Candelalides A-C: novel diterpenoid pyrones from fermentations of Sesquicillium candelabrum as blockers of the voltage-gated potassium channel Kv1.3.

[figure: see text] Blockers of the voltage-gated potassium channel Kv1.3 are potential immunosuppressants. Candelalides A-C are three novel diterpenoid pyrones that block this channel. The structure, stereochemistry, and activity against Kv1.3 are described.

Binding of correolide to K(v)1 family potassium channels. Mapping the domains of high affinity interaction.

Correolide, a novel nortriterpene natural product, potently inhibits the voltage-gated potassium channel, K(v)1.3, and [(3)H]dihydrocorreolide (diTC) binds with high affinity (K(d) approximately 10 nM) to membranes from Chinese hamster ovary cells that express K(v)1.3 (Felix, J. P., Bugianesi, R. M., Schmalhofer, W. A., Borris, R., Goetz, M. A., Hensens, O. D., Bao, J.-M., Kayser, F. , Parsons, W. H., Rupprecht, K., Garcia, M. L., Kaczorowski, G. J., and Slaughter, R. S. (1999) Biochemistry 38, 4922-4930). Mutagenesis studies were used to localize the diTC binding site and to design a high affinity receptor in the diTC-insensitive channel, K(v)3.2. Transferring the pore from K(v)1.3 to K(v)3.2 produces a chimera that binds peptidyl inhibitors of K(v)1.3 with high affinity, but not diTC. Transfer of the S(5) region of K(v)1.3 to K(v)3.2 reconstitutes diTC binding at 4-fold lower affinity as compared with K(v)1.3, whereas transfer of the entire S(5)-S(6) domain results in a normal K(v)1.3 phenotype. Substitutions in S(5)-S(6) of K(v)1.3 with nonconserved residues from K(v)3.2 has identified two positions in S(5) and one in S(6) that cause significant alterations in diTC binding. High affinity diTC binding can be conferred to K(v)3.2 after substitution of these three residues with the corresponding amino acids found in K(v)1.3. These results suggest that lack of sensitivity of K(v)3.2 to diTC is a consequence of the presence of Phe(382) and Ile(387) in S(5), and Met(458) in S(6). Inspection of K(v)1.1-1.6 channels indicates that they all possess identical S(5) and S(6) domains. As expected, diTC binds with high affinity (K(d) values 7-21 nM) to each of these homotetrameric channels. However, the kinetics of binding are fastest with K(v)1.3 and K(v)1.4, suggesting that conformations associated with C-type inactivation will facilitate entry and exit of diTC at its binding site. Taken together, these findings identify K(v)1 channel regions necessary for high affinity diTC binding, as well as, reveal a channel conformation that markedly influences the rate of binding of this ligand.

Identification and biochemical characterization of a novel nortriterpene inhibitor of the human lymphocyte voltage-gated potassium channel, Kv1.3.

A novel nortriterpene, termed correolide, purified from the tree Spachea correae, inhibits Kv1.3, a Shaker-type delayed rectifier potassium channel present in human T lymphocytes. Correolide inhibits 86Rb+ efflux through Kv1.3 channels expressed in CHO cells (IC50 86 nM; Hill coefficient 1) and displays a defined structure-activity relationship. Potency in this assay increases with preincubation time and with time after channel opening. Correolide displays marked selectivity against numerous receptors and voltage- and ligand-gated ion channels. Although correolide is most potent as a Kv1.3 inhibitor, it blocks all other members of the Kv1 family with 4-14-fold lower potency. C20-29-[3H]dihydrocorreolide (diTC) was prepared and shown to bind in a specific, saturable, and reversible fashion (Kd = 11 nM) to a single class of sites in membranes prepared from CHO/Kv1.3 cells. The molecular pharmacology and stoichiometry of this binding reaction suggest that one diTC site is present per Kv1.3 channel tetramer. This site is allosterically coupled to peptide and potassium binding sites in the pore of the channel. DiTC binding to human brain synaptic membranes identifies channels composed of other Kv1 family members. Correolide depolarizes human T cells to the same extent as peptidyl inhibitors of Kv1.3, suggesting that it is a candidate for development as an immunosuppressant. Correolide is the first potent, small molecule inhibitor of Kv1 series channels to be identified from a natural product source and will be useful as a probe for studying potassium channel structure and the physiological role of such channels in target tissues of interest.

Complex subunit assembly of neuronal voltage-gated K+ channels. Basis for high-affinity toxin interactions and pharmacology.

