV, respectively, and strong inactivation during the test pulse. In addition, blocker-resistant Ca(2+) currents in CA1 neurons displayed a characteristically rapid deactivation. Application of mock action potentials revealed that charge transfer through blocker-resistant Ca(2+) channels is highly sensitive to action potential shape and changes in resting membrane voltage. Pharmacological experiments showed that these currents were highly sensitive to the divalent cation Ni(2+) (half-maximal block at 28 microM), but were relatively resistant to the spider toxin SNX-482 (8% and 52% block at 0.1 and 1 microM, respectively).In addition to the functional analysis, we examined the expression of pore-forming and accessory Ca(2+) channel subunits on the messenger RNA level in isolated CA1 neurons using quantitative real-time polymerase chain reaction. Of the pore-forming alpha subunits encoding high-threshold Ca(2+) channels, Ca(v)2.1, Ca(v)2.2 and Ca(v)2.3 messenger RNA levels were most prominent, corresponding to the high proportion of N-, P/Q- and R-type currents in these neurons.In summary, CA1 neurons display blocker-resistant Ca(2+) currents with distinctive biophysical and pharmacological properties similar to R-type currents in other neuron types, and express Ca(2+) channel messenger RNAs that give rise to R-type Ca(2+) currents in expression systems.     

Differential facilitation of N- and P/Q-type calcium channels during trains of action potential-like waveforms

Inhibition of presynaptic voltage-gated calcium channels by direct G-protein βγ subunit binding is a widespread mechanism that regulates neurotransmitter release. Voltage-dependent relief of this inhibition (facilitation), most likely to be due to dissociation of the G-protein from the channel, may occur during bursts of action potentials. In this paper we compare the facilitation of N- and P/Q-type Ca²⁺ channels during short trains of action potential-like waveforms (APWs) using both native channels in adrenal chromaffin cells and heterologously expressed channels in tsA201 cells. While both N- and P/Q-type Ca²⁺ channels exhibit facilitation that is dependent on the frequency of the APW train, there are important quantitative differences. Approximately 20 % of the voltage-dependent inhibition of N-type I _Ca was reversed during a train while greater than 40 % of the inhibition of P/Q-type I _Ca was relieved. Changing the duration or amplitude of the APW dramatically affected the facilitation of N-type channels but had little effect on the facilitation of P/Q-type channels. Since the ratio of N-type to P/Q-type Ca²⁺ channels varies widely between synapses, differential facilitation may contribute to the fine tuning of synaptic transmission, thereby increasing the computational repertoire of neurons.     

Unique properties of R-type calcium currents in neocortical and neostriatal neurons

Whole cell recordings from acutely dissociated neocortical pyramidal neurons and striatal medium spiny neurons exhibited a calcium-channel current resistant to known blockers of L-, N-, and P/Q-type Ca(2+) channels. These R-type currents were characterized as high-voltage-activated (HVA) by their rapid deactivation kinetics, half-activation and half-inactivation voltages, and sensitivity to depolarized holding potentials. In both cell types, the R-type current activated at potentials relatively negative to other HVA currents in the same cell type and inactivated rapidly compared with the other HVA currents. The main difference between cell types was that R-type currents in neocortical pyramidal neurons inactivated at more negative potentials than R-type currents in medium spiny neurons. Ni(2+) sensitivity was not diagnostic for R-type currents in either cell type. Single-cell RT-PCR revealed that both cell types expressed the alpha1E mRNA, consistent with this subunit being associated with the R-type current.     

