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.     

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.     

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.     

Expression of recombinant calcium channels support secretion in a mouse pheochromocytoma cell line

We have characterized a recently established mouse pheochromocytoma cell line (MPC 9/3L) as a useful model for studying neurotransmitter release and neuroendocrine secretion. MPC 9/3L cells express many of the proteins involved in Ca2+-dependent neurotransmitter release but do not express functional endogenous Ca2+-influx pathways. When transfected with recombinant N-type Ca2+ channel subunits alpha1B,beta2a,alpha2delta (Cav2.2), the cells expressed robust Ca2+ currents that were blocked by omega-conotoxin GVIA. Activation of N-type Ca2+ currents caused rapid increases in membrane capacitance of the MPC 9/3L cells, indicating that the Ca2+ influx was linked to exocytosis of vesicles similar to that reported in chromaffin or PC12 cells. Synaptic protein interaction (synprint) sites, like those found on N-type Ca2+ channels, are thought to link voltage-dependent Ca2+ channels to SNARE proteins involved in synaptic transmission. Interestingly, MPC 9/3L cells transfected with either LC-type (alpha1C, beta2a, alpha2delta, Cav1.2) or T-type (alpha1G, beta2a, alpha2delta, Cav3.1) Ca2+ channel subunits, which do not express synprint sites, expressed appropriate Ca2+ currents that supported rapid exocytosis. Thus MPC 9/3L cells provide a unique model for the study of exocytosis in cells expressing specific Ca2+ channels of defined subunit composition without complicating contributions from endogenous channels. This model may help to distinguish the roles that different Ca2+ channels play in Ca2+-dependent secretion.     

T-Type calcium channel alpha1G and alpha1H subunits in human retinoblastoma cells and their loss after differentiation

Human retinoblastoma cells are multipotent retinal precursor cells capable of differentiating into photoreceptors, neurons, and glia. The current-voltage relation of the undifferentiated cells is dominated by a transient inward current that disappears shortly after differentiation. In 20 mM Ba(2+)-containing bath solutions, the current has an activation midpoint near -25 mV and appears to be fully inactivated at -20 mV. Sr(2+) and Ca(2+) are preferred charge carriers relative to Ba(2+), and the current vanishes in the absence of these divalent cations. Cd(2+) blocks the current with an IC(50) of 160 microM, and Ni(2+) blocks in a biphasic manner with IC(50)s of 22 and 352 microM. The current is unaffected when sodium is replaced with other monovalent cations, and it is insensitive to nifedipine, omega-conotoxin GVIA, omega-agatoxin IVA, and omega-conotoxin MVIIC. RT-PCR revealed the presence of alpha 1G and alpha 1H mRNA in undifferentiated cells, but following differentiation, a striking reduction of both alpha 1G and alpha 1H mRNA was found, and this was paralleled by the loss of T-type Ca channel currents. alpha 1I subunit mRNA levels were low in undifferentiated and differentiated cells. These results suggest that T-type Ca channels could play a role in undifferentiated retinoblastoma cell physiology since alpha 1G and alpha 1H Ca channel subunit expression is reduced in cells that have differentiated and exited the cell cycle.     

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.     

Reconstitution of neurotransmission by determining communication between differentiated PC12 pheochromocytoma and HEL 92.1.7 erythroleukemia cells

Neurotransmitter release was monitored using fura-2-loaded HEL 92.1.7 cells dispersed among differentiated PC12 cells (loaded with another Ca2+ indicator fluo-3) and immobilised using transparent polycarbonate membrane filters with uniform pore size. Depolarisation with K+ caused a rapid rise in Ca2+ concentration in the PC12 cells, followed by a delayed secondary Ca2+ response in simultaneously monitored nearby HEL cells. There was a lag period of about 20 s between the responses of the two cell types. Voltage-gated Ca2+ channels in PC12 cells were inhibited by the P/Q-type (omega-conotoxin MVIIC, omega-agatoxin IVA), N-type (omega-conotoxin GVIA) and L-type channel blockers (nifedipine) as determined using fura-2 or whole-cell patch-clamp recordings. The communication between the cell types on the other hand was sensitive to P/Q- and N-type but not to L-type channel blockers. This suggests that, as in neurons, P/Q- and N-type Ca2+ channels mediate the release of neurotransmitters acting on HEL cells. Theoretically, the procedure employed should be sensitive enough to detect single exocytotic events. Our results demonstrate that a random distribution between effector and target cells is sufficient to allow communication between cells in a manner similar to extrasynaptic transmission.     