Neurons require specific patterns of K+ channel subunit expression as well as the precise coassembly of channel subunits into heterotetrameric structures for proper integration and transmission of electrical signals. In vivo subunit coassembly was investigated by studying the pharmacological profile, distribution, and subunit composition of voltage-gated Shaker family K+ (Kv1) channels in rat cerebellum that are labeled by 125I-margatoxin (125I-MgTX; Kd, 0.08 pM). High-resolution receptor autoradiography showed spatial receptor expression mainly in basket cell terminals (52% of all cerebellar sites) and the molecular layer (39% of sites). Sequence-directed antibodies indicated overlapping expression of Kv1. 1 and Kv1.2 in basket cell terminals, whereas the molecular layer expressed Kv1.1, Kv1.2, Kv1.3, and Kv1.6 proteins. Immunoprecipitation experiments revealed that all 125I-MgTX receptors contain at least one Kv1.2 subunit and that 83% of these receptors are heterotetramers of Kv1.1 and Kv1.2 subunits. Moreover, 33% of these Kv1.1/Kv1.2-containing receptors possess either an additional Kv1.3 or Kv1.6 subunit. Only a minority of the 125I-MgTX receptors (<20%) seem to be homotetrameric Kv1.2 channels. Heterologous coexpression of Kv1.1 and Kv1.2 subunits in COS-1 cells leads to the formation of a complex that combines the pharmacological profile of both parent subunits, reconstituting the native MgTX receptor phenotype. Subunit assembly provides the structural basis for toxin binding pharmacology and can lead to the association of as many as three distinct channel subunits to form functional K+ channels in vivo.

Margatoxin binds to a homomultimer of K(V)1.3 channels in Jurkat cells. Comparison with K(V)1.3 expressed in CHO cells.

Voltage-gated potassium (K(V)) channels play key roles in setting the resting potential and in the activation cascade of human peripheral T lymphocytes. Margatoxin (MgTX), a 39-amino acid peptide from Centruroides margaritatus, is a potent inhibitor of lymphocyte K(V) channels. The binding of monoiodotyrosinyl margatoxin ([125I]MgTX) to plasma membranes prepared from either Jurkat cells, a human leukemic T cell line, or CHO cells stably transfected with the Shaker-type voltage-gated K+ channel, K(V)1.3, has been used to investigate the properties of lymphocyte K(V) channels. These data were compared with [125I]MgTX binding to heterotetrameric K(V) channels in rat brain synaptic plasma membranes [Knaus, H. G., et al. (1995) Biochemistry 34, 13627-13634]. The affinity for [125I]MgTX is 100-200 fM in either Jurkat or CHO/K(V)1.3 membranes, and the receptor density is 20-120 fmol/mg in Jurkat membranes or 1000 fmol/mg in CHO/K(V)1.3 membranes. In contrast to rat brain, [125I]MgTX binding to Jurkat and CHO/K(V)1.3 membranes exhibits an absolute requirement for K+, with no potentiation of binding by Na+. K(V)1.3 was the only K(V)1 series channel present in either CHO/K(V)1.3 or Jurkat plasma membranes as determined by immunoprecipitation of [125I]MgTX binding or by Western blot analyses using sequence-specific antibodies prepared against members of the K(V)1 family. The relative potencies of a series of peptidyl K(V) channel inhibitors was essentially the same for inhibition of [125I]MgTX binding to Jurkat, CHO, or rat brain membranes and for blocking 86Rb+ efflux from the CHO/K(V)1.3 cells, except that alpha-dendrotoxin was more potent at blocking binding to rat brain membranes than in the other assays. The characteristics of [125I]MgTX binding, the antibody profiles, and the effects of the peptidyl K(V) inhibitors all indicate that the [125I]MgTX receptor in Jurkat lymphocytes is comprised of a homomultimer of K(V)1.3, unlike the heteromultimeric arrangement of the receptor in rat brain.

[125I]margatoxin, an extraordinarily high affinity ligand for voltage-gated potassium channels in mammalian brain.