Mitral cell presynaptic Ca(2+) influx and synaptic transmission in frog amygdala

Dextran-conjugated Ca(2+) indicators were injected into the accessory olfactory bulb of frogs in vivo to selectively fill presynaptic terminals of mitral cells at their termination in the ipsilateral amygdala. After one to three days of uptake and transport, the forebrain hemisphere anterior to the tectum was removed and maintained in vitro for simultaneous electrophysiological and optical measurements. Ca(2+) influx into these terminals was compared to synaptic transmission between mitral cells and amygdala neurons under conditions of reduced Ca(2+) influx resulting from reduced extracellular [Ca(2+)], blockade of N- and P/Q-type channels, and application of the cholinergic agonist carbachol. Reducing extracellular [Ca(2+)] had a non-linear effect on release; release was proportional to Ca(2+) influx raised to the power of approximately 3.6, as observed at numerous other synapses. The N-type Ca(2+) channel blocker, omega-conotoxin-GVIA (1 microM), blocked 77% of Ca(2+) influx and 88% of the postsynaptic field potential. The P/Q-type Ca(2+) channel blocker, omega-agatoxin-IVA (200 nM), blocked 19% of Ca(2+) influx and 25% of the postsynaptic field, while the two toxins combined to block 92% of Ca(2+) influx and 97% of the postsynaptic field. The relationship between toxin blockade of Ca(2+) influx and synaptic transmission was therefore only slightly non-linear; release was proportional to Ca(2+) influx raised to the power approximately 1.4. Carbachol (100 microM) acting via muscarinic receptors had no effect on the afferent volley, but rapidly and reversibly reduced Ca(2+) influx through both N- and P/Q-type channels by 51% and postsynaptic responses by 78%, i.e. release was proportional to Ca(2+) raised to the power approximately 2.5.The weak dependence of release on changes in Ca(2+) when channel toxins block channels suggests little overlap between Ca(2+) microdomains from channels supporting release or substantial segregation of channel subtypes between terminals. The proportionately greater reduction of transmission by muscarinic receptors compared to Ca(2+) channel toxins suggests that they directly affect the release machinery in addition to reducing Ca(2+) influx.     

Altered regulation of calcium channels and exocytosis in single human pheochromocytoma cells

We established primary cultures of human pheochromocytoma chromaffin cells. We then tried to find what mechanism of their secretory apparatus could be altered to produce the massive release of catecholamines into the circulation and the subsequent hypertensive crisis observed in patients suffering this type of tumor. Their whole-cell Ca2+ channel currents could be pharmacologically separated into components similar to those found in normal human adrenal chromaffin cells: 20% L-type, 30% N-type, and 50% P/Q-type Ca2+ channels. However, modulation of the channels by exogenous or endogenous ATP and opioids, via a G-protein membrane-delimited pathway, was deeply altered; some cells having no modulation or very little modulation alternated with others having normal modulation. This may be the cause of the uncontrolled secretory response, measured amperometrically at the single-cell level. Some cells secreted for long time periods and were insensitive to nifedipine (L-type channel blocker) or to omega-conotoxin MVIIC (N/P/Q-type channel blocker), while others were highly sensitive to nifedipine and partially sensitive to omega-conotoxin MVIIC. Alteration of the autocrine/paracrine modulation of Ca2+ channels may lead to indiscriminate Ca2+ entry and exacerbate catecholamine release responses in human pheochromocytoma cells.     

Characterization of voltage-gated ionic channels in cholinergic amacrine cells in the mouse retina

Recent studies have shown that cholinergic amacrine cells possess unique membrane properties. However, voltage-gated ionic channels in cholinergic amacrine cells have not been characterized systematically. In this study, using electrophysiological and immunohistochemical techniques, we examined voltage-gated ionic channels in a transgenic mouse line the cholinergic amacrine cells of which were selectively labeled with green fluorescent protein (GFP). Voltage-gated K(+) currents contained a 4-aminopyridine-sensitive current (A current) and a tetraethylammonium-sensitive current (delayed rectifier K(+) current). Voltage-gated Ca(2+) currents contained a omega-conotoxin GVIA-sensitive component (N-type) and a omega-Aga IVA-sensitive component (P/Q-type). Tetrodotoxin-sensitive Na(+) currents and dihydropyridine-sensitive Ca(2+) currents (L-type) were not observed. Immunoreactivity for the Na channel subunit (Pan Nav), the K channel subunits (the A-current subunits [Kv. 3.3 and Kv 3.4]) and the Ca channel subunits (alpha1(A) [P/Q-type], alpha1(B) [N-type] and alpha1(C) [L-type]) was detected in the membrane fraction of the mouse retina by Western blot analysis. Immunoreactivity for the Kv. 3.3, Kv 3.4, alpha1(A) [P/Q-type], and alpha1(B) [N-type] was colocalized with the GFP signals. Immunoreactivity for alpha1(C) [L-type] was not colocalized with the GFP signals. Immunoreactivity for Pan Nav did not exist on the membrane surface of the GFP-positive cells. Our findings indicate that signal propagation in cholinergic amacrine cells is mediated by a combination of two types of voltage-gated K(+) currents (the A current and the delayed rectifier K(+) current) and two types of voltage-gated Ca(2+) currents (the P/Q-type and the N-type) in the mouse retina.     