Presynaptic GABA(B) receptors regulate retinohypothalamic tract synaptic transmission by inhibiting voltage-gated Ca2+ channels

Presynaptic GABA(B) receptor activation inhibits glutamate release from retinohypothalamic tract (RHT) terminals in the suprachiasmatic nucleus (SCN). Voltage-clamp whole cell recordings from rat SCN neurons and optical recordings of Ca2+-sensitive fluorescent probes within RHT terminals were used to examine GABA(B)-receptor modulation of RHT transmission. Baclofen inhibited evoked excitatory postsynaptic currents (EPSCs) in a concentration-dependent manner equally during the day and night. Blockers of N-, P/Q-, T-, and R-type voltage-dependent Ca2+ channels, but not L-type, reduced the EPSC amplitude by 66, 36, 32, and 18% of control, respectively. Joint application of multiple Ca2+ channel blockers inhibited the EPSCs less than that predicted, consistent with a model in which multiple Ca2+ channels overlap in the regulation of transmitter release. Presynaptic inhibition of EPSCs by baclofen was occluded by omega-conotoxin GVIA (< or = 72%), mibefradil (< or = 52%), and omega-agatoxin TK (< or = 15%), but not by SNX-482 or nimodipine. Baclofen reduced both evoked presynaptic Ca2+ influx and resting Ca2+ concentration in RHT terminals. Tertiapin did not alter the evoked EPSC and baclofen-induced inhibition, indicating that baclofen does not inhibit glutamate release by activation of Kir3 channels. Neither Ba2+ nor high extracellular K+ modified the baclofen-induced inhibition. 4-Aminopyridine (4-AP) significantly increased the EPSC amplitude and the charge transfer, and dramatically reduced the baclofen effect. These data indicate that baclofen inhibits glutamate release from RHT terminals by blocking N-, T-, and P/Q-type Ca2+ channels, and possibly by activation of 4-AP-sensitive K+ channels, but not by inhibition of R- and L-type Ca2+ channels or by Kir3 channel activation.     

Effects of the dihydropyridine receptor subunits γ and α2δ on the kinetics of heterologously expressed L-type Ca2+ channels

Ba2+ currents through L-type Ca2+ channels were measured in tsA201 cells transiently transfected with expression vectors encoding the dihydropyridine (DHP) receptor subunits alpha1C, beta1a-GFP, alpha2delta and gamma. The subunit effect on channel function was studied by omitting either alpha2delta or gamma from the transfection mixture and analyzing the voltage dependence and kinetics of activation, inactivation and recovery from inactivation. Activation could be described by a single exponential function while the time course of inactivation of the Ba2+ current followed a double exponential function. Progressively longer depolarization led to increasingly slower recovery, indicating the successive occupancy of several inactive states. Activation parameters remained largely unaffected in y-deficient cells whereas the voltage dependence of inactivation was shifted by 16 mV to more positive potentials and the larger one of the two inactivation time constants was increased by one-third. On the other hand, alpha2delta-deficient cells showed decreased current density and slowed activation and inactivation. Recovery from inactivation was significantly slowed by gamma coexpression. This and the effect of the gamma subunit on steady-state inactivation were independent of the presence of alpha2delta. We conclude that y stabilizes L-type Ca2+ channel inactivation in a way similar to certain Ca(2+)-antagonistic drugs. Alpha2delta is not needed for this effect.     