Monoiodotyrosine margatoxin ([125I]MgTX) specifically and reversibly labels a maximum of 0.8 pmol of sites/mg of protein in purified rat brain synaptic plasma membrane vesicles with a dissociation constant of 0.1 pM under equilibrium binding conditions. This Kd value was confirmed by kinetic experiments (Kd of 0.07 pM), competition assays employing native margatoxin (MgTX) (Ki of 0.15 pM), and receptor saturation studies (Kd of 0.18 pM). Thus, this toxin represents the highest affinity, reversible radioligand for any membrane-bound receptor or ion channel described to date. [125I]MgTX binding in this system is modulated by charybdotoxin (Ki of 5 pM), kaliotoxin (Ki of 1.5 pM), and the agitoxins I and II (Ki's of 0.1 and 0.3 pM, respectively), in a noncompetitive manner. Moreover, alpha-dendrotoxin displayed a Ki value of 0.5 pM. Iberiotoxin was without any effect, suggesting that the receptor site is likely to be associated with a voltage-gated K+ channel complex. [125I]MgTX binding is inhibited by cations that are established blockers of voltage-dependent K+ channels (Ba2+, Ca2+, Cs+). The monovalent cations Na+ and K+ stimulate binding at low concentrations before producing complete inhibition as their concentrations are increased. Stimulation of binding results from an allosteric interaction that decreases Kd, whereas inhibition is due to an ionic strength effect. Affinity labeling of the binding site in rat brain synaptic plasma membranes employing [125I]MgTX and the bifunctional cross-linking reagent, disuccinimidyl suberate, causes specific and covalent incorporation of toxin into a glycoprotein of an apparent molecular weight (M(r)) of 74,000. Deglycosylation studies reveal an M(r) for the core polypeptide of the MgTX receptor of 63,000.(ABSTRACT TRUNCATED AT 250 WORDS)

Charybdotoxin and its effects on potassium channels.

Over the last few years, a considerable amount of information has been obtained regarding K+ channels. Different areas of research have contributed to knowledge in this field. Charybdotoxin (ChTX), a 37-amino acid peptide isolated from venom of the scorpion Leiurus quinquestriatus var. hebraeus, represents a remarkable tool for studying K+ channels. With its use, it has been possible to purify the high-conductance Ca(2+)-activated K+ (maxi-K) channel to homogeneity and determine the subunit composition of this channel. This has led to the discovery of an auxiliary beta-subunit that, when coexpressed with the pore-forming subunit, mSlo, alters the biophysical and pharmacological properties of this latter subunit. With the feasibility of producing large amounts of ChTX by recombinant techniques and the knowledge of the three-dimensional structure of the peptide, it has been possible to carry out site-directed mutagenesis studies and obtain a picture of the interaction surface of the toxin with two channels, maxi-K and Shaker, and to derive a picture of the complementary surface of the receptor in these two channels. Finally, ChTX, and the more selective K+ channel toxins that were subsequently discovered, have provided us with unique tools not only to determine the functional role that K+ channels play in target tissues but also to develop the molecular pharmacology of these channels.

Voltage-gated potassium channels regulate calcium-dependent pathways involved in human T lymphocyte activation.

The role that potassium channels play in human T lymphocyte activation has been investigated by using specific potassium channel probes. Charybdotoxin (ChTX), a blocker of small conductance Ca(2+)-activated potassium channels (PK,Ca) and voltage-gated potassium channels (PK,V) that are present in human T cells, inhibits the activation of these cells. ChTX blocks T cell activation induced by signals (e.g., anti-CD2, anti-CD3, ionomycin) that elicit a rise in intracellular calcium ([Ca2+]i) by preventing the elevation of [Ca2+]i in a dose-dependent manner. However, ChTX has no effect on the activation pathways (e.g., anti-CD28, interleukin 2 [IL-2]) that are independent of a rise in [Ca2+]i. In the former case, both proliferative response and lymphokine production (IL-2 and interferon gamma) are inhibited by ChTX. The inhibitory effect of ChTX can be demonstrated when added simultaneously, or up to 4 h after the addition of the stimulants. Since ChTX inhibits both PK,Ca and PK,V, we investigated which channel is responsible for these immunosuppressive effects with the use of two other peptides, noxiustoxin (NxTX) and margatoxin (MgTX), which are specific for PK,V. These studies demonstrate that, similar to ChTX, both NxTX and MgTX inhibit lymphokine production and the rise in [Ca2+]i. Taken together, these data provide evidence that blockade of PK,V affects the Ca(2+)-dependent pathways involved in T lymphocyte proliferation and lymphokine production by diminishing the rise in [Ca2+]i that occurs upon T cell activation.

Bis(benzylisoquinoline) analogs of tetrandrine block L-type calcium channels: evidence for interaction at the diltiazem-binding site.

