Bcl2 mitigates Ca2+ entry and mitochondrial Ca2+ overload through downregulation of L-type Ca2+ channels in PC12 cells

Altered calcium homeostasis and increased cytosolic calcium concentrations ([Ca(2+)](c)) are linked to neuronal apoptosis in epilepsy and in cerebral ischemia, respectively. Apoptotic programmed cell death is regulated by the antiapoptotic Bcl2 family of proteins. Here, we investigated the role of Bcl2 on calcium (Ca(2+)) homeostasis in PC12 cells, focusing on L-type voltage-dependent calcium channels (VDCC). Cytosolic Ca(2+) transients ([Ca(2+)](c)) and changes of mitochondrial Ca(2+) concentrations ([Ca(2+)](m)) were monitored using cytosolic and mitochondrially targeted aequorins of control PC12 cells and PC12 cells stably overexpressing Bcl2. We found that: (i) the [Ca(2+)](c) and [Ca(2+)](m) elevations elicited by K(+) pulses were markedly depressed in Bcl2 cells, with respect to control cells; (ii) such depression of [Ca(2+)](m) was not seen either in digitonin-permeabilized cells or in intact cells treated with ionomycin; (iii) the [Ca(2+)](c) transient depression seen in Bcl2 cells was reversed by shRNA transfection, as well as by the Bcl2 inhibitor HA14-1; (iv) the L-type Ca(2+) channel agonist Bay K 8644 enhanced K(+)-evoked [Ca(2+)](m) peak fourfold in Bcl2, and twofold in control cells; (v) in current-clamped cells the depolarization evoked by K(+) generated a more hyperpolarized voltage step in Bcl2, as compared to control cells. Taken together, our experiments suggest that the reduction of the [Ca(2+)](c) and [Ca(2+)](m) transients elicited by K(+), in PC12 cells overexpressing Bcl2, is related to the reduction of Ca(2+) entry through L-type Ca(2+) channels. This may be due to the fact that Bcl2 mitigates cell depolarization, thus diminishing the recruitment of L-type Ca(2+) channels, the subsequent Ca(2+) entry, and mitochondrial Ca(2+) overload.     

Properties of Na+ currents conducted by a skeletal muscle L-type Ca2+ channel pore mutant (SkEIIIK)

Four glutamate residues residing at corresponding positions within the four conserved membrane-spanning repeats of L-type Ca(2+) channels are important structural determinants for the passage of Ca(2+) across the selectivity filter. Mutation of the critical glutamate in Repeat III in the a 1S subunit of the skeletal L-type channel (Ca(v)1.1) to lysine virtually eliminates passage of Ca(2+) during step depolarizations. In this study, we examined the ability of this mutant Ca(v)1.1 channel (SkEIIIK) to conduct inward Na(+) current. When 150 mM Na(+) was present as the sole monovalent cation in the bath solution, dysgenic (Ca(v)1.1 null) myotubes expressing SkEIIIK displayed slowly-activating, non-inactivating, nifedipine-sensitive inward currents with a reversal potential (45.6 ± 2.5 mV) near that expected for Na(+). Ca(2+) block of SkEIIIK-mediated Na(+) current was revealed by the substantial enhancement of Na(+) current amplitude after reduction of Ca(2+) in the external recording solution from 10 mM to near physiological 1 mM. Inward SkEIIIK-mediated currents were potentiated by either ±Bay K 8644 (10 mM) or 200-ms depolarizing prepulses to +90 mV. In contrast, outward monovalent currents were reduced by ±Bay K 8644 and were unaffected by strong depolarization, indicating a preferential potentiation of inward Na(+) currents through the mutant Ca(v)1.1 channel. Taken together, our results show that SkEIIIK functions as a non-inactivating, junctionally-targeted Na(+) channel when Na(+) is the sole monvalent cation present and urge caution when interpreting the impact of mutations designed to ablate Ca(2+) permeability mediated by Ca(v) channels on physiological processes that extend beyond channel gating and permeability.     

Hyperpolarization, but not depolarization, increases intracellular Ca(2+) level in cultured chick myoblasts.