Human adrenal chromaffin cell calcium channels: drastic current facilitation in cell clusters, but not in isolated cells

 Human adrenal medullary chromaffin cells were prepared and cultured from a cystic tumoral adrenal gland whose medullary tissue was unaffected. Adrenaline-containing and noradrenaline-containing cells were identified using a confocal fluorescence microscope and antibodies against dopamine beta-hydroxylase (DBH) and phenylethanolamine N-methyltransferase (PNMT). Current/voltage (I/V) curves performed with the voltage-clamped cells bathed in 10 mM Ba²⁺ (holding potential, V _h=–80 mV) revealed the presence of only high-threshold voltage-dependent Ca²⁺ channels; T-type Ca²⁺ channels were not seen. By using supramaximal concentrations of selective Ca²⁺ channel blockers, the whole-cell I _Ba could be fractionated into various subcomponents. Thus, I _Ba had a 25% fraction sensitive to 1 μM nifedipine (L-type channels), 21% sensitive to 1 μM ω-conotoxin GVIA (N-type channels), and 60% sensitive to 2 μM ω-agatoxin IVA (P/Q-type channels). The activation of I _Ba was considerably slowed down, and the peak current was inhibited upon superfusion with 10 μM ATP. The slow activation and peak current blockade were reversed by strong depolarizing pre-pulses to +100 mV (facilitation). A drastic facilitation of I _Ba was also observed in voltage-clamped human chromaffin cell surrounded by other unclamped cells; in contrast, in voltage-clamped cells not immersed in a cell cluster, facilitation was scarce. So, facilitation of Ca²⁺ channels in a voltage-clamped cell seems to depend upon the exocytotic activity of neighbouring unclamped cells, which is markedly increased by Ba²⁺. It is concluded that human adrenal chromaffin cells mostly express P/Q-types of voltage-dependent Ca²⁺ channels (60%). L-Type channels and N-type channels are also expressed, but to a considerably minor extent (around 20% each). This dominance of P/Q-type channels in human chromaffin cells clearly contrasts with the relative proportion of each channel type expressed by chromaffin cells of five other animal species studied previously, where the P/Q-type channels accounted for 5–50%. The results also provide strong support for the hypothesis that Ca²⁺ channels of human chromaffin cells are regulated in an autocrine/paracrine fashion by materials co-secreted with the catecholamines, i.e. ATP and opiates. Received: 1 May 1998 / Received after revision and accepted: 21 May 1998     

Expression of voltage-dependent calcium channel subunits in the rat retina

The expression patterns of different Ca(2+) channel alpha(1) subunits (alpha(1A-E)) were immunohistochemically studied in the rat retina. Intense immunoreactivity (IR) for alpha(1A) (P/Q-type) and alpha(1B) (N-type) Ca(2+) channels was observed in both the outer and inner plexiform layers (OPL and IPL). In addition, alpha(1B)-IR was found in the outer and inner nuclear layers. Staining for alpha(1E) (R-type) was diffusely distributed in all three nuclear layers and in the IPL. The alpha(1C) and alpha(1D), two L-type Ca(2+) channel subunits, exhibited distinct expression patterns, with alpha(1C) being almost exclusively expressed on bipolar cells, and alpha(1D) mainly on photoreceptor cell bodies and in the OPL. Staining for alpha(1D) was also observed on Müller cells. The differential expression pattern of the alpha(1) subunits suggests that these Ca(2+) channel subtypes may be associated with different retinal functions.     

A tctex1-Ca2+ channel complex for selective surface expression of Ca2+ channels in neurons

Voltage-gated Ca(2+) channels (VGCCs) are important in regulating a variety of cellular functions in neurons. It remains poorly understood how VGCCs with different functions are sorted within neurons. Here we show that the t-complex testis-expressed 1 (tctex1) protein, a light-chain subunit of the dynein motor complex, interacts directly and selectively with N- and P/Q-type Ca(2+) channels, but not L-type Ca(2+) channels. The interaction is insensitive to Ca(2+). Overexpression in hippocampal neurons of a channel fragment containing the binding domain for tctex1 significantly decreases the surface expression of endogenous N- and P/Q-type Ca(2+) channels but not L-type Ca(2+) channels, as determined by immunostaining. Furthermore, disruption of the tctex1-Ca(2+) channel interaction significantly reduces the Ca(2+) current density in hippocampal neurons. These results underscore the importance of the specific tctex1-channel interaction in determining sorting and trafficking of neuronal Ca(2+) channels with different functionalities.     