Bis(benzylisoquinoline) alkaloids block Ca2+ uptake through the L-type Ca2+ channel and modulate binding of ligands to four distinct sites (dihydropyridine, benzothiazepine, aralkylamine, and (diphenylbutyl)piperidine) in the Ca2+ entry blocker receptor complex of the channel. These alkaloids are structural analogs of tetrandrine, which has previously been demonstrated to block the L-type Ca2+ channel through interaction at the benzothiazepine (diltiazem) site (King et al., 1988). Different alkaloid conformational classes display either alpha-beta, beta-alpha, alpha-alpha, or beta-beta stereochemistry at the two chiral isoquinoline carbons. Compounds from all four classes were tested for their ability to interact with Ca2+ entry blocker ligands. All analogs completely inhibit diltiazem binding, but many only partially inhibit D-600 and fluspirilene binding. For dihydropyridine binding, the compounds show either stimulation or inhibition or exhibit no effect. This profile is quite different from the interaction displayed by diltiazem or tetrandrine. Scatchard analyses show effects predominantly on Kd for diltiazem, D-600, and PN200-110 binding. Representative conformers do not effect diltiazem dissociation rates but alter dissociation kinetics of ligands which bind to the other three sites. A correlation of the ability of these compounds to inhibit Ca2+ uptake through the L-type Ca2+ channel in GH3 cells exists only with their inhibition of diltiazem binding but not with inhibition of binding of ligands representing other classes of Ca2+ entry blockers. These data, taken together, indicate that a variety of bis(benzylisoquinoline) congeners act to block the L-type Ca2+ channel by binding to the benzothiazepine site on the channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective blockers of voltage-gated K+ channels depolarize human T lymphocytes: mechanism of the antiproliferative effect of charybdotoxin.

Charybdotoxin (ChTX), a K+ channel blocker, depolarizes human peripheral T lymphocytes and renders them insensitive to activation by mitogen. We observed four types of K+ channels in human T cells: one voltage-activated, and three Ca(2+)-activated. To discern the mechanism by which ChTX depolarizes T cells, we examined the sensitivity of both the voltage-activated and Ca(2+)-activated K+ channels to ChTX and other peptide channel blockers. All four types were blocked by ChTX, whereas noxiustoxin and margatoxin blocked only the voltage-activated channels. All three toxins, however, produced equivalent depolarization in human T cells. We conclude that the membrane potential of resting T cells is set by voltage-activated channels and that blockade of these channels is sufficient to depolarize resting human T cells and prevent activation.

Amiloride analogs inhibit L-type calcium channels and display calcium entry blocker activity.

Three structural classes of commonly used amiloride analogs, molecules derivatized at the terminal guanidino-nitrogen, the five-position pyrazinoyl-nitrogen, or di-substituted at both of these positions, inhibit binding of the L-type Ca2+ channel modulators diltiazem, gallopamil, and nitrendipine to porcine cardiac sarcolemmal membrane vesicles. The rank order of inhibitory potencies among the various derivatives tested is well defined with amiloride being the least potent. Saturation binding studies indicate that inhibition of ligand binding results primarily from effects on Kd. Ligand dissociation measurements suggest that amiloride derivatives do not associate directly at any of the known sites in the Ca2+ entry blocker receptor complex. In addition, these compounds do not compete at the "Ca2+ coordination site" within the channel. However, studies with inorganic and substituted diphenylbutylpiperidine Ca2+ entry blockers reveal that amiloride analogs interact at a site on the channel where metal ions bind and occlude the pore. Photolysis experiments performed with amiloride photoaffinity reagents confirm that a specific interaction occurs between such probes and the channel protein. Upon photolysis, these agents produce concentration- and time-dependent irreversible inactivation of Ca2+ entry blocker binding activities, which can be protected against by either verapamil or diltiazem. 45Ca2+ flux and voltage-clamp experiments performed with GH3 anterior pituitary cells demonstrate that amiloride-like compounds inhibit L-type Ca2+ channels directly. Moreover, these compounds block contraction of isolated vascular tissue in pharmacological assays. Electrophysiological experiments indicate that they also inhibit T-type Ca2+ channels in GH3 cells. Taken together, these results demonstrate unequivocally that amiloride analogs display significant Ca2+ entry blocker activity in both ligand binding and functional assays. This property, therefore, can seriously complicate the interpretation of many in vitro and in vivo studies where amiloride analogs are used to elicit inhibition of other transport systems (e.g. Na-Ca and Na-H exchange).