Ca(2+) influx appears to be important for triggering myoblast fusion. It remains, however, unclear how Ca(2+) influx rises prior to myoblast fusion. The present study examines a possible involvement of the voltage-dependent Ca(2+) influx pathways. Treatment with the L-type Ca(2+) channel blockers, diltiazem, and nifedipine did not alter cytosolic Ca(2+) levels. Depolarization with high K(+) solution and activation of Ca(2+) channel with Bay K 8644, and agonist of voltage dependent Ca(2+) channels, failed to elicit increases intracellular Ca(2+) level, indicating the absence of depolarization-operated mechanisms. In contrast, phloretin, an agonist of Ca(2+)-activated potassium (K(Ca)) channels, was able to hyperpolarize membrane potential and promoted Ca(2+) influx. These effects were completely abolished by treatment of charybdotoxin, a specific inhibitor of K(Ca) channels. In addition, gadolinium, a potent stretch-activated channel (SAC) blocker, prevented the phloretin-mediated Ca(2+) increase, indicating the involvement of SACs in Ca(2+) influx. Furthermore, phloretin stimulated precocious myoblast fusion and this effect was blocked with gadolinium or charybdotoxin. Taken together, these results suggest that induced hyperpolarization, but not depolarization increases Ca(2+) influx through stretch-activated channels, and in turn triggers myoblast fusion.     

Role of N- and L-type calcium channels in depolarization-induced activation of tyrosine hydroxylase and release of norepinephrine by sympathetic cell bodies and nerve terminals.

Multiple types of voltage-activated calcium (Ca(2+)) channels are present in all nerve cells examined so far; however, the underlying functional consequences of their presence is often unclear. We have examined the contribution of Ca(2+) influx through N- and L- type voltage-activated Ca(2+) channels in sympathetic neurons to the depolarization-induced activation of tyrosine hydroxylase (TH), the rate-limiting enzyme in norepinephrine (NE) synthesis, and the depolarization-induced release of NE. Superior cervical ganglia (SCG) were decentralized 4 days prior to their use to eliminate the possibility of indirect effects of depolarization via preganglionic nerve terminals. The presence of both omega-conotoxin GVIA (1 microM), a specific blocker of N-type channels, and nimodipine (1 microM), a specific blocker of L-type Ca(2+) channels, was necessary to inhibit completely the stimulation of TH activity by 55 mM K(+), indicating that Ca(2+) influx through both types of channels contributes to enzyme activation. In contrast, K(+) stimulation of TH activity in nerve fibers and terminals in the iris could be inhibited completely by omega-conotoxin GVIA alone and was unaffected by nimodipine as previously shown. K(+) stimulation of NE release from both ganglia and irises was also blocked completely when omega-conotoxin GVIA was included in the medium, while nimodipine had no significant effect in either tissue. These results indicate that particular cellular processes in specific areas of a neuron are differentially dependent on Ca(2+) influx through N- and L-type Ca(2+) channels.     

Aspartate residues of the Glu-Glu-Asp-Asp (EEDD) pore locus control selectivity and permeation of the T-type Ca(2+) channel alpha(1G)

The structural determinant of the permeation and selectivity properties of high voltage-activated (HVA) Ca(2+) channels is a locus formed by four glutamate residues (EEEE), one in each P-region of the domains I-IV of the alpha(1) subunit. We tested whether the divergent aspartate residues of the EEDD locus of low voltage-activated (LVA or T-type) Ca(2+) channels account for the distinctive permeation and selectivity features of these channels. Using the whole-cell patch-clamp technique in the HEK293 expression system, we studied the properties of the alpha(1G) T-type, the alpha(1C) L-type Ca(2+) channel subunits, and alpha(1G) pore mutants, containing aspartate-to-glutamate conversions in domain III, domain IV, or both. Three characteristic features of HVA Ca(2+) channel permeation, i.e. (a) Ba(2+) over Ca(2+) permeability, (b) Ca(2+)/Ba(2+) anomalous mole fraction effect (AMFE), and (c) high Cd(2+) sensitivity, were conferred on the domain III mutant (EEED) of alpha(1G). In contrast, the relative Ca(2+)/Ba(2+) permeability and the lack of AMFE of the alpha(1G) wild type channel were retained in the domain IV mutant (EEDE). The double mutant (EEEE) displayed AMFE and a Cd(2+) sensitivity similar to that of alpha(1C), but currents were larger in Ca(2+)- than in Ba(2+)-containing solutions. The mutation in domain III, but not that in domain IV, consistently displayed outward fluxes of monovalent cations. H(+) blocked Ca(2+) currents in all mutants more efficiently than in alpha(1G). In addition, activation curves of all mutants were displaced to more positive voltages and had a larger slope factor than in alpha(1G) wild type. We conclude that the aspartate residues of the EEDD locus of the alpha(1G) Ca(2+) channel subunit not only control its permeation properties, but also affect its activation curve. The mutation of both divergent aspartates only partially confers HVA channel permeation properties to the alpha(1G) Ca(2+) channel subunit.     

