The devapamil-binding site of the purified skeletal muscle receptor for organic-calcium channel blockers is modulated by micromolar and millimolar concentrations of Ca2+.

The interaction of 2,7-dimethyl-3-(3,4-dimethoxyphenyl)-3-cyan-7-aza-9-(3- methoxyphenyl) nonahydrochloride (devapamil), a stereospecific analog of (3-[2-(3,4-dimethoxyphenyl)ethyl]- methylaminopropyl-3,4-dimethoxy-(1-methylethyl)benzeneacetonitr ile (verapamil), with the purified skeletal muscle receptor for calcium channel blockers (CaCB) was studied at 4 degrees C and 30 degrees C in the absence and presence of calcium. The purified CaCB receptor bound 0.9 mol devapamil/mol calcium-channel alpha 1 subunit, with an apparent Kd of 13 +/- 2.6 nM at 4 degrees C in the presence of 0.4 microM Ca2+. The affinity, and not the density, of the devapamil-binding site was decreased by lowering the pH from 8.5-6.5, or by increasing the Ca2+ concentration from 0.4 microM to 100 mM. The same results were obtained at 30 degrees C, although the ligand-receptor complex was not stable at Ca2+ concentrations below 10 microM. These binding data were confirmed by kinetic experiments. The rate constants calculated for a pseudo-first-order and a second-order reactions were identical and yielded fourfold lower k-1/k+1 (KD) values than the equilibrium experiments performed using 1 nM and 0.4 microM Ca2+, but the same values using 1 mM Ca2+. 1 mM Ca2+ increased the k-1/k+1 (KD) by decreasing 10-fold the association rate at 4 degrees C. The dissociation rate was increased about 10-fold by 5 microM devapamil or 100 microM D-cis-diltiazem, suggesting that the high affinity site is negatively regulated allosterically by millimolar Ca2+ concentrations and by the occupation of a second low-affinity site. Incubation of the CaCB receptors in the absence of Ca2+ and devapamil at 30 degrees C, but not at 4 degrees C, resulted in an apparent loss of devapamil-binding sites. The decrease in binding sites was caused by a reduced affinity. This apparent loss of binding sites was prevented by the addition of Ca2+ with an apparent median effective concentration of 0.4 microM. The apparent half-maximal inactivation times of the devapamil-binding site were 90 s and 12 min in the presence of 1 nM and 0.4 microM Ca2+, respectively. These results show that micromolar Ca2+ concentrations stabilize the CaCB receptor in a conformation which allows high-affinity binding of phenylalkylamines. Millimolar Ca2+ concentrations induce a low-affinity state of the devapamil-binding site on a stable CaCB receptor.     

Sequence differences between alpha1C and alpha1S Ca2+ channel subunits reveal structural determinants of a guarded and modulated benzothiazepine receptor

The molecular basis of the Ca2+ channel block by (+)-cis-diltiazem was studied in class A/L-type chimeras and mutant alpha1C-a Ca2+ channels. Chimeras consisted of either rabbit heart (alpha1C-a) or carp skeletal muscle (alpha1S) sequence in transmembrane segments IIIS6, IVS6, and adjacent S5-S6 linkers. Only chimeras containing sequences from alpha1C-a were efficiently blocked by (+)-cis-diltiazem, whereas the phenylalkylamine (-)-gallopamil efficiently blocked both constructs. Carp skeletal muscle and rabbit heart Ca2+ channel alpha1 subunits differ with respect to two nonconserved amino acids in segments IVS6. Transfer of a single leucine (Leu1383, located at the extracellular mouth of the pore) from IVS6 alpha1C-a to IVS6 of alpha1S significantly increased the (+)-cis-diltiazem sensitivity of the corresponding mutant L1383I. An analysis of the role of the two heterologous amino acids in a L-type alpha1 subunit revealed that corresponding amino acids in position 1487 (outer channel mouth) determine recovery of resting Ca2+ channels from block by (+)-cis-diltiazem. The second heterologous amino acid in position 1504 of segment IVS6 (inner channel mouth) was identified as crucial inactivation determinant of L-type Ca2+ channels. This residue simultaneously modulates drug binding during membrane depolarization. Our study provides the first evidence for a guarded and modulated benzothiazepine receptor on L-type channels.     

