Pharmacological properties of Ca(V)3.2, a low voltage-activated Ca2+ channel cloned from human heart.

Three genes encoding T-type Ca2+ channels have been described but their correspondence to the various native T-type Ca2+ currents remains uncertain. In particular, Ca(V)3.2 (or alpha1H) was cloned from a human heart library, its message was found abundantly in cardiac tissue, and expressed Ca(V)3.2 was shown to conduct low voltage-activated currents, which inactivate rapidly and are sensitive to Ni2+ and mibefradil. These observations suggested that Ca(V)3.2 might encode native cardiac T-type Ca2+ channels but more information on the pharmacology of Ca(V)3.2 was needed to confirm this hypothesis. In the present study, we compare the pharmacology of Ca(V)3.2 expressed in HEK293 cells and of native T-type Ca2+ channels in guinea pig atrial myocytes ("native-T"). (1) Ca(V)3.2 and native-T are insensitive to TTX and to toxins selective for N-, P-, or Q-type Ca2+ channels (omega-CTx-GVIA, omega-Aga-IVA, omega-CTx-MVIIC). (2) The half-maximal blocking concentration (IC50) of mibefradil on Ca(V)3.2 is near that on native-T and the block is similarly voltage-dependent. (3) Ca(V)3.2 is five- to sixfold less sensitive than native-T to the 1,4-dihydropyridine (DHP) amlodipine, suggesting a difference in the DHP binding site. (4) Both channels display similar (but not identical) sensitivities to the inorganic blockers Ni2+ and Cd2+ and the IC50s are in the range of values found for T-type Ca2+ currents in other cell types. (5) Ni2+ shifts the voltage dependence of Ca(V)3.2 activation but not that of native-T. The many similarities between the two channels support the contention that Ca(V)3.2 encodes cardiac T-type Ca2+ channels. The slight differences may be due to species variations and/or to the choice of splice variant.

Nodulisporic acid opens insect glutamate-gated chloride channels: identification of a new high affinity modulator.

Nodulisporic acid (NA) is an indole diterpene fungal product with insecticidal activity. NA activates a glutamate-gated chloride channel (GluCl) in grasshopper neurons and potentiates channel opening by glutamate. The endectocide ivermectin (IVM) induces a similar, but larger current than NA. Using Drosophila melanogaster head membranes, a high affinity binding site for NA was identified. Equilibrium binding studies show that an amide analogue, N-(2-hydroxyethyl-2,2-(3)H)nodulisporamide ([(3)H]NAmide), binds to a single population of sites in head membranes with a K(D) of 12 pM and a B(max) of 1.4 pmol/mg of protein. A similar K(D) is determined from the kinetics of ligand binding and dissociation. Four lines of evidence indicate that the binding site is a GluCl. First, NA potentiates opening of a glutamate-gated chloride current in grasshopper neurons. Second, glutamate inhibits the binding of [(3)H]NAmide by increasing the rate of dissociation 3-fold. Third, IVM potently inhibits the binding of [(3)H]NAmide and IVM binds to GluCls. Finally, the binding of [(3)H]IVM is inhibited by NA. The B(max) of [(3)H]IVM is twice that of [(3)H]NAmide, and about half of the [(3)H]IVM binding sites are inhibited by NA with high affinity (K(I) = 25 pM). In contrast, [(3)H]IVM binding to Caenorhabditis elegans membranes is not inhibited by NA at 100 nM, and there are no high affinity binding sites for NA on these membranes. Thus, half of the Drosophila IVM receptors and all of the NA receptors are associated with GluCl. NA distinguishes between nematode and insect GluCls and identifies subpopulations of IVM binding sites.

Effects of the T-type Ca(2+) channel blocker mibefradil on repolarization of guinea pig, rabbit, dog, monkey, and human cardiac tissue.