Role of voltage-gated calcium channels in potassium-stimulated aldosterone secretion from rat adrenal zona glomerulosa cells

The mineralocorticoid aldosterone plays an important role in the regulation of plasma electrolyte homeostasis. Exposure of acutely isolated rat adrenal zona glomerulosa cells to elevated K(+) activates voltage-gated calcium channels and initiates a calcium-dependent increase in aldosterone synthesis. We developed a novel 96-well format aldosterone secretion assay to rapidly evaluate the effect of known T- and L-type calcium channel antagonists on K(+)-stimulated aldosterone secretion and better define the role of voltage-gated calcium channels in this process. Reported T-type antagonists, mibefradil and Ni(2+), and selected L-type antagonist dihydropyridines, inhibited K(+)-stimulated aldosterone synthesis. Dihydropyridine-mediated inhibition occurred at concentrations which had no effect on rat alpha1H T-type Ca(2+) currents. In contrast, below 10 microM, the L-type antagonists verapamil and diltiazem showed only minimal inhibitory effects. To examine the selectivity of the calcium channel antagonist-mediated inhibition, we established an aldosterone secretion assay in which 8Br-cAMP stimulates aldosterone secretion independent of extracellular calcium. Mibefradil remained inhibitory in this assay, while the dihydropyridines had only limited effects. Taken together, these data demonstrate a role for the L-type calcium channel in K(+)-stimulated aldosterone secretion. Further, they confirm the need for selective T-type calcium channel antagonists to better address the role of T-type channels in K(+)-stimulated aldosterone secretion.     

Calbindin-D28k decreases L-type calcium channel activity and modulates intracellular calcium homeostasis in response to K+ depolarization in a rat beta cell line RINr1046-38

Calbindin-D(28k), acts as a modulator of depolarization induced calcium transients in the pancreatic beta cell. However, specific mechanisms have not been defined. Here we show for the first time that the calcium binding protein calbindin-D(28k) acts by affecting calcium influx through voltage-dependent calcium channels in RIN pancreatic beta cells. Whole-cell patch-clamp recordings revealed that Ca(2+) current amplitudes of calbindin-D(28k) expressing RINr1046-38 beta cells were smaller than the Ca(2+) current amplitudes in control cells in response to depolarizing pulses. The peak current was observed at +20mV and the average amplitude was approximately 50pA in the calbindin expressing cells compared to approximately 250pA in control cells. In calbindin-D(28k) expressing cells, the channels had enhanced sensitivity to Ca(2+) dependent inactivation and currents decayed much more rapidly than in control cells. The Ca(2+) channels affected by calbindin were found to have biophysical properties consistent with dihydropyridine-sensitive L-type calcium channels. In response to depolarizing concentrations of K(+), calbindin expression caused a five-fold decrease in the rate of rise of [Ca(2+)](i) and decay was slower in the calbindin expressing cells. Application of verapamil resulted in a drop in the [Ca(2+)](i) signal to pre-stimulation levels indicating that the Ca(2+) channel responsible for the depolarization evoked Ca(2+) entry, modulated by calbindin, is the L-type. Co-immunoprecipitation and GST pull-down assays indicate that calbindin-D(28k) can interact with the alpha(1) subunit of Ca(v)1.2. We thus conclude that calbindin-D(28k) can regulate calcium influx via L-type calcium channels. Our findings suggest a role for calbindin-D(28k) in the beta cell in modulating Ca(2+) influx via L-type voltage-dependent calcium channels.     