Characterization of high affinity binding sites for charybdotoxin in sarcolemmal membranes from bovine aortic smooth muscle. Evidence for a direct association with the high conductance calcium-activated potassium channel.

Charybdotoxin (ChTX), a peptidyl inhibitor of the high conductance Ca2+-activated K+ channel (PK,Ca), has been radiolabeled to high specific activity with 125I, and resulting derivatives have been well separated. The monoiodotyrosine adduct blocks PK,Ca in vascular smooth muscle with slightly reduced potency compared with the native peptide under defined experimental conditions. [125I]ChTX, representing this derivative, binds specifically and reversibly to a single class of sites in sarcolemmal membrane vesicles prepared from bovine aortic smooth muscle. These sites display a Kd of 100 pM for the iodinated toxin, as determined by either equilibrium or kinetic binding analyses. Binding site density is about 500 fmol/mg of protein in isolated membranes. The addition of low digitonin concentrations to disrupt the vesicle permeability barrier increases the maximum receptor concentration to 1.5 pmol/mg of protein, correlating with the observations that ChTX binds only at the external pore of PK,Ca and that the membrane preparation is of mixed polarity. Competition studies with ChTX yield a Ki of about 20 pM for native toxin. Binding of [125I]ChTX is modulated by ionic strength as well as by metal ions that are known to interact with PK,Ca. Moreover, tetraethylammonium ion, which blocks PK,Ca with moderately high affinity when applied at the external membrane surface, inhibits [125I]ChTX binding in an apparently competitive fashion with a Ki similar to that found for channel inhibition. In marked contrast, agents that do not inhibit PK,Ca in smooth muscle (e.g. tetrabutylammonium ion, other toxins homologous with ChTX, and pharmacological agents that modulate the activity of dissimilar ion channels) have no effect on [125I]ChTX binding in this tissue. Taken together, these results suggest that the binding sites for ChTX which are present in vascular smooth muscle are directly associated with PK,Ca, thus identifying [125I]ChTX as a useful probe for elucidating the biochemical properties of these channels.

High levels of sodium-calcium exchange in vascular smooth muscle sarcolemmal membrane vesicles.

Membrane vesicles which exhibit high levels of Nai-dependent Ca2+ uptake have been prepared from either porcine or bovine aortic smooth muscle. These membranes are identified as being of sarcolemmal origin by enrichment of marker activities associated with the sarcolemma (e.g., binding of the ligands PN 200-110, iodocyanopindolol, and ouabain). The Vmax of Na-Ca exchange in the two aortic sarcolemmal preparations [0.5-3.5 nmol s-1 (mg of protein)-1] is significantly higher than that previously reported with membrane preparations derived from visceral and vascular smooth muscle and compares favorably with maximal values recorded in cardiac sarcolemmal membrane vesicles [5-20 nmol-1 s-1 (mg of protein)-1] under identical experimental conditions. The Km of Ca2+ (15 +/- 5 microM) and the Km of Na+ (15 +/- 7 mM) are similar values as determined in heart. Aortic and cardiac Na-Ca exchange activities are equivalent in their sensitivity to inhibition by La3+ and two known classes of mechanism-based organic blockers of transport activity (i.e., amiloride analogues and bepridil-like agents). Both also display electrogenic behavior. However, Li+, K+, and choline all inhibit the smooth muscle transporter with markedly greater potency than found in heart, and intravesicular Ca2+ does not affect transport activity in smooth muscle membranes as it does in the cardiac system. When maximal transport velocities are compared, aortic membrane vesicles have 3-6-fold higher Na-Ca exchange than sarcolemmal Ca2+-ATPase Ca2+ transporting capacities.(ABSTRACT TRUNCATED AT 250 WORDS)

Substituted diphenylbutylpiperidines bind to a unique high affinity site on the L-type calcium channel. Evidence for a fourth site in the cardiac calcium entry blocker receptor complex.