The role of region IVS5 of the human cardiac calcium channel in establishing inactivated channel conformation: use-dependent block by benzothiazepines

The role of inactivated channel conformation and use dependence for diltiazem, a specific benzothiazepine calcium channel inhibitor, was studied in chimeric constructs and point mutants created in the IVS5 transmembrane segment of the L-type cardiac calcium channel. All mutations, chimeric or point mutations, were restricted to IVS5, while the YAI-containing segment in IVS6, i.e. the primary interaction site with benzothiazepines, remained intact. Slowed inactivation rate and incomplete steady state inactivation, a behavior of some mutants, were accompanied by a reduced or by a complete loss of use-dependent block by diltiazem. Single channel properties of mutants that lost use dependence toward diltiazem were characterized by drastically elongated mean open times and distinctly slower time constants of open time distribution. Mutation of individual residues of the IVMLF segment in IVS5 did not mimic the complete loss of use dependence as observed for the replacement of the whole stretch. These results establish evidence that amino acids that govern inactivation and the drug-binding site and other amino acids that are located distal from the putative drug-binding site contribute significantly to the function of the benzothiazepine receptor region. The data are consistent with a complex "pocket" conformation that is responsive to a specific class of L-type calcium channel inhibitors. The data allow for a concept that multiple sites within regions of the alpha(1) subunit contribute to auto-regulation of the L-type Ca(2+) channel.     

Molecular mechanism of calcium channel block by isradipine. Role of a drug-induced inactivated channel conformation

The role of the inactivated channel conformation in the molecular mechanism of Ca(2+) channel block by the 1,4-dihydropyridine (DHP) (+)-isradipine was analyzed in L-type channel constructs (alpha(1Lc); Berjukow, S., Gapp, F., Aczel, S., Sinnegger, M. J., Mitterdorfer, J., Glossmann, H., and Hering, S. (1999) J. Biol. Chem. 274, 6154-6160) and a DHP-sensitive class A Ca(2+) channel mutant (alpha(1A-DHP); Sinnegger, M. J., Wang, Z., Grabner, M., Hering, S., Striessnig, J., Glossmann, H., and Mitterdorfer, J. (1997) J. Biol. Chem. 272, 27686-27693) carrying the high affinity determinants of the DHP receptor site but inactivating at different rates. Ca(2+) channel inactivation was modulated by coexpressing the alpha(1A-DHP)- or alpha(1Lc)-subunits in Xenopus oocytes with either the beta(2a)- or the beta(1a)-subunit and amino acid substitutions in L-type segment IVS6 (I1497A, I1498A, and V1504A). Contrary to a modulated receptor mechanism assuming high affinity DHP binding to the inactivated state we observed no clear correlation between steady state inactivation and Ca(2+) channel block by (+)-isradipine: (i) a 3-fold larger fraction of alpha(1A-DHP)/beta(1a) channels in steady state inactivation at -80 mV (compared with alpha(1A-DHP)/beta(2a)) did not enhance the block by (+)-isradipine; (ii) different steady state inactivation of alpha(1Lc) mutants at -30 mV did not correlate with voltage-dependent channel block; and (iii) the midpoint-voltages of the inactivation curves of slowly inactivating L-type constructs and more rapidly inactivating alpha(1Lc)/beta(1a) channels were shifted to a comparable extent to more hyperpolarized voltages. A kinetic analysis of (+)-isradipine interaction with different L-type channel constructs revealed a drug-induced inactivated state. Entry and recovery from drug-induced inactivation are modulated by intrinsic inactivation determinants, suggesting a synergism between intrinsic inactivation and DHP block.     

A molecular dynamics study of an L-type calcium channel model

In this work, we propose a molecular model of the L-type calcium channel pore from the human cardiac alpha1 subunit. Four glutamic acid residues, the EEEE locus, located at highly conserved P loops (also called SS1-SS2 segments) of the alpha1 subunit, molecularly express the calcium channel selectivity. The proposed alpha-helix structure for the SS1 segment, analyzed through molecular dynamics simulations in aqueous-phase, was validated by the plotting of Ramachandran diagrams for the averaged structures and by the analysis of i and i + 4 helical hydrogen bonding between the amino acid residues. The results of the simulation of the calcium channel model with one and two Ca2+ ions at the binding site are in accordance with mutation studies which suggest that the EEEE locus in the L-type calcium channel must form a single high-affinity binding site. These results suggest that the Ca2+ permeation through the channel would be derived from competition between two ions for the only high-affinity binding site. Furthermore, the experimentally observed blocking of the Na+ flux at micromolar Ca2+ concentrations, probably due to the occupancy of the single high-affinity binding site for one Ca2+, was also reproduced by our model.     