大鼠胰腺β细胞离子通道的一些特性

实验以单个Ｗｉｓｔａｒ大鼠胰腺β细胞为对象,用穿孔膜片箝和细胞贴附式记录技术研究ＡＴＰ敏感Ｋ＾＋通道（ＫＡＴＰ）、延迟整流型Ｋ＾＋通道（ＫＤＲ）、Ｃａ＾２＋通道和Ｎａ＾＋通道的有关特性。结果表明：⑴ＫＡＴＰ通道的内流电导约６５ｐＳ,外流电导约３１ｐＳ,反转电位在－６０ｍＶ左右;⑵ＫＤＲ通道在延迟２０ｍｓ后达到最大激活,ＫＤＲ电流约为ＫＡＴＰ的１／３;⑶钙电流在０ｍＶ左右达到４０－６０ｐＡ的峰值,Ｌ     

Ca(2+)-dependent inactivation of cardiac L-type Ca2+ channels does not affect their voltage sensor

Inactivation of currents carried by Ba2+ and Ca2+, as well as intramembrane charge movement from L-type Ca2+ channels were studied in guinea pig ventricular myocytes using the whole-cell patch clamp technique. Prolonged (2 s) conditioning depolarization caused substantial reduction of charge movement between -70 and 10 mV (charge 1, or charge from noninactivated channels). In parallel, the charge mobile between -70 and -150 mV (charge 2, or charge from inactivated channels) was increased. The availability of charge 2 depended on the conditioning pulse voltage as the sum of two Boltzmann components. One component had a central voltage of -75 mV and a magnitude of 1.7 nC/microF. It presumably is the charge movement (charge 2) from Na+ channels. The other component, with a central voltage of approximately - 30 mV and a magnitude of 3.5 nC/microF, is the charge 2 of L-type Ca2+ channels. The sum of charge 1 and charge 2 was conserved after different conditioning pulses. The difference between the voltage dependence of the activation of L-type Ca2+ channels (half-activation voltage, V, of approximately -20 mV) and that of charge 2 (V of -100 mV) made it possible to record the ionic currents through Ca2+ channels and charge 2 in the same solution. In an external solution with Ba2+ as sole metal the maximum available charge 2 of L-type Ca2+ channels was 10-15% greater than that in a Ca(2+)-containing solution. External Cd2+ caused 20-30% reduction of charge 2 both from Na+ and L-type Ca2+ channels. Voltage- and Ca(2+)-dependent inactivation phenomena were compared with a double pulse protocol in cells perfused with an internal solution of low calcium buffering capacity. As the conditioning pulse voltage increased, inactivation monitored with the second pulse went through a minimum at about 0 mV, the voltage at which conditioning current had its maximum. Charge 2, recorded in parallel, did not show any increase associated with calcium entry. Two alternative interpretations of these observations are: (a) that Ca(2+)- dependent inactivation does not alter the voltage sensor, and (b) that inactivation affects the voltage sensor, but only in the small fraction of channels that open, and the effect goes undetected. A model of channel gating that assumes the first possibility is shown to account fully for the experimental results. Thus, extracellular divalent cations modulate voltage-dependent inactivation of the Ca2+ channel. Intracellular Ca2+ instead, appears to cause inactivation of the channel without affecting its voltage sensor.     

Single-channel Ba2+ currents in insulin-secreting cells are activated by glyceraldehyde stimulation

Single-channel Ba2+ current recordings have been made from the insulin-secreting cell line RINm5F with the patch-clamp technique. Depolarization evokes opening of Ca2+ (Ba2+) channels with a relatively high conductance (30 pS) and during the 200 ms depolarizing pulses there is no inactivation. The threshold is high as 50 mV depolarization from the resting membrane potential of -70 mV is required for any channel opening to occur. Glyceraldehyde, a substance evoking insulin secretion from the RINm5F cells, enhances the voltage-activated Ca2+ channel opening by increasing the mean open time and decreasing the longer of the two mean shut times and also decreases the voltage threshold for channel opening.     

Ca2+ channel modulation and kinase-C activation in a pituitary cell line: induction of immediate early genes and inhibition of proliferation.

