Both T- and L-type Ca2+ channels can contribute to excitation-contraction coupling in cardiac Purkinje cells.

Although L-type Ca2+ channels have been shown to play a central role in cardiac excitation-contraction (E-C) coupling, little is known about the role of T-type Ca2+ channels in this process. We used the amphotericin B perforated patch method to study the possible role of T-type Ca2+ current in E-C coupling in isolated canine Purkinje myocytes where both Ca2+ currents are large. T-type Ca2+ current was separated from L-type Ca2+ current using protocols employing the different voltage dependencies of the channel types and their different sensitivities to pharmacological blockade. We showed that Ca2+ admitted through either T- or L-type Ca2+ channels is capable of initiating contraction and that the contractions depended on Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR). The contractions, however, had different properties. Those initiated by Ca2+ entry through T-type Ca2+ channels had a longer delay to the onset of shortening, slower rates of shortening and relaxation, lower peak shortening, and longer time to peak shortening. These differences were present even when L-type Ca2+ current amplitude, or charge entry, was less than that of T-type Ca2+ current, suggesting that Ca2+ entry through the T-type Ca2+ channel is a less effective signal transduction mechanism to the SR than is Ca2+ entry through the L-type Ca2+ channel. We conclude that under our experimental conditions in cardiac Purkinje cells Ca2+ entry through the T-type Ca2+ channel can activate cell contraction. However, Ca2+ entry through the L-type Ca2+ channel is a more effective signal transduction mechanism. Our findings support the concept that different structural relationships exist between these channel types and the SR Ca2+ release mechanism.     

Targets for calcium channel blockers in mammalian skeletal muscle and their respective functions in excitation-contraction coupling

The L-type Ca2+ channel is blocked by 1,4-dihydropyridines (DHP), by phenylalkylamines, by diphenylbutylpiperidines or by benzolactams. We first show with mouse muscle cells in culture that all these L-type Ca2+ channel blockers block contraction. However, voltage-clamp analysis associated to contraction measurements also clearly show that Ca2+ influx through L-type Ca2+ channels is not required for contraction. Therefore, there is a need for a voltage-sensor which would be responsible for the excitation-contraction (E-C) coupling. We are showing here that the voltage-sensor involved in E-C coupling and the L-type Ca2+ channel have a similar pharmacology. Some of the blockers used are more active on the voltage sensor, others on the L-type Ca2+ channel.     

Diversity and novel pharmacological properties of Ca2+ channels in Drosophila brain membranes.

Binding studies as well as affinity labelling and immunoblot techniques were used to identify and characterize the receptors for Ca2+ channel blockers in Drosophila brain membranes. Despite structural analogies with mammalian receptors, Drosophila binding sites for phenylalkylamines and 1,4-dihydropyridines, unlike those described in skeletal and cardiac muscle, were found to be located on separate Ca2+ channels. Single-channel bilayer recordings from reconstituted membranes revealed the presence of eight distinct cobalt-sensitive Ba2+-conducting channels in Drosophila brain membrane preparations. In good agreement with binding studies, the most frequently observed Ca2+ channel type (Ba2+ conductance of 13 pS) was extremely sensitive to phenylalkylamines but not affected by micromolar concentrations of 1,4-dihydropyridines. Distinct 1,4-dihydropyridine-sensitive and phenylalkylamine-insensitive channels were also identified. They had unitary Ba2+ conductances of 21 and 31 pS. A detailed analysis of drug action showed that both 1,4-dihydropyridines and phenylalkylamines first increased channel open state probability before fully blocking channel activity. Other types of channels have been identified with unitary Ba2+ conductances of 9, 41, 53, 64 and 81 pS. They were insensitive to the previously described organic Ca2+ channel blockers. The Drosophila system seems to be a unique model to analyse the properties of several different types of Ca2+ channels and particularly those of channel types that are uniquely blocked by phenylalkylamines or uniquely blocked by 1,4-dihydropyridines.     