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)     

Nimodipine block of calcium channels in rat vascular smooth muscle cell lines. Exceptionally high-affinity binding in A7r5 and A10 cells

Calcium channel currents were studied in the A10 and A7r5 cell lines derived from rat thoracic aorta muscle cells. The whole-cell variation of the patch voltage clamp technique was used. Results with each cell line were nearly identical. Two types of Ca channels were found in each cell line that are similar to the L-type and T-type Ca channels found in excitable cells. Nimodipine block of the L-type Ca channels in both cell lines is more potent than in previously studied tissues. The kinetics of nimodipine block are accounted for by a model that postulates 1:1 drug binding to open Ca channels with an apparent dissociation constant (KO) of 16-45 pM. In A7r5 cells, the rate of onset of nimodipine block increases with the test potential, in quantitative agreement with the model of open channel block. The apparent association rate (f) is 1.4 x 10(9) M-1 s-1; the dissociation rate (b) is about 0.024 s-1. In anterior pituitary cells (GH4C1 cells), KO is 30 times larger; b is only twice as fast, but f is 15 times slower. The comparative kinetic analysis indicates that the high-affinity binding site for nimodipine is similar in both GH4C1 and A7r5 cells, but nimodipine diffuses much faster or has a larger partition coefficient into the plasmalemma of A7r5 cells than for GH4C1 cells. Unusually high-affinity binding was not observed in earlier 45Ca flux studies with A10 and A7r5 cells. The model of open channel block accounts for the discrepancy; only a small fraction of the Ca channels are in the high affinity open state under the conditions used in 45Ca flux studies, so an effective binding constant is measured that is much greater than the dissociation constant for high-affinity binding.     

Probing the architecture of an L-type calcium channel with a charged phenylalkylamine: evidence for a widely open pore and drug trapping

Voltage-gated calcium channels are in a closed conformation at rest and open temporarily when the membrane is depolarized. To gain insight into the molecular architecture of Ca(v)1.2, we probed the closed and open conformations with the charged phenylalkylamine (-)devapamil ((-)qD888). To elucidate the access pathway of (-)D888 to its binding pocket from the intracellular side, we used mutations replacing a highly conserved Ile-781 by threonine/proline in the pore-lining segment IIS6 of Ca(v)1.2 (1). The shifted channel gating of these mutants (by 30-40 mV in the hyperpolarizing direction) enabled us to evoke currents with identical kinetics at different potentials and thus investigate the effect of the membrane potentials on the drug access per se. We show here that under these conditions the development of channel block by (-)qD888 is not affected by the transmembrane voltage. Recovery from block at rest was, however, accelerated at more hyperpolarized voltages. These findings support the conclusion that Ca(v)1.2 must be opening widely to enable free access of the charged (-)D888 molecule to its binding site, whereas drug dissociation from the closed channel conformation is restricted by bulky channel gates. The functional data indicating a location of a trapped (-)D888 molecule close to the central pore region are supported by a homology model illustrating that the closed Ca(v)1.2 is able to accommodate a large cation such as (-)D888.     

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.     

Positive cooperativity of ryanodine binding to the calcium release channel of sarcoplasmic reticulum from heart and skeletal muscle

Ryanodine is a specific ligand for the calcium release channel which mediates calcium release in excitation-contraction coupling in muscle. In this study, ryanodine binding in sarcoplasmic reticulum from heart muscle and skeletal muscle is further compared and correlated with function. The new findings include the following: (1) Two types of binding, high affinity (KD1 approximately 5-10 nM) and low affinity (KD2 approximately 3 microM), can now be discerned for the skeletal muscle receptor. KD1 is approximately the same as and KD2 of similar magnitude to that previously reported for heart. (2) The dissociation rates for the high-affinity binding have been directly measured for both heart and skeletal muscle (t1/2 approximately 30-40 min). These rates are more rapid than previously reported (t1/2 approximately 14 h). (3) KD1's obtained from the ratio of the dissociation and association rate constants agree with the dissociation constant measured by equilibrium binding Scatchard analysis. (4) Ryanodine binding to the low-affinity site can be correlated with a decrease in the dissociation rate constant (k-1) of the high-affinity site, and thereby in the apparent dissociation constant (KD1). The inhibition constant (KI) for inhibiting the high-affinity off rate obtained from a double-reciprocal plot of the change in off rate vs [ryanodine] is practically the same in heart (0.66 microM) and skeletal muscle (0.64 microM) and in the range of the KD2. The binding of cold ryanodine to the low-affinity site appears to lock the bound [3H]ryanodine onto the high-affinity site rather than to exchange with it. Thus, in this sense, the ryanodine receptor exhibits "positive cooperativity".(ABSTRACT TRUNCATED AT 250 WORDS)     

Roles of Leu249, Lys252, and Leu253 in membrane segment M3 of sarcoplasmic reticulum Ca2+-ATPase in control of Ca2+ migration and long-range intramolecular communication