We have studied the interaction between dihydropyridine (DHP) Ca2+ modulators and the phorbol ester phorbol 12-myristate 13-acetate (PMA) on whole cell Ca2+ currents, 45Ca2+ uptake, immediate early gene (IEG) expression, and proliferation in the rat pituitary GH4C1 cell line. When short (3- to 5-msec) depolarizing voltage clamp steps were used to activate L-type Ca2+ channels, the DHP Ca2+ agonist (-)Bay K 8644 markedly enhanced Ca2+ entry by slowing channel closing upon repolarization. In contrast, the Ca2+ agonist induced only small and inconsistent increases in c-fos mRNA and did not measurably increase NGFI-A. Ca2+ channel activation by depolarization with 50 mM KCl in the presence of (-)Bay K 8644 induced large increases in 45Ca2+ uptake, but failed to markedly induce either of the IEGs. The phorbol ester PMA did not alter T- or L-type Ca2+ current or 45Ca2+ uptake by GH4C1 cells, but triggered large increases in both c-fos and NGFI-A mRNA. In combination, PMA and (-)Bay K 8644 acted synergistically to increase mRNAs for both IEGs. The effect of the DHPs was stereospecific; (+)Bay K 8644, a Ca2+ antagonist, inhibited PMA-induced increases in c-fos and NGFI-A mRNAs. Both PMA and (-)Bay K 8644 inhibited the proliferation of GH4C1 cells, measured by cell count or [3H]thymidine incorporation. The inhibition by the Ca2+ agonist was stereoselective and approximately additive to that of PMA. These results indicate that the expression of c-fos IEG and that of NGFI-A IEG are differentially regulated by separate second messenger pathways in GH4C1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of voltage- and Ca2+-dependent gating of CaV1.3 L-type calcium channels by alternative splicing of a C-terminal regulatory domain

Low voltage activation of Ca(V)1.3 L-type Ca(2+) channels controls excitability in sensory cells and central neurons as well as sinoatrial node pacemaking. Ca(V)1.3-mediated pacemaking determines neuronal vulnerability of dopaminergic striatal neurons affected in Parkinson disease. We have previously found that in Ca(V)1.4 L-type Ca(2+) channels, activation, voltage, and calcium-dependent inactivation are controlled by an intrinsic distal C-terminal modulator. Because alternative splicing in the Ca(V)1.3 alpha1 subunit C terminus gives rise to a long (Ca(V)1.3(42)) and a short form (Ca(V)1.3(42A)), we investigated if a C-terminal modulatory mechanism also controls Ca(V)1.3 gating. The biophysical properties of both splice variants were compared after heterologous expression together with beta3 and alpha2delta1 subunits in HEK-293 cells. Activation of calcium current through Ca(V)1.3(42A) channels was more pronounced at negative voltages, and inactivation was faster because of enhanced calcium-dependent inactivation. By investigating several Ca(V)1.3 channel truncations, we restricted the modulator activity to the last 116 amino acids of the C terminus. The resulting Ca(V)1.3(DeltaC116) channels showed gating properties similar to Ca(V)1.3(42A) that were reverted by co-expression of the corresponding C-terminal peptide C(116). Fluorescence resonance energy transfer experiments confirmed an intramolecular protein interaction in the C terminus of Ca(V)1.3 channels that also modulates calmodulin binding. These experiments revealed a novel mechanism of channel modulation enabling cells to tightly control Ca(V)1.3 channel activity by alternative splicing. The absence of the C-terminal modulator in short splice forms facilitates Ca(V)1.3 channel activation at lower voltages expected to favor Ca(V)1.3 activity at threshold voltages as required for modulation of neuronal firing behavior and sinoatrial node pacemaking.     