Isolation of myocardial L-type calcium channel gating currents with the spider toxin omega-Aga-IIIA

The peptide omega-agatoxin-IIIA (omega-Aga-IIIA) blocks ionic current through L-type Ca channels in guinea pig atrial cells without affecting the associated gating currents. omega-Aga-IIIA permits the study of L- type Ca channel ionic and gating currents under nearly identical ionic conditions. Under conditions that isolate L-type Ca channel currents, omega-Aga-IIIA blocks all ionic current during a test pulse and after repolarization. This block reveals intramembrane charge movements of equal magnitude and opposite sign at the beginning of the pulse (Q(on)) and after repolarization (Q(off)). Q(on) and Q(off) are suppressed by 1 microM felodipine, saturate with increasing test potential, and are insensitive to Cd. The decay of the transient current associated with Q(on) is composed of fast and slow exponential components. The slow component has a time constant similar to that for activation of L-type Ca channel ionic current, over a broad voltage range. The current associated with Q(off) decays monoexponentially and more slowly than ionic current. Similar charge movements are found in guinea pig tracheal myocytes, which lack Na channels and T-type Ca channels. The kinetic and pharmacological properties of Q(on) and Q(off) indicate that they reflect gating currents associated with L-type Ca channels. omega-Aga-IIIA has no effect on gating currents when ionic current is eliminated by stepping to the reversal potential for Ca or by Cd block. Gating currents constitute a significant component of total current when physiological concentrations of Ca are present and they obscure the activation and deactivation of L-type Ca channels. By using omega- Aga-IIIA, we resolve the entire time course of L-type Ca channel ionic and gating currents. We also show that L- and T-type Ca channel ionic currents can be accurately quantified by tail current analysis once gating currents are taken into account.     

Modulation of the T-type cardiac Ca channel by changes in proton concentration

Single-channel measurements and whole-cell experiments with the two suction electrode, voltage clamp technique were used to investigate the effects of external and internal proton concentrations on T-type Ca channels in heart muscle cells of the guinea pig. As in the L-type Ca channel, an increase in the external proton concentration decreases T-type currents, while external alkalinization enlarges the currents. In contrast to the L-type Ca channel, however, a change in the internal proton concentration does not modulate T-type Ca currents. The T-type Ca channel is much more sensitive to variations in pHo than the L-type Ca channel. By the combination of single-channel and whole-cell experiments we can conclude that the observed changes in macroscopic currents are due to (a) changes in the single-channel conductance and in the probability of the T-type Ca channel being open, and (b) the titration of the negative surface charges in the neighborhood of the T-type Ca channel with shifts of both the activation and inactivation processes of the channel. The pHo-induced changes in the maximal conductance (gmax) of the T-type Ca channel show an apparent pKa in the range of 7.1-7.5, while the titration of the negative surface charges near the channel shows an apparent pKa of 7.1 with a concomitant surface potential of -24.6 mV at 5.4 mM [Ca]o. These pKa values, less acid than the pKa values found for the pHo-induced, L-type Ca channel modulation, might imply a physiological importance of this novel type of channel modulation.     

Heparin binds with high affinity to voltage-dependent L-type Ca2+ channels. Evidence for an agonistic action