Point mutants with alterations to Leu249, Lys252, Leu253, Asp254, and Glu255 in membrane segment M3, and Pro824, Lys825, and Glu826 in loop L6-7, of the sarcoplasmic reticulum Ca2+-ATPase were analyzed functionally by steady-state and transient kinetic methods. In mutants Leu249Ala, Lys252Glu, and Leu253Ala, the rate of Ca2+ dissociation from the cytoplasmically facing high-affinity Ca2+ sites was increased 4- to 7-fold relative to wild type, and in Leu249Ala and Lys252Glu the rate of Ca2+ binding was increased as well. Substitution of Lys252 with arginine, alanine, glutamine, or methionine affected Ca2+ interaction much less, indicating that the negative charge of the glutamate is particularly disturbing. These findings may be understood on the basis of the hypothesis that a water-accessible channel leading between membrane segments M1 and M3 in the thapsigargin-bound Ca2+-free structure [Toyoshima, C., and Nomura, H. (2002) Nature 418, 605-611] is closely related to the migration pathway for Ca2+. The effects of alanine mutations to Leu249 and Leu253 on Ca2+ dissociation may arise from destabilization of the hydrophobic wall lining the pathway. In mutant Lys252Glu, unfavorable interaction between the glutamate and L6-7 may open the pathway. In addition, Leu253Ala, and to a lesser extent some of the other mutations, reduced the rate of the E1PCa2 to E2P transition of the phosphoenzyme, enhanced the rate of dephosphorylation of E2P, and reduced the apparent affinity for vanadate, suggesting interference with the conformational change of the phosphoenzyme and the function of the catalytic site in E2 and E2P.     

Design of a functional calcium channel protein: inferences about an ion channel-forming motif derived from the primary structure of voltage-gated calcium channels.

To identify sequence-specific motifs associated with the formation of an ionic pore, we systematically evaluated the channel-forming activity of synthetic peptides with sequence of predicted transmembrane segments of the voltage-gated calcium channel. The amino acid sequence of voltage-gated, dihydropyridine (DHP)-sensitive calcium channels suggests the presence in each of four homologous repeats (I-IV) of six segments (S1-S6) predicted to form membrane-spanning, alpha-helical structures. Only peptides representing amphipathic segments S2 or S3 form channels in lipid bilayers. To generate a functional calcium channel based on a four-helix bundle motif, four-helix bundle proteins representing IVS2 (T4CaIVS2) or IVS3 (T4CaIVS3) were synthesized. Both proteins form cation-selective channels, but with distinct characteristics: the single-channel conductance in 50 mM BaCl2 is 3 pS and 10 pS. For T4CaIVS3, the conductance saturates with increasing concentration of divalent cation. The dissociation constants for Ba2+, Ca2+, and Sr2+ are 13.6 mM, 17.7 mM, and 15.0 mM, respectively. The conductance of T4CaIVS2 does not saturate up to 150 mM salt. Whereas T4CaIVS3 is blocked by microM Ca2+ and Cd2+, T4CaIVS2 is not blocked by divalent cations. Only T4CaIVS3 is modulated by enantiomers of the DHP derivative BayK 8644, demonstrating sequence requirement for specific drug action. Thus, only T4CaIVS3 exhibits pore properties characteristic also of authentic calcium channels. The designed functional calcium channel may provide insights into fundamental mechanisms of ionic permeation and drug action, information that may in turn further our understanding of molecular determinants underlying authentic pore structures.     

A region in IVS5 of the human cardiac L-type calcium channel is required for the use-dependent block by phenylalkylamines and benzothiazepines

Mutations in motif IVS5 and IVS6 of the human cardiac calcium channel were made using homologous residues from the rat brain sodium channel 2a. [3H]PN200-110 and allosteric binding assays revealed that the dihydropyridine and benzothiazepine receptor sites maintained normal coupling in the chimeric mutant channels. Whole cell voltage clamp recording from Xenopus oocytes showed a dramatically slowed inactivation and a complete loss of use-dependent block for mutations in the cytoplasmic connecting link to IVS5 (HHT-5371) and in IVS5 transmembrane segment (HHT-5411) with both diltiazem and verapamil. However, the use-dependent block by isradipine was retained by these two mutants. For mutants HHT-5411 and HHT-5371, the residual current appeared associated with a loss of voltage dependence in the rate of inactivation indicating a destabilization of the inactivated state. Furthermore, both HHT-5371 and -5411 recovered from inactivation significantly faster after drug block than that of the wild type channel. Our data demonstrate that accelerated recovery of HHT-5371 and HHT-5411 decreased accumulation of these channels in inactivation during pulse trains and suggest a close link between inactivation gating of the channel and use-dependent block by phenylalkylamines and benzothiazepines and provide evidence of a role for the transmembrane and cytoplasmic regions of IVS5 in the use-dependent block by diltiazem and verapamil.     

Voltage-dependent calcium channels

Voltage-activated