The Janus-faced atracotoxins are specific blockers of invertebrate K(Ca) channels

The Janus-faced atracotoxins are a unique family of excitatory peptide toxins that contain a rare vicinal disulfide bridge. Although lethal to a wide range of invertebrates, their molecular target has remained enigmatic for almost a decade. We demonstrate here that these toxins are selective, high-affinity blockers of invertebrate Ca(2+)-activated K(+) (K(Ca)) channels. Janus-faced atracotoxin (J-ACTX)-Hv1c, the prototypic member of this toxin family, selectively blocked K(Ca) channels in cockroach unpaired dorsal median neurons with an IC(50) of 2 nm, but it did not significantly affect a wide range of other voltage-activated K(+), Ca(2+) or Na(+) channel subtypes. J-ACTX-Hv1c blocked heterologously expressed cockroach large-conductance Ca(2+)-activated K(+) (pSlo) channels without a significant shift in the voltage dependence of activation. However, the block was voltage-dependent, indicating that the toxin probably acts as a pore blocker rather than a gating modifier. The molecular basis of the insect selectivity of J-ACTX-Hv1c was established by its failure to significantly inhibit mouse mSlo currents (IC(50) approximately 10 mum) and its lack of activity on rat dorsal root ganglion neuron K(Ca) channel currents. This study establishes the Janus-faced atracotoxins as valuable tools for the study of invertebrate K(Ca) channels and suggests that K(Ca) channels might be potential insecticide targets.     

Calcium flux in turtle ventricular myocytes

The relative contribution of the sarcoplasmic reticulum (SR), the L-type Ca(2+) channel and the Na(+)/Ca(2+) exchanger (NCX) were assessed in turtle ventricular myocytes using epifluorescent microscopy and electrophysiology. Confocal microscopy images of turtle myocytes revealed spindle-shaped cells, which lacked T-tubules and had a large surface area-to-volume ratio. Myocytes loaded with the fluorescent Ca(2+)-sensitive dye Fura-2 elicited Ca(2+) transients, which were insensitive to ryanodine and thapsigargin, indicating the SR plays a small role in the regulation of contraction and relaxation in the turtle ventricle. Sarcolemmal Ca(2+) currents were measured using the perforated-patch voltage-clamp technique. Depolarizing voltage steps to 0 mV elicited an inward current that could be blocked by nifedipine, indicating the presence of Ca(2+) currents originating from L-type Ca(2+) channels (I(Ca)). The density of I(Ca) was 3.2 +/- 0.5 pA/pF, which led to an overall total Ca(2+) influx of 64.1 +/- 9.3 microM/l. NCX activity was measured as the Ni(+)-sensitive current at two concentrations of intracellular Na(+) (7 and 14 mM). Total Ca(2+) influx through the NCX during depolarizing voltage steps to 0 mV was 58.5 +/- 7.7 micromol/l and 26.7 +/- 3.2 micromol/l at 14 and 7 mM intracellular Na(+), respectively. In the absence of the SR and L-type Ca(2+) channels, the NCX is able to support myocyte contraction independently. Our results indicate turtle ventricular myocytes are primed for sarcolemmal Ca(2+) transport, and most of the Ca(2+) used for contraction originates from the L-type Ca(2+) channel.     

Mechanism of generation of spontaneous miniature outward currents (SMOCs) in retinal amacrine cells

A subtype of retinal amacrine cells displayed a distinctive array of K(+) currents. Spontaneous miniature outward currents (SMOCs) were observed in the narrow voltage range of -60 to -40 mV. Depolarizations above approximately -40 mV were associated with the disappearance of SMOCs and the appearance of transient (I(to)) and sustained (I(so)) outward K(+) currents. I(to) appeared at about -40 mV and its apparent magnitude was biphasic with voltage, whereas I(so) appeared near -30 mV and increased linearly. SMOCs, I(to), and a component of I(so) were Ca(2+) dependent. SMOCs were spike shaped, occurred randomly, and had decay times appreciably longer than the time to peak. In the presence of cadmium or cobalt, SMOCs with pharmacologic properties identical to those seen in normal Ringer's could be generated at voltages of -20 mV and above. Their mean amplitude was Nernstian with respect to [K(+)](ext) and they were blocked by tetraethylammonium. SMOCs were inhibited by iberiotoxin, were insensitive to apamin, and eliminated by nominally Ca(2+)-free solutions, indicative of BK-type Ca(2+)-activated K(+) currents. Dihydropyridine Ca(2+) channel antagonists and agonists decreased and increased SMOC frequencies, respectively. Ca(2+) permeation through the kainic acid receptor had no effect. Blockade of organelle Ca(2+) channels by ryanodine, or intracellular Ca(2+) store depletion with caffeine, eradicated SMOCs. Internal Ca(2+) chelation with 10 mM BAPTA eliminated SMOCs, whereas 10 mM EGTA had no effect. These results suggest a mechanism whereby Ca(2+) influx through L-type Ca(2+) channels and its subsequent amplification by Ca(2+)-induced Ca(2+) release via the ryanodine receptor leads to a localized elevation of internal Ca(2+). This amplified Ca(2+) signal in turn activates BK channels in a discontinuous fashion, resulting in randomly occurring SMOCs.     