At supratherapeutic doses (2- to 5-fold), the T-type Ca(2+) antagonist mibefradil modifies the T/U wave of the human ECG. In this study, we show that this effect is observed in conscious monkeys and is duplicated by verapamil or diltiazem. We then evaluate the proarrhythmic risk of such alterations of cardiac repolarization by examining the actions of mibefradil on cardiac action potentials (APs). In isolated cardiomyocytes from guinea pigs or humans, mibefradil dose dependently shortens the plateau of the AP; this effect is similar to other Ca(2+) antagonists and opposite to drugs having class III antiarrhythmic properties. The metabolites of mibefradil, singly or in combination, also shorten APs. In isolated rabbit hearts, noncardiodepressant concentrations of mibefradil have no effect on monophasic action potentials (MAPs), whereas cardiodepressant levels produce a slight nonsignificant lengthening. In hearts of open-chest bradycardic dogs, mibefradil has no effect on MAP dispersion or on QT interval and shortens MAPs slightly; although high doses produce atrioventricular block, likely through Ca(2+) antagonism, arrhythmias are never observed. In contrast, d-sotalol lengthens QT interval and MAPs, increases dispersion, and produces arrhythmias. Together, these in vitro and in vivo results suggest that mibefradil carries no proarrhythmic risk despite changes in T/U wave morphology. Although these changes resemble those observed with class III compounds, they also are seen with nonproarrhythmic compounds such as verapamil and diltiazem. In conclusion, the classical models used in the present study could not link the changes in T/U wave morphology produced by mibefradil and verapamil to any experimental marker of proarrhythmic liability.

Voltage-gated T-type Ca2+ channels and heart failure.

In the cardiovascular system, two types of voltage-gated Ca2+ channels are present: the L-type and the T-type. Under normal conditions, T-type Ca2+ channels are involved in the maintenance of vascular tone and cardiac automaticity but, since they are not present in contractile myocardial cells, they do not contribute significantly to myocardial contraction. In experimental models of cardiac hypertrophy, myocardial T-type Ca2+ channels are upregulated, which could contribute to the increased incidence of ventricular arrhythmia. In addition, T-type Ca2+ channels participate in the regulation of cell proliferation and neurohormonal secretion; through these pathways, T-type Ca2+ channels might participate in myocardial remodeling. The pathophysiological role of T-type Ca2+ channels in heart failure has been investigated using mibefradil, a Ca2+ antagonist that is 10-50 times more potent at blocking T-type than L-type Ca2+ channels. In contrast with classic L-type Ca2+ channel antagonists, miberfradil appears beneficial in many animal models of heart failure; in particular, it does not exert negative inotropic effects nor does it stimulate the neurohormonal system. Furthermore, in the Pfeffer rat model, blockade of T-type Ca2+ channels with mibefradil is associated with an improved survival rate. In humans, however, major metabolic drug interactions independent of T-type Ca2+ channel blockade made it impossible to determine the efficacy of mibefradil in treating heart failure; indeed, these interactions led to the withdrawal of the drug from the market.

Inhibitory action of mibefradil on calcium signaling and aldosterone synthesis in bovine adrenal glomerulosa cells.

Mibefradil is a new cardiovascular drug with peculiar Ca++ antagonistic properties. The most remarkable feature of mibefradil is its unique relative selectivity for T type calcium channels, a property that has been proposed to explain in part the beneficial pharmacological and clinical profiles of this drug. In adrenal glomerulosa cells, aldosterone biosynthesis and secretion in response to angiotensin II or extracellular potassium is dependent on a sustained influx of Ca++ through T type Ca++ channels. The effect of mibefradil on the steroidogenic function of glomerulosa cells was therefore investigated. Using the patch clamp technique, we found that mibefradil inhibits selectively and in a concentration-dependent manner (IC50 = 3 microM)++ T type currents in bovine glomerulosa cells. In addition to this tonic (voltage independent) inhibition, the drug also induced a shift of the steady-state inactivation curve of these channels toward hyperpolarized voltages, contributing to its efficacy to prevent Ca++ influx into the cell through T type channels. Concomitantly, mibefradil reduced the cytosolic calcium responses to potassium and angiotensin II (as assessed with fluorescent probes), without affecting the capacitative Ca++ influx, and inhibited pregnenolone and aldosterone formation. This inhibition of steroidogenesis was not exclusively due to mibefradil action on voltage-operated Ca++ channels, because this agent also partially reduced steroid synthesis induced by adrenocorticotropic hormone or forskolin, two activators of the cyclic AMP pathway. In conclusion, mibefradil is highly effective in adrenal glomerulosa cells in reducing T type channel activity and aldosterone biosynthesis, two actions that should contribute to the beneficial effect of the drug in the treatment of hypertension.