Fluspirilene binds with high affinity to a single class of sites in purified porcine cardiac sarcolemmal membrane vesicles at a Kd of 0.6 nM and a Bmax that is in approximately 1:1 stoichiometry with other Ca2+ entry blocker receptors. Fluspirilene binding is modulated by various classes of L-type Ca2+ channel effectors. Metal ion channel inhibitors (e.g. Cd2+) stimulate binding primarily by increasing ligand affinity, whereas channel substrates (e.g. Ca2+) inhibit binding. Dihydropyridine, aralkylamine, and benzothiazepine Ca2+ entry blockers partially inhibit binding with Ki values equivalent to their respective Kd values, indicating close coupling between binding sites for the former agents and the diphenylbutylpiperidine site. All of these agents function as mixed inhibitors and affect both Kd and Bmax of fluspirilene binding. Only other substituted diphenylbutylpiperidines (e.g. pimozide) inhibit binding competitively. Diphenylbutylpiperidines, on the other hand, block nitrendipine, D-600, and diltiazem binding through a noncompetitive mechanism with Ki values much reduced from their measured Kd values, suggesting that coupling between the diphenylbutylpiperidine site and receptors for diverse Ca2+ entry blockers is more indirect. In addition, high affinity sites have been detected for fluspirilene in bovine aortic sarcolemmal vesicles, rat brain synaptic membranes, and GH3 rat anterior pituitary cell plasma membranes. Fluspirilene also effectively blocks Ca2+ flux through L-type Ca2+ channels in GH3 cells. Together, these results suggest that fluspirilene binds with high affinity to a unique fourth site in the Ca2+ entry blocker receptor complex and that substituted diphenylbutylpiperidines represent a new structural class of potent L-type Ca2+ channel inhibitors.

Inhibition of sodium-calcium exchange in cardiac sarcolemmal membrane vesicles. 1. Mechanism of inhibition by amiloride analogues.

The mechanism by which terminal guanidino nitrogen substituted analogues of amiloride inhibit Na-Ca exchange in purified cardiac sarcolemmal membrane vesicles has been investigated. These inhibitors block both Nai-dependent Ca2+ uptake and Nao-dependent Ca2+ efflux. Inhibition of Na-Ca exchange monitored in K+ is noncompetitive vs Ca2+ but competitive vs Na+. Substitution of sucrose for K+ results in mixed kinetics of inhibition vs Ca2+, suggesting a complex interaction between inhibitor and carrier under this condition. Amiloride derivatives also block two other modes of carrier action: Na-Na exchange is inhibited in a competitive fashion with Na+ and kinetics of Ca-Ca exchange inhibition are mixed vs Ca2+ in either sucrose or K+. However, Ca-Ca exchange inhibition can be alleviated by increasing K+ concentration. Dixon analyses of Na-Ca exchange block with mixtures of inhibitors suggest that these agents are interacting at more than one site. In addition, Hill plots of inhibition are biphasic with Hill coefficients of 1 and 2 at low and high inhibitor concentrations, respectively. These results indicate that amiloride derivatives are mechanism-based inhibitors that interact at two classes of substrate-binding sites on the carrier; at low concentration they bind preferentially to a site that is exclusive for Na+, while at higher concentration they also interact at a site that is common for Na+, Ca2+, and K+.

Inhibition of sodium-calcium exchange in cardiac sarcolemmal membrane vesicles. 2. Mechanism of inhibition by bepridil.

Bepridil, an antiarrhythmic agent, inhibits Na-Ca exchange in cardiac sarcolemmal membrane vesicles (Ki = 30 microM) by a novel mechanism, different from that determined for amiloride analogues [Slaughter, R. S., Garcia, M. L., Cragoe, E. J., Jr., Reeves, J. P., & Karczorowski, G. J. (1988) Biochemistry (preceding paper in this issue)]. Bepridil causes partial inhibition of Nai-dependent Ca2+ uptake but complete block of Nao-dependent Ca2+ efflux. Inhibition of Na-Ca exchange is noncompetitive vs Ca2+ but competitive vs Na+ in both K+ and sucrose. Bepridil also blocks Ca-Ca exchange, with or without K+ present. However, K+ has two effects on inhibition: it reduces the potency of bepridil and causes inhibition to become partial. Inhibition of Ca-Ca exchange displays noncompetitive kinetics vs Ca2+ in either sucrose or K+. Dixon analyses of Na-Ca exchange inhibition caused by mixtures of bepridil and amiloride analogues demonstrate that these compounds produce a competitive interaction at a common carrier site that is evident only at low concentrations of amiloride inhibitors. Hill plots of bepridil inhibition of Na-Ca and Ca-Ca exchange display unitary Hill coefficients. These results indicate that bepridil interacts at only one substrate-binding site, the site selective for Na+, where amiloride analogues also preferentially interact. However, unlike amiloride, bepridil does not interact at the common Na+, Ca2+-binding site of the carrier.(ABSTRACT TRUNCATED AT 250 WORDS)