The gating and conductance properties of Cav3.2 low-voltage-activated T-type calcium channels

Calcium channels are essential for excitation-contraction coupling and pacemaker potentials in cardiac muscle cells. Whereas L-type Ca(2+) channels have been extensively studied, T-type channels have been poorly characterized in cardiac myocytes. We describe here the functional properties of recombinant Ca(V)3.2 T-type Ca(2+) channels expressed in mammalian cell lines. The T-type Ca(2+) current showed a rapid activation and an inactivation phase in response to depolarization, and it displayed a window current over the voltage range from -60 to -40 mV in 1 to 10 mM external Ca(2+). Barium (Ba(2+)) and strontium (Sr(2+)) permeate the channel with similar activation kinetics. On the other hand, monovalent cations, Li(+) and Na(+), permeate the T-type Ca(2+) channel more easily than the L-type Ca(2+) channel. The permeability order of the Ca(V)3.2 T-type Ca(2+) channel among monovalent and divalent cations was determined as Ba(2+)>Mn(2+)>Ca(2+)>Sr(2+)>Li(+1)>Na(+) with the permeability order of 1.39:1.25:1.00:0.95:0.55:0.29. The ionic conductance sequence for cations relative to calcium was Sr(2+)>Ba(2+)>Ca(2+)>Li(+1)>Mn(2+)>Na(+) with the conductance ratio of 1.39:1.21:1.00:0.40:0.23:0.11. The permeation profile of manganese (Mn(2+)) is complex. Mn(2+) permeates the Ca(2+) channel with a permeability similar to Ca(2+) or Ba(2+), but with a much smaller current density, resulting in a much smaller conductance. The properties relating to progression and recovery from inactivation in the Ca(V)3.2 channel are substantially identical with either Ca(2+) or Ba(2+) as the charge carrier.     

Characterization of voltage-gated calcium currents in freshly isolated smooth muscle cells from rat tail main artery.

The aim of the present study was to characterize voltage-gated Ca2+ currents in smooth muscle cells freshly isolated from rat tail main artery in the presence of 5 mmol L(-1) external Ca2+. Calcium currents were identified on the basis of their voltage dependencies and sensitivity to nifedipine, Ni2+ and cinnarizine. In the majority of the cells studied, T- and L-type currents were observed, while the remaining cells showed predominantly L-type currents. In the latter group of cells, holding potential change from -50 to either -70 or -90 mV increased the corresponding inward current amplitude while its voltage activation threshold remained unchanged. The steady state inactivation of L-type Ca2+ channels showed half-maximal inactivation at -38 mV. A Ca2+-dependent inactivation was also evident. Nifedipine (3 micromol L(-1)) blocked L-type but not T-type Ca2+ currents. Ni2+ (50 micromol L(-1)) as well as cinnarizine (1 micromol L(-1)) suppressed the nifedipine-resistant, T-type component of the currents. At higher concentrations, both Ni2+ (0.3-1 mmol L(-1)) and cinnarizine (10 micromol L(-1)) blocked the net inward current. Replacement of Ca2+ with 10 mmol L(-)1 Ba2+ significantly increased the amplitude of L-type Ca2+ currents. These results demonstrate that smooth muscle cells freshly isolated from rat tail main artery may be divided into two populations, one expressing both L- and T-type and the other only L-type Ca2+ channels. Furthermore, this report shows that in arterial smooth muscle cells cinnarizine potently inhibited T-type currents at low concentrations (1 micromol L(-1)) but also blocked L-type Ca2+ currents at higher concentrations (10 micromol L(-1)).     

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.     

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.     

Voltage-dependent ion channel currents in putative neuroendocrine cells dissociated from the ventral prostate of rat