Low-voltage-activated T-type Ca2+ channels.

Although progress in our understanding of T channels and their physiological role has been slower than with other Ca2+ channels, it was clear during this two-day workshop that interest and research in the field remain very intense. Advances have been hampered by many factors: small current amplitude, lack of pharmacological tools, apparent heterogeneity, and lack of a cloned channel. Nevertheless, many interesting roles for T channels have been described, which point to a generally subtle modulatory action. Furthermore, recent results suggest that the above barriers might soon be abolished: new pharmacological tools (mibefradil and newer generation compounds) with T-channel selectivity are being developed and many groups claim to be close to cloning a T channel.

Discovery and main pharmacological properties of mibefradil (Ro 40-5967), the first selective T-type calcium channel blocker.

UNLABELLED: PROPERTIES OF MIBEFRADIL: Mibefradil is a novel calcium channel antagonist with structural and pharmacological characteristics clearly distinct from those of classical calcium antagonists. It is a potent vasodilator with a high selectivity for the coronary vasculature over the peripheral vasculature and the myocardium. Most importantly, this compound can relax vascular muscle and slow the heart rate without reducing cardiac contractility. In addition, it does not stimulate neurohormonal reflexes and it exhibits a good pharmacological profile characterized by a long duration of action. MECHANISM OF ACTION: The mechanism of action of mibefradil is characterized by the selective blockade of transient, low-voltage-activated (T-type) calcium channels over long-lasting, high-voltage-activated (L-type) calcium channels, which is probably responsible for many of its unique properties. CLINICAL USE OF MIBEFRADIL: Although calcium antagonists are mainly used for the treatment of hypertension and angina pectoris, there is strong preclinical evidence that mibefradil may also be beneficial in the treatment of congestive heart failure.

T-type Ca2+ channels and pharmacological blockade: potential pathophysiological relevance.

Low-voltage-activated T-type Ca2+ channels are present in most excitable tissues including the heart (mainly pacemaker cells), smooth muscle, central and peripheral nervous systems, and endocrine tissues, but also in non-excitable cells, such as osteoblasts, fibroblasts, glial cells, etc. Although they comprise a slightly heterogeneous population, these channels share many defining characteristics: small conductance (< 10 pS), similar Ca2+ and Ba2+ permeabilities, slow deactivation, and a voltage-dependent inactivation rate. In addition, activation at low voltages, rapid inactivation, and blockade by Ni2+ are classical properties of T-type Ca2+ channels, which are less specific. T-type Ca2+ channels are weakly blocked by standard Ca2+ antagonists. Pharmacological blockers are scarce and often lack specificity and/or potency. The physiological modulation of T-type Ca2+ currents is complex: they are enhanced by endothelin-1, angiotensin II (AT1-receptor), ATP, and isoproterenol (cAMP-independent), but are reduced by angiotensin II (AT2-receptor), somatostatin and atrial natriuretic peptide. Norepinephrine enhances these currents in some cells but decreases them in others. T-type Ca2+ currents have many known or suggested physiological and pathophysiological roles in growth (protein synthesis, cell differentiation, and proliferation), neuronal firing regulation, some aspects of genetic hypertension, cardiac hypertrophy, cardiac fibrosis, cardiac rhythm (normal and abnormal), and atherosclerosis. Mibefradil is a new Ca2+ antagonist that is effective in hypertension and angina pectoris. Its favorable pharmacological profile and limited side effects appear to be related to selective block of T-type Ca2+ channels: mibefradil reduces vascular resistance and heart rate without negative inotropy or neurohormonal stimulation, and it also has significant antiproliferative actions.