Calcium channels involved in synaptic transmission from reticulospinal axons in lamprey

The pharmacology of calcium channels involved in glutamatergic synaptic transmission from reticulospinal axons in the lamprey spinal cord was analyzed with specific agonists and antagonists of different high-voltage activated calcium channels. The N-type calcium channel blocker omega-conotoxin GVIA (omega-CgTx) induced a large decrease of the amplitude of reticulospinal-evoked excitatory postsynaptic potentials (EPSPs). The P/Q-type calcium channel blocker omega-agatoxin IVA (omega-Aga) also reduced the amplitude of the reticulospinal EPSPs, but to a lesser extent than omega-CgTx. The dihydropyridine agonist Bay K and antagonist nimodipine had no effect on the amplitude of the reticulospinal EPSP. Combined application of omega-CgTx and omega-Aga strongly decreased the amplitude the EPSPs but was never able to completely block them, indicating that calcium channels insensitive to these toxins (R-type) are also involved in synaptic transmission from reticulospinal axons. We have previously shown that the group III metabotropic glutamate receptor agonist L(+)-2-amino-4-phosphonobutyric acid (L-AP4) mediates presynaptic inhibition at the reticulospinal synapse. To test if this presynaptic effect is mediated through inhibition of calcium influx, the effect of L-AP4 on reticulospinal transmission was tested before and after blockade of N-type channels, which contribute predominantly to transmitter release at this synapse. Blocking the N-type channels with omega-CgTx did not prevent inhibition of reticulospinal synaptic transmission by L-AP4. In addition, L-AP4 had no affect on the calcium current recorded in the somata of reticulospinal neurons or on the calcium component of action potentials in reticulospinal axons. These results show that synaptic transmission from reticulospinal axons in the lamprey is mediated by calcium influx through N-, P/Q- and R-type channels, with N-type channels playing the major role. Furthermore, presynaptic inhibition of reticulospinal transmission by L-AP4 appears not to be mediated through inhibition of presynaptic calcium channels.     

Calcium channels coupled to depolarization-evoked glutamate release in the myenteric plexus of guinea-pig ileum

Glutamate is the major excitatory neurotransmitter in the CNS. The recent characterization of glutamate as a neurotransmitter in the enteric nervous system opened a new line of investigation concerning the role of glutamate in that system. The present study aimed to further characterize the enteric glutamate release and the calcium channels coupled to it. For this study the myenteric plexus-longitudinal muscle of guinea-pig ileum was stimulated with potassium chloride or with electrical pulses. The released glutamate was detected by spectrofluorimetry. Laser scanning confocal microscopy was used for analysis of immunolabeled enteric tissue for co-localization studies of calcium channels (N- and P/Q-type) and glutamate transporters (EAAC1).Here we report the effects of known Ca(2+)-channel blockers on glutamate release evoked by KCl-depolarization or electrical stimulation in the myenteric plexus. We find that N-type Ca(2+) channels control a major portion of evoked glutamate release from this system, with a very small contribution from L-type Ca(2+) channels. Moreover, alpha(1A)-like (P-type Ca(2+) channel) and alpha(1B)-like (N-type Ca(2+ )channel) immunoreactivity co-localized with glutamate transporters in the myenteric plexus. In addition, KCl-evoked or electrically stimulated glutamate release was sensitive to omega-agatoxin IVA, in a frequency-dependent manner, suggesting that P-type channels are also coupled to the release of glutamate. We, thus, conclude that both N-type and P-type Ca(2+) channels control most of the evoked glutamate release from the enteric nervous system, as also occurs in some parts of the CNS.     

Pharmacological and biophysical characterization of voltage-gated calcium currents in the endopiriform nucleus of the guinea pig

The endopiriform nucleus (EPN) is a well-defined structure that is located deeply in the piriform region at the border with the striatum and is characterized by dense intrinsic connections and prominent projections to piriform and limbic cortices. The EPN has been proposed to promote synchronization of large populations of neurons in the olfactory cortices via the activation of transient depolarizations possibly mediated by Ca(2+) spikes. It is known that principal cells in the EPN express both a low- and high-voltage-activated (HVA) Ca(2+) currents. We further characterized HVA conductances possibly related to Ca(2+)-spike generation in the EPN with a whole cell, patch-clamp study on neurons acutely dissociated from the EPN of the guinea pig. To study HVA currents in isolation, experiments were performed from a holding potential of -60 mV, using Ba(2+) as the permeant ion. Total Ba(2+) currents (I(Ba)) evoked by depolarizing square pulses peaked at 0/+10 mV and were completely abolished by 200 microM Cd(2+). The pharmacology of HVA I(Ba)s was analyzed by applying saturating concentrations of specific Ca(2+)-channel blockers. The L-type blocker nifedipine (10 microM; n = 11), the N-type-channel blocker omega-conotoxin GVIA (0.5 microM; n = 24), and the P/Q-type blocker omega-conotoxin MVIIC (1 microM; n = 16) abolished fractions of total I(Ba)s equal on average to 24.7 +/- 5.4%, 27.1 +/- 3.4%, and 22.2 +/- 2.4%, respectively (mean +/- SE). The simultaneous application of the three blockers reduced I(Ba) by 68.5 +/- 6.6% (n = 10). Nifedipine-sensitive currents and most N- and P/Q-type currents were slowly decaying, the average fractional persistence after 300 ms of steady depolarization being 0.77 +/- 0.02, 0.60 +/- 0.06, and 0.68 +/- 0.04, respectively. The residual, blocker-resistant (R-type) currents were consistently faster inactivating, with an average fractional persistence after 300 ms of 0.30 +/- 0.08. Fast-decaying R-type currents also displayed a more negative threshold of activation (by about 10 mV) than non-R-type HVA currents. These results demonstrate that EPN neurons express multiple pharmacological components of the HVA Ca(2+) currents and point to the existence of an R-type current with specific functional properties including fast inactivation kinetics and intermediate threshold of activation.     