Prostate neuroendocrine (NE) cells play important roles in the growth and differentiation of the prostate. Following enzymatic digestion of rat ventral prostate, the whole-cell patch-clamp technique was applied to dark, round cells that exhibited chromogranin-A immunoreactivity, a representative marker of NE cells. Under zero current-clamp conditions, putative NE cells showed hyperpolarized resting membrane potentials of some -70 mV, and spontaneous action potentials were induced by an increase in external [K+] or by the injection of current. Using a CsCl pipette solution, step-like depolarization activated high-voltage-activated Ca2+ current (HVA I(Ca)) and tetrodotoxin-resistant voltage-activated Na+ current. The HVA I(Ca) was blocked by nifedipine and omega-conotoxin GVIA, L-type and N-type Ca2+ channel blockers, respectively. Using a KCl pipette solution, the transient outward K+ current (I(to)), Ca2+ -activated K+ currents (I(K,Ca)), the non-inactivating outward current and an inwardly rectifying K+ current (I(Kir)) were identified. I(K,Ca) was suppressed by charybdotoxin (50 nM), iberiotoxin (10 nM) or clotrimazol (1 microM), but not by apamine (100 nM). I(to) was inhibited by 4-aminopyridine (5 mM). I(Kir) was identified as a Ba2+ -sensitive inwardly rectifying current in the presence of a high-K+ bath solution. The voltage- and Ca2+ -activated ion channels could play significant roles in the regulation of neurohormonal secretion in the prostate.     

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.     

Identification of T-type alpha1H Ca2+ channels (Ca(v)3.2) in major pelvic ganglion neurons

Among autonomic neurons, sympathetic neurons of the major pelvic ganglia (MPG) are unique by expressing low-voltage-activated T-type Ca2+ channels. To date, the T-type Ca2+ channels have been poorly characterized, although they are believed to be potentially important for functions of the MPG neurons. In the present study, thus we investigated characteristics and molecular identity of the T-type Ca2+ channels using patch-clamp and RT-PCR techniques. When the external solution contained 10 mM Ca2+ as a charge carrier, T-type Ca2+ currents were first activated at -50 mV and peaked around -20 mV. Besides the low-voltage activation, T-type Ca2+ currents displayed typical characteristics including transient activation/inactivation and voltage-dependent slow deactivation. Overlap of the activation and inactivation curves generated a prominent window current around resting membrane potentials. Replacement of the external Ca2+ with 10 mM Ba2+ did not affect the amplitudes of T-type Ca2+ currents. Mibefradil, a known T-type Ca2+ channel antagonist, depressed T-type Ca2+ currents in a concentration-dependent manner (IC50 = 3 microM). Application of Ni2+ also produced a concentration-dependent blockade of T-type Ca2+ currents with an IC50 of 10 microM. The high sensitivity to Ni2+ implicates alpha1H in generating the T-type Ca2+ currents in MPG neurons. RT-PCR experiments showed that MPG neurons predominantly express mRNAs encoding splicing variants of alpha1H (called pelvic Ta and Tb, short and long forms of alpha1H, respectively). Finally, we tested whether the low-threshold spikes could be generated in sympathetic MPG neurons expressing T-type Ca2+ channels. When hyperpolarizing currents were injected under a current-clamp mode, sympathetic neurons produced postanodal rebound spikes, while parasympathetic neurons were silent. The number of the rebound spikes was reduced by 10 microM Ni2+ that blocked 50% of T-type Ca2+ currents and had a little effect on HVA Ca2+ currents in sympathetic MPG neurons. Furthermore, generation of the rebound spikes was completely prevented by 100 microM Ni2+ that blocked most of the T-type Ca2+ currents. In conclusions, T-type Ca2+ currents in MPG neurons mainly arise from alpha1H among the three isoforms (alpha1G, alpha1H, and alpha1I) and may contribute to generation of low-threshold spikes in sympathetic MPG neurons.     