Heparin and related polyanions are a new class of compounds interacting with 1,4-dihydropyridine-sensitive L-type Ca2+ channels in a tissue-specific manner. Labeling of membrane-bound Ca2+ channels in rabbit skeletal muscle transverse tubules at the phenylalkylamine, benzothiazepine, and 1,4-dihydropyridine-selective domains was inhibited reversibly by a noncompetitive mechanism as shown by equilibrium saturation analysis and kinetic studies. (+)-cis-diltiazem but not (-)-cis-diltiazem reduced the inhibitory potency of heparin for 1,4-dihydropyridines. Antagonistic but not agonistic 1,4-dihydropyridines reversed heparin inhibition at the benzothiazepine site. Heparin forms a tight complex with the purified Ca2+ channel which is highly sensitive with respect to heparin inhibition (IC50 value: 0.05 microgram/ml) of 1,4-dihydropyridine binding. Reconstituted channel complexes have completely lost 1,4-dihydropyridine binding-inhibition by heparin and are not retained by lectin or heparin affinity columns. In whole cell patch clamp experiments with guinea-pig cardiac myocytes heparin increased the current through L-type Ca2+ channels when applied extracellulary. Synthetic peptides (representing putative heparin binding domains) which were derived from the rabbit skeletal muscle alpha 1-subunit reversed the inhibitory effects of heparin on 1,4-dihydropyridine receptors. Reversal for a peptide representing an extracellular domain occurred by an apparently competitive mechanism. It is suggested that heparin and related polyanions may interact with an evolutionary conserved cluster of basic amino acids in the large putative extracellular domain connecting the fifth and sixth putative transmembrane segment in the first motif of the ionic pore-forming alpha 1-subunit from skeletal muscle.     

T-type Ca2+ channels in vascular smooth muscle: multiple functions

Vascular smooth muscle is a major constituent of the blood vessel wall, and its many functions depend on type and location of the vessel, developmental or pathological state, and environmental and chemical factors. Vascular smooth muscle cells (VSMCs) use calcium as a signal molecule for multiple functions. An important component of calcium signaling pathways is the entry of extracellular calcium via voltage-gated Ca2+ channels, which in vascular smooth muscle cells (VSMCs) are of two main types, the high voltage-activated (HVA) L-type and low voltage-activated (LVA) T-type channels. Whereas L-type channels function primarily to regulate Ca2+ entry for contraction, it is generally accepted that T-type Ca2+ channels do not contribute significantly to arterial vasoconstriction, with the possible exception of the renal microcirculation. T-type Ca2+ channels are also present in some veins that display spontaneous contractile activity, where they likely generate pacemaker activity. T-type Ca2+ channel expression has also been associated with normal and pathological proliferation of VSMCs, often stimulated by external cues in response to insult or injury. Expression of T-type channels has been linked to the G1 and S phases of the cell cycle, a period important for the signaling of gene expression necessary for cell growth, progression of the cell cycle and ultimately cell division. To better understand T-type Ca2+ channel functions in VSM, it will be necessary to develop new approaches that are specifically targeted to this class of Ca2+ channels and its individual members.     

Participation of transient-type Ca2+ channels in the sustained increase of Ca2+ level in GH3 cells

Participation of two types of Ca2+ channels (T- and L-types) in the sustained increase of cytosolic-free Ca2+ concentration [( Ca2+]i) was studied in thyrotropin-releasing hormone (TRH)-stimulated clonal GH3 pituitary cells. The effects of Ca2+ channel blockers were analyzed by measuring Ca2+ channel current and [Ca2+]i, using whole-cell voltage-clamp and Fura-2 fluorometry, respectively. Phenytoin (100 microM) and Ni2+ (100 microM) selectively blocked T-type Ca2+ channels and suppressed the TRH-induced sustained [Ca2+]i increase in single cells. Synthetic omega-conotoxin (omega-CgTX, 2 microM) preferentially blocked L-type Ca2+ channels, but it did not suppress the TRH-induced sustained [Ca2+]i increase. The present results suggest that the sustained elevations of [Ca2+]i triggered by TRH may be mediated by T-type Ca2+ channels in GH3 cells.     