Block of T-type Ca channels in guinea pig atrial cells by antiarrhythmic agents and Ca channel antagonists

Myocardial cells have two types of Ca channels commonly called T-type and L-type. Whole cell Ca channel currents in guinea pig atrial myocytes can be separated and quantitated by analyzing channel closing kinetics after a brief depolarization (tail current analysis). L-type Ca channels deactivate rapidly when the membrane is repolarized and T-type Ca channels deactivate relatively slowly. Ca channel block by the therapeutically useful Ca channel antagonists is voltage dependent, so it is desirable to study block of both channel types over an extended voltage range. Tail current analysis allows this and was used to study block of both types of Ca channels under identical conditions. Amiodarone, bepridil, and cinnarizine block T-type Ca channels more potently than L-type Ca channels when binding equilibrates at normal diastolic potentials (approximately -90 mV). None of these drugs is a selective blocker of T-type Ca channels because block of L-type Ca channels is enhanced when cells are almost completely depolarized. Although weak block of T-type Ca channels by 1,4-dihydropyridines has usually been reported, we found that felodipine blocks these channels with high affinity. When most T-type Ca channels are inactivated, the apparent dissociation constant (KI) is 13 nM. Felodipine also blocks T-type Ca channels in GH3 cells (a cell line derived from rat anterior pituitary), but KI = 700 nM. Thus, T-type Ca channels in different cell types are pharmacologically distinct. Felodipine can block L-type Ca channels in atrial cells more potently than T-type Ca channels, but block of L-type Ca channels is potent only at depolarized potentials; block of both channel types is comparable at normal diastolic membrane potentials. Felodipine and the 1,4-dihydropyridines isradipine and (-)-202-791 are approximately equipotent at blocking T-type Ca channels, but differ substantially in potency for block of L-type Ca channels. Block of T-type Ca channels may account for some of the pharmacological effects of 1,4-dihydropyridines and for the antiarrhythmic activity of amiodarone and bepridil.     

Calcium entry through L-type calcium channels causes mitochondrial disruption and chromaffin cell death

Sustained, mild K+ depolarization caused bovine chromaffin cell death through a Ca(2+)-dependent mechanism. During depolarization, Ca(2+) entered preferentially through L-channels to induce necrotic or apoptotic cell death, depending on the duration of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) signal, as proven by the following. (i) The L-type Ca(2+) channel activators Bay K 8644 and FPL64176, more than doubled the cytotoxic effects of 30 mm K+; (ii) the L-type Ca(2+) channel blocker nimodipine suppressed the cytotoxic effects of K+ alone or K+ plus FPL64176; (iii) the potentiation by FPL64176 of the K+ -evoked [Ca(2+)](c) elevation was totally suppressed by nimodipine. Cell exposure to K+ plus the L-type calcium channel agonist FPL64176 caused an initial peak rise followed by a sustained elevation of the [Ca(2+)](c) that, in turn, increased [Ca(2+)](m) and caused mitochondrial membrane depolarization. Cyclosporin A, a blocker of the mitochondrial transition pore, and superoxide dismutase prevented the apoptotic cell death induced by Ca(2+) overload through L-channels. These results suggest that Ca(2+) entry through L-channels causes both calcium overload and mitochondrial disruption that will lead to the release of mediators responsible for the activation of the apoptotic cascade and cell death. This predominant role of L-type Ca(2+) channels is not shared by other subtypes of high threshold voltage-dependent neuronal Ca(2+) channels (i.e. N, P/Q) expressed by bovine chromaffin cells.