Modulation of recombinant T-type Ca2+ channels by hypoxia and glutathione

T-type Ca2+ channels are expressed in a wide variety of central and peripheral neurons and play an important role in neuronal firing and rhythmicity. Here we examined the effects of hypoxia on the recently cloned T-type Ca2+ channel alpha1G, alpha1H and alpha1I subunits, stably expressed in HEK 293 cells. In cells expressing the human alpha1H or the rat alpha1I subunit, Ca2+ channel currents were inhibited reversibly by hypoxia (PO2<110 mm Hg). The degree of inhibition was more marked in cells expressing the a1H subunit. This hypoxic inhibition was not voltage dependent. In cells expressing the rat alpha1G subunit, hypoxia caused no detectable reduction in Ca2+ channel activity. Regardless of the channel type examined, hypoxia was without effect on the kinetic properties of the Ca2+ current (activation, inactivation and deactivation) or on steady-state inactivation. Ca2+ current through the alpha1H subunit was enhanced by the reducing agent reduced glutathione (GSH; 2 mM) and inhibited by oxidised glutathione (GSSG; 2 mM). In contrast, Ca2+ current through the alpha1G subunit was unaffected by GSH. In alpha1H cells, neither GSH nor GSSG had any effect on the ability of hypoxia to reduce Ca2+ current amplitudes. Thus, different members of the T-type Ca2+ channel family are differently regulated by hypoxia and redox agents. Hypoxic regulation of the alpha1H subunit appears to be independent of changes in levels of the intracellular redox couple GSSG:GSH.     

Age-related changes in the subtypes of voltage-dependent calcium channels in rat brain cortical synapses

Age-related changes in the relative contribution of voltage-dependent calcium channel (VDCC) subtypes to depolarization-induced Ca(2+) influx and in the density of VDCC subtypes in cortical synapses were investigated using synaptosomes and their membrane preparations from brain cortices of Wistar rats. The relative contribution of VDCC subtypes to Ca(2+) influx was determined by measuring the inhibition of depolarization-induced Ca(2+) influx with four VDCC subtype-specific peptide blockers. In adult rat synaptosomes, L-, N-, P- and Q-type channels accounted for 24, 32, 27 and 12% of the total Ca(2+) influx, respectively. Brain aging significantly reduced the relative contributions of N- and P-type channels and increased the contribution of the channels resistant to the four blockers used. The densities of VDCC subtypes, determined by binding experiments using radiolabeled PN200 -110, omega-conotoxin GVIA and omega-conotoxin MVIIC, were found to be significantly decreased in aged synaptic plasma membranes. On the contrary, the dissociation constants of the blockers were not changed except for PN200-110-sensitive L-type channels. These results suggest that aging alters the relative contributions of each VDCC subtype to depolarization-induced Ca(2+) influx and decreases the number of VDCCs in rat brain cortical synapses. These changes in VDCCs may lead to age-related hypofunction of synaptic neurotransmission in brain cortices.     

Age-related functional changes of high-voltage-activated calcium channels in different neuronal subtypes of mouse striatum

By means of whole-cell patch-clamp recordings, we characterized the developmental profile of high-voltage-activated (HVA) calcium (Ca(2+)) channel subtypes in distinct neuronal populations of mouse striatum. Acutely dissociated medium spiny neurons (MSNs) and cholinergic interneurons (ChIs) were recorded from mice at five developmental stages: postnatal-days (PD) 14, 23, 40, 150 and 270. During ageing, total HVA Ca(2+) current recorded from both MSNs and ChIs was unchanged. However, the pharmacological analysis of the differential contribution of HVA Ca(2+) channel subtypes showed a significant rearrangement of each component. In both neuronal subtypes, a large fraction of the total HVA current recorded from PD14 mice was inhibited by the L-type HVA channel blocker nifedipine. This dihydropyridine-sensitive component accounted for nearly 50%, in MSNs, and 35%, in ChIs, of total current at PD14, but its contribution was down-regulated up to 20-25% at 9 months. Likewise, the N-type, omega-conotoxin GVIA-sensitive component decreased from 35% to 40% to about 25% in MSNs and 15% in ChIs. The P-type, omega-agatoxin-sensitive fraction did not show significant changes in both neuronal subtypes, whereas the Q-type, omega-conotoxin MVIIC-sensitive channels did show a significant up-regulation at 9 months. As compared with striatal neurons, we recorded pyramidal neurons dissociated from cortical layers IV-V and found no significant developmental change in the different components of HVA Ca(2+) currents. In conclusion, our data demonstrate a functional reconfiguration of HVA Ca(2+) channels in striatal but not cortical pyramidal neurons during mouse development. Such changes might have profound implications for physiological and pathophysiological processes of the striatum.