Frequency-dependent blockade of T-type Ca2+ current by efonidipine in cardiomyocytes

Efonidipine is a dihydropyridine Ca2+ antagonist with inhibitory effects on both L-type and T-type Ca2+ channels and potent bradycardiac activity especially in patients with high heart rate. In the present study, we examined the frequency dependence of efonidipine action on the T-type Ca2+ channel in isolated guinea-pig ventricular myocytes. The potency of efonidipine to inhibit the T-type Ca2+ current was higher under higher stimulation frequencies. The IC50 values were 1.3 x 10(-8), 2.0 x 10(-6) and 6.3 x 10(-6) M under stimulation frequencies of 1, 0.2 and 0.05 Hz, respectively. The reduction of T-type Ca2+ current amplitude was not accompanied by change in the time course of current decay. Efonidipine (10 microM) inhibited T-type Ca2+ current elicited by depolarization from holding potentials ranging from -90 to -30 mV by about 30%; the voltage-dependence of steady-state inactivation was not changed by the drug. Efonidipine slowed the recovery from inactivation following an inactivating prepulse. In conclusion, efonidipine was shown to have frequency-dependent inhibitory effects on the T-type Ca2+ channel, which could be explained by slow dissociation of the drug from the inactivated state of the channel.     

Gamma-dendrotoxin blocks large conductance Ca2+-activated K+ channels in neuroblastoma cells

In N1E 115 neuroblastoma cells, gamma-dendrotoxin (DTX, 200 nM) blocked the outward K(+) current by 31.1 +/- 3.5% (n = 4) with approximately 500 nM Ca(2+) in the pipet solution, but had no effect on the outward K(+) current when internal Ca(2+) was reduced. Using a ramp protocol, iberiotoxin (IbTX, 100 nM) inhibited a component of the whole cell current, but in the presence of 200 nM gamma-DTX, no further inhibition by IbTX was observed. Two types of single channels were seen using outside-out patches when the pipette free Ca(2+) concentration was approximately 500 nM; a 63 pS and a 187 pS channel. The 63 pS channel was TEA-, IbTX- and gamma-DTX-insensitive, while the 187 pS channel was blocked by 1 mM TEA, 100 nM IbTX or 200 nM gamma-DTX. Both channels were activated by external application of ionomycin, when the pipet calcium concentration was reduced. gamma-DTX (200 nM) reduced the probability of openings of the 187 pS channel, with an IC(50) of 8.5 nM. In GH(3) cells gamma-DTX (200 nM) also blocked an IbTX-sensitive component of whole-cell K(+) currents. These results suggest that gamma-DTX blocks a large conductance Ca(2+) activated K(+) current in N1E 115 cells. This is the first indication that any of the dendrotoxins, which have classically been known to block voltage-gated (Kv) channels, can also block Ca(2+) activated K(+) channels.     

TRPA1 channels: novel targets of 1,4-dihydropyridines

Transient receptor potential type A1 (TRPA1) channels are cation permeable channels activated by irritant chemicals and pungent natural compounds. Their location in peptidergic sensory terminals innervating the skin and blood vessels makes them important effectors of vasodilator responses of neural origin. 1,4-dihydropyridines are a class of L-type calcium channel antagonists commonly used in the treatment of hypertension and ischemic heart disease. Here we show that four different 1,4-dihydropyridines (nifedipine, nimodipine, nicardipine and nitrendipine), and the structurally related L-type calcium channel agonist BayK8644, exert powerful excitatory effects on TRPA1 channels. The activation does not depend on elevated Ca2+ levels and cross-desensitizes with that produced by other TRPA1 agonists. The activation produced by nifedipine was reduced by camphor and the selective TRPA1 antagonist HC03001. In a subclass of mouse nociceptors expressing TRPA1 channels, assessed by responses to the TRPA1 agonist mustard oil, nifedipine also produced large elevations in [Ca2+](i). These responses were fully abrogated in TRPA1(-/-) mice. These findings identify TRPA1 channels as a new molecular target for the 1,4-dihydropyridine class of calcium channel modulators.     

Inhibition of Transiently Expressed Low- and High-Voltage-Activated Calcium Channels by Trivalent Metal Cations

Calcium channels are important regulators of neuronal excitability and contribute to transmitter release, calcium dependent gene expression, and oscillatory behavior in many cell types. Under physiological conditions, native low-voltage (T-type)- and high-voltage-activated (HVA) currents are potently inhibited by trivalent cations. However, the presence of multiple calcium channel isoforms has hampered our ability to unequivocally assess the effects of trivalent cations on channel activity. Here, we describe the actions of nine trivalent metal ions on transiently expressed alpha1G (Cav3.1) T-type calcium channels cloned from human brain. In 2 mM external barium solution, yttrium most potently inhibited alpha1G current (IC50 = 28 nM), followed by erbium > gadolinium ~ cerium > holmium > ytterbium > neodymium > lanthanum > scandium. With the exception of scandium, blocking affinity was loosely correlated with decreasing ionic radius. A detailed characterization of yttrium block revealed a 25-fold decrease in blocking affinity when the external concentration of charge carrier was increased from 2 mM to 20 mM. In 20 mM barium, yttrium also effectively inhibited various types of cloned HVA channels indicating that this ion is a nonselective blocker. For all calcium channels examined, yttrium preferentially inhibited inward over outward current, but block was otherwise voltage independent. In addition to peak current inhibition, P/Q- and L-type channels underwent a unique speeding of the macroscopic time course of inactivation. Whereas peak current block of alpha1A channels was highly sensitive to the external charge carrier concentration, the inactivation effects mediated by yttrium were not, suggesting that the two effects are due to distinct mechanisms. Moreover, the speeding effect was greatly attenuated by manipulations that slowed the inactivation kinetics of the channels. Thus, our evidence suggests that yttrium effects are mediated by two distinct events: peak current block likely occurring by occlusion of the pore, and kinetic speeding arising from yttrium interactions with the channel that alter the state of the inactivation gate.     

L-type voltage-operated Ca(+2) channels modulate transient Ca(+2) influx triggered by activation of Sertoli cell surface L-selectin

Near the base of mammalian seminiferous epithelium, Sertoli cells are joined by tight junctions, which constitute the blood-testis barrier. Differentiating germ cells are completely enveloped by Sertoli cells and must traverse the tight junctions during spermatogenic cycle. Following the specific ligand activation of L-selectin, the up-regulated Rho family small G-proteins have been implicated as important modulators of tight junctional dynamics. Although the activation of L-selectin transmits subsequent intracellular signals in a Ca(+2)-dependent fashion in various cell types, little is understood regarding the signaling pathways utilized by L-selectin in Sertoli cells. Therefore, we have examined the possible resultant calcium influx triggered by specific ligand-activation of cell surface L-selectin receptors or by cross-linking of L-selectin with anti-L-selectin. Spectrofluorimetric studies demonstrate increase of intracellular Ca(+2) levels immediately after the treatment of the L-selectin ligands, fucoidan and sialyl Lewis-a, or after treatment with anti-L-selectin antibody. We then determined the mechanism of Ca(+2) influx by investigating L- and T-type voltage-operated Ca(+2) channels, which have been suggested to present in the membranes of Sertoli cells. Data demonstrate that Sertoli cells treated with L-type voltage-operated Ca(+2) channel antagonists, nifedipine, diltiazem, or verapamil, lead to dose-dependent blockage of L-selectin-induced Ca(+2) influx. Cells treated with mibedradil, a T-type voltage-operated Ca(+2) channel antagonist, results in little or no blocking effect. Therefore, we conclude that activation of Sertoli cell L-selectin induces Ca(+2) influx, which is at least partially regulated by L-type voltage-operated Ca(+2) channels.     