R-type voltage-gated Ca(2+) channel interacts with synaptic proteins and recruits synaptotagmin to the plasma membrane of Xenopus oocytes

It is well established that syntaxin 1A, synaptosomal-associated protein of 25 kDa (SNAP-25) and synaptotagmin either alone or in combination, modulate the kinetic properties of voltage-gated Ca(2+) channels Ca(v)1.2 (Lc-channel) Ca(v)2.2 (N-type) and Ca(v)2.1 (P/Q-type). The interaction interface was found to reside at the cytosolic II-III domain of the alpha1 subunit of the channels. In this study, we demonstrated a functional coupling of human neuronal Ca(v)2.3 (R-type channel) with syntaxin 1A, SNAP-25 and synaptotagmin in BAPTA injected Xenopus oocytes. The kinetic properties of Ca(v)2.3 assembled with syntaxin 1A, SNAP-25 or synaptotagmin individually differed from Ca(v)2.3 associated with binary complexes syntaxin 1A/SNAP-25, syntaxin 1A/synaptotagmin or SNAP-25/synaptotagmin. Co-expression of Ca(v)2.3 with syntaxin 1A, SNAP-25 and synaptotagmin together, produced a channel with distinctive kinetic properties analogous to excitosome multiprotein complex generated by Ca(v)1.2 and Ca(v)2.2. Exchanging the current-carrying ions altered the kinetics of channel/synaptic proteins interaction, indicating a tight crosstalk formed between the permeation pathway of Ca(v)2.3 and the fusion apparatus during membrane depolarization. This putative coupling could predict how the release site might be organized to allow a rapid communication between the channel and the release machinery. In vivo confocal imaging of oocytes revealed GFP-synaptotagmin at the plasma membrane when the channel was present, as opposed to random distribution in its absence, consistent with Ca(2+)-independent molecular link of synaptotagmin and the channel. Synaptotagmin was detected at the membrane also in oocytes co-expressing the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). Both imaging studies and protein-protein interactions in Xenopus oocytes show that channel linkage to synaptotagmin precedes Ca(2+) influx. Altogether, the R-type channel appears to associate with synaptic proteins to generate a multiprotein excitosome complex prior to Ca(2+)-entry. We propose that the distinct kinetics of the Ca(2+)-channel acquired by the close association with the vesicle and the t-SNAREs within the excitosome complex may be essential for depolarization evoked transmitter release.     

Low-threshold L-type calcium channels in rat dopamine neurons

Ca(2+) channel subtypes expressed by dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) were studied using whole cell patch-clamp recordings and blockers selective for different channel types (L, N, and P/Q). Nimodipine (Nim, 2 microM), omega-conotoxin GVIA (Ctx, 1 microM), or omega-agatoxin IVA (Atx, 50 nM) blocked 27, 36, and 37% of peak whole cell Ca(2+) channel current, respectively, indicating the presence of L-, N-, and P-type channels. Nim blocked approximately twice as much Ca(2+) channel current near activation threshold compared with Ctx or Atx, suggesting that small depolarizations preferentially opened L-type versus N- or P-type Ca(2+) channels. N- and L-channels in DA neurons opened over a significantly more negative voltage range than those in rat dorsal root ganglion cells, recorded from using identical conditions. These data provide an explanation as to why Ca(2+)-dependent spontaneous oscillatory potentials and rhythmic firing in DA neurons are blocked by L-channel but not N-channel antagonists and suggest that pharmacologically similar Ca(2+) channels may exhibit different thresholds for activation in different types of neurons.