Voltage-gated calcium currents have two opposing effects on the secretion of aldosterone.

Using Ca2+ channel blockers with different specificities for L- and T-type Ca2+ channels, we have investigated the roles of these two channel types in K(+)-induced aldosterone secretion. In whole cell voltage-clamp experiments, the spider toxin omega-agatoxin-IIIA (omega-Aga-IIIA) completely blocks L-type Ca2+ channels but has no effect on T-type Ca2+ channels. In contrast, Ni2+ and 1,4-dihydropyridines block both L- and T-type Ca2+ channels. Secretion induced by 7 mM extracellular K+ concentration ([K+]o) is unaffected by omega-Aga-IIIA but is strongly inhibited by Ni2+ or the 1,4-dihydropyridine, nitrendipine. This suggests that physiological increases in [K+]o stimulate aldosterone secretion primarily by enhancing Ca2+ entry through T-type Ca2+ channels. Surprisingly, secretion induced by 60 mM [K+]o is enhanced by omega-Aga-IIIA or Ni2+ and is inhibited by the L-type Ca2+ channel activator BAY K 8644. Nitrendipine (1 nM) also stimulates such secretion, although higher concentrations are inhibitory (concentration inhibiting 50% of maximal response approximately 30 nM). If extracellular Ca2+ concentration is reduced from 1.25 to 0.5 mM, secretion induced by 60 mM [K+]o is enhanced, and Ni2+ or low nitrendipine become inhibitory. Together, these results that L-type Ca2+ currents can reduce steroidogenesis and that the role of these currents was previously misconstrued because 1,4-dihydropyridines modify secretion by multiple mechanisms. Thus Ca2+ entry can function as a negative modulator of steroid secretion.

Modulation of guanylate cyclase and phosphodiesterase by monovalent cations and nucleoside triphosphates in light-sensitive excised patches of rod outer segments.

Excised inside-out patches of vertebrate rod outer segment can support phototransduction. I have examined how ionic and metabolic conditions influence the functional properties of light-sensitive patches from Gekko gekko. I find that such patches retain a variable level of basal phosphodiesterase activity, which lowers the cyclic guanosine monophosphate (cGMP) concentration reaching the channels and reduces the dark current. The dose/response relationship for channel opening by cGMP varies among patches and this variability is only reduced by working in darkness with the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX), suggesting that it is only partially due to phosphodiesterase activity. MgATP or MgGTP, but not Mg or ATP separately, increase this activity but a kinase does not appear to be involved. Intracellular monovalent cations also influence dark current intensity and light response kinetics. With 5 mM MgGTP, 1 mM IBMX, and 144 mM Li+, Na+, K+, or Rb+, dark current intensity and recovery time follow the respective sequences K+ > Rb+ > Na+ > Li+ and K+ < Rb+ < Li+ < Na+. Without IBMX, a dark current develops with K+ but not with Na+. These effects are not due to altered channel permeability (P) [PLi+:Na+:K+:Rb+:guanidinium)/PNa+ = 0.84:1.00:1.01:1.09:0.42], or differential Mg2+ block, but to modulation of guanylate cyclase, which overcomes phosphodiesterase when the major cation is K+ but not when it is Na+.

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.

Type III omega-agatoxins: a family of probes for similar binding sites on L- and N-type calcium channels.