Fast exocytosis mediated by T- and L-type channels in chromaffin cells: distinct voltage-dependence but similar Ca<Superscript>2+</Superscript>-dependence

Expression, spatial distribution and specific roles of different Ca(2+) channels in stimulus-secretion coupling of chromaffin cells are intriguing issues still open to discussion. Most of the evidence supports a role of high-voltage activated (HVA) Ca(2+) channels (L-, N-, P/Q- and R-types) in the control of exocytosis: some suggesting a preferential coupling of specific Ca(2+) channel subunits with the secretory apparatus, others favoring the idea of a contribution to secretion proportional to the expression density and gating properties of Ca(2+) channels. In this work we review recent findings and bring new evidence in favor of the hypothesis that also the LVA (low-voltage-activated, T-type) Ca(2+) channels effectively control fast exocytosis near resting potential in adrenal chromaffin cells of adult rats. T-type channels recruited after long-term treatments with pCPT-cAMP (or chronic hypoxia) are shown to control exocytosis with the same efficacy of L-type channels, which are the dominant Ca(2+) channel types expressed in rodent chromaffin cells. A rigorous comparison of T- and L-type channel properties shows that, although operating at different potentials and with different voltage-sensitivity, the two channels possess otherwise similar Ca(2+)-dependence of exocytosis, size and kinetics of depletion of the immediately releasable pool and mobilize vesicles of the same quantal size. Thus, T- and L-type channels are coupled with the same Ca(2+)-efficiency to the secretory apparatus and deplete the same number of vesicles ready for release. The major difference of the secretory signals controlled by the two channels appear to be the voltage range of operation, suggesting the idea that stressful conditions (hypoxia and persistent beta-adrenergic stimulation) can lower the threshold of cell excitability by recruiting new Ca(2+) channels and activate an additional source of catecholamine secretion.     

A Ca(v)3.2/syntaxin-1A signaling complex controls T-type channel activity and low-threshold exocytosis

T-type calcium channels represent a key pathway for Ca(2+) entry near the resting membrane potential. Increasing evidence supports a unique role of these channels in fast and low-threshold exocytosis in an action potential-independent manner, but the underlying molecular mechanisms have remained unknown. Here, we report the existence of a syntaxin-1A/Ca(v)3.2 T-type calcium channel signaling complex that relies on molecular determinants that are distinct from the synaptic protein interaction site (synprint) found in synaptic high voltage-activated calcium channels. This interaction potently modulated Ca(v)3.2 channel activity, by reducing channel availability. Other members of the T-type calcium channel family were also regulated by syntaxin-1A, but to a smaller extent. Overexpression of Ca(v)3.2 channels in MPC 9/3L-AH chromaffin cells induced low-threshold secretion that could be prevented by uncoupling the channels from syntaxin-1A. Altogether, our findings provide compelling evidence for the existence of a syntaxin-1A/T-type Ca(2+) channel signaling complex and provide new insights into the molecular mechanism by which these channels control low-threshold exocytosis.     

L, P-/Q- and T-type Ca2+ channels in smooth muscle cells from human umbilical artery.

The electrophysiological and pharmacological properties of Ca(2+) current (I(Ca)) were determined by the whole-cell configuration of the patch-clamp technique in smooth muscle cells from human umbilical artery. Using 5 mM extracellular Ca(2+), depolarizing step pulses from -60 to 50 mV from a holding membrane potential of -80 mV evoked an I(Ca) which activated at membrane potentials more positive than -50 mV and exhibited a maximum current density in a range of 10-20 mV. Steady-state inactivation protocols using a V(test) of 10 mV gave a voltage at one-half inactivation and a slope factor of -35.6 mV and 9.5 mV, respectively. Nifedipine (1 microM), an L-type Ca(2+) channels antagonist, completely inhibited I(Ca), while the L-type Ca(2+) channels agonist Bay-K 8644 (1 microM) significantly increased I(Ca) amplitude. Moreover, the selective blocker of P-/Q-type Ca(2+) channels omega-agatoxin IVA partially blocked I(Ca) (about 40 % inhibition at +20 mV by 20 nM). These pharmacological results suggest that L- and P-/Q-type Ca(2+) channels, both nifedipine-sensitive, underlie the I(Ca) registered using low extracellular Ca(2+). The presence of the P-/Q-type Ca(2+) channels was confirmed by immunoblot analysis. When I(Ca) was recorded in a high concentration (30 mM) of extracellular Ca(2+) or Ba(2+) as current carrier, it was evident the presence of a nifedipine-insensitive component which completely inactivated during the course of the voltage-step (75 ms) at all potentials tested, and was blocked by the T-type Ca(2+) channels blocker mibefradil (10 microM). Summarizing, this work shows for the first time the electrophysiological and pharmacological properties of voltage-activated Ca(2+) currents in human umbilical artery smooth muscle cells.     

Modulation of L-type Ca2+ channels in UMR 106 cells by parathyroid hormone-related protein.

The effects of rat parathyroid hormone-related protein (rPTHrP) and bovine and rat parathyroid hormone (bPTH and rPTH) on L-type Ca2+ channels in UMR 106 cells were investigated using the patch clamp technique. rPTHrP increased the whole cell L-type Ca2+ channel currents and the increase was concentration dependent. rPTHrP, at a concentration of 62.5 nM, increased the L-type Ca2+ channel current by 122+/-25%. bPTH was less potent. A concentration of 7.5 microM bPTH increased the current by 99+/-24%. Results obtained with rPTH were similar to those obtained using bPTH. Single channel measurements, using the cell-attached version of the patch clamp technique, showed an increase in both the number of channel openings and the mean open time when the cells were exposed to rPTHrP. This suggested that rPTHrP affected the gating of L-type Ca2+ channels in UMR 106 cells. This study demonstrates that the actions of bPTH and rPTHrP in UMR cells are mediated in part by extracellular Ca2+ entry. PTHrP, a paracrine agent important in development, is more potent in regulating Ca2+ entry than PTH.     

Positive heterotropic allosteric regulators of dihydropyridine binding increase the Ca2+ affinity of the L-type Ca2+ channel. Stereoselective reversal by the novel Ca2+ antagonist BM 20.1140.

The alpha 1-subunit of the voltage-dependent L-type Ca2+ channel has distinct, allosterically coupled binding domains for drugs from different chemical classes (dihydropyridines, benzothiazepines, phenylalkylamines, diphenylbutylpiperidines). (-)-BM 20.1140 (ethyl-2,2-di-phenyl-4-(1-pyrrolidino)-5-(2-picolyl)- oxyvalerate) is a novel Ca2+ channel blocker which potently stimulates dihydropyridine binding (K0.5 = 2.98 nM) to brain membranes. This property is shared by (+)-cis-diltiazem, (+)-tetrandrine, fostedil and trans-diclofurime, but (-)-BM 20.1140 does not bind in a competitive manner to the sites labeled by (+)-cis-[3H]diltiazem. (+)-cis-Diltiazem and (-)-BM 20.1140 have differential effects on the rate constants of dihydropyridine binding. (+)-BM 20.1140 reverses the stimulation of the positive allosteric regulators (pA2 value for reversal of (-)-BM 20.1140 stimulation = 7.4, slope 0.72). The underlying molecular mechanism of the potentiation of dihydropyridine binding has been clarified. The K0.5 for free Ca2+ to stabilize a high affinity binding domain for dihydropyridines on purified L-type channels from rabbit skeletal muscle is 300 nM. (+)-Tetrandine (10 microM) increases the affinity 8-fold (K0.5 for free Ca2+ = 30.1 nM) and (+)-BM 20.114 (10 microM) inhibits the affinity increase (K0.5 for free Ca2+ = 251 nM). Similar results were obtained with membrane-bound Ca(2+)-channels from brain tissue which have higher affinity for free Ca2+ (K0.5 for free Ca2+ = 132 nM) and for dihydropyridines compared with skeletal muscle. It is postulated that the dihydropyridine and Ca(2+)-binding sites are interdependent on the alpha 1-subunit, that the different positive heterotropic allosteric regulators (by their differential effects on Ca2+ rate constants) optimize coordination for Ca2+ in the channel pore and, in turn, increase affinity for the dihydropyridines.