The peptide omega-agatoxin-IIIA (omega-Aga-IIIA) from venom of the funnel web spider Agelenopsis aperta is the only known agent that blocks L-type and N-type Ca channels with equal high potency (IC50 < or = 1 nM). From the same venom, we have purified and sequenced a family of peptides which are homologous to omega-Aga-IIIA but vary over 100-fold in their relative affinity for L-type versus N-type Ca channels. One of these, omega-Aga-IIIB, is 76 amino acids long and identical to omega-Aga-IIIA in 66 positions. We identified two other similar peptides, omega-Aga-IIIC and omega-Aga-IIID, as well as one single amino acid variant of omega-Aga-IIIA and two of omega-Aga-IIIB. The type III omega-agatoxins exhibit similar but distinct activities on voltage-gated Ca channels. omega-Aga-IIIA, omega-Aga-IIIB, and omega-Aga-IIID are nearly indistinguishable in their actions at the insect neuromuscular junction (no effect at 0.1 microM), on atrial T-type Ca channels (no effect at 0.5 microM), and in two assays for synaptosomal Ca channels: they are nearly equipotent inhibitors of 125I-omega-conotoxin GVIA binding to rat brain synaptic membranes (IC50 = 0.17-0.33 nM) and blockers of the K(+)-induced 45Ca2+ influx into chick brain synaptosomes (omega-Aga-IIIB, 1.2 nM; omega-Aga-IIIA, 2.4 nM). In contrast, omega-Aga-IIIA is a better blocker of locust Ca channels (IC50 approximately 10-50 nM) than is omega-Aga-IIIB. Finally, although omega-Aga-IIIA, omega-Aga-IIIB, and omega-Aga-IIID all block atrial L-type Ca channels, omega-Aga-IIIA is over 100-fold more potent. Thus, although type III omega-agatoxins appear to recognize a binding site common to L- and N-type Ca channels, omega-Aga-IIIB and omega-Aga-IIID identify differences between the two channels.

High affinity block of myocardial L-type calcium channels by the spider toxin omega-Aga-toxin IIIA: advantages over 1,4-dihydropyridines.

The peptide omega-agatoxin IIIA (omega-Aga-IIIA) from venom of the funnel web spider Agelenopsis aperta blocks L-type Ca2+ channels in neurons and myocardial cells with high affinity. We report that omega-Aga-IIIA also blocks whole-cell Ca2+ channel currents in guinea pig atrial myocytes. Although other high affinity blockers of L-type Ca2+ channels are available (such as the 1,4-dihydropyridines), omega-Aga-IIIA is a valuable pharmacological tool; omega-Aga-IIIA is the only known ligand that blocks L-type Ca2+ channels with high affinity at all voltages (IC50 approximately 1 nM) and it causes little or no block of T-type Ca2+ channels, unlike the 1,4-dihydropyridines. We use omega-Aga-IIIA to selectively eliminate L-type Ca2+ currents and we show that felodipine blocks T-type Ca2+ currents. Consequently, the toxin is better than dihydropyridines for separating ionic currents through voltage-dependent Ca2+ channels and defining their physiological function.

Excised patches of plasma membrane from vertebrate rod outer segments retain a functional phototransduction enzymatic cascade.

Ion channels in excised patches of plasma membrane are generally considered to be isolated from any intracellular regulation mechanisms. For example, in excised patches of vertebrate rod outer segment plasma membrane, the cGMP-activated cation channels have traditionally been studied in room light because the enzyme cascade linking photon absorption to channel closure was assumed to be inoperative. To investigate the possibility that, in fact, such excised patches retain a functional phototransduction enzymatic cascade, this same preparation was studied in darkness. Patches excised in the dark were found to retain the light sensitivity of their cGMP-induced conductance and the ability to synthesize cGMP. In the presence of guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]), a nonhydrolyzable GTP analog, light suppresses the cGMP-induced conductance irreversibly. Furthermore, inhibitors of phosphodiesterase activity reduce light sensitivity, whereas activated phosphodiesterase or activated transducin does not directly affect the channels. These results (i) establish that excised patches from rod outer segment retain functional phototransduction enzymes, (ii) support the classical view that channel opening is modulated by phosphodiesterase-mediated cGMP hydrolysis, and, most surprisingly, (iii) demonstrate that diffusion in excised patches is so restricted that local enzymes can induce variations in the concentration of small molecules. The indication that excised patches are not as simple as usually surmised opens the possibility of using them to study other intracellular transduction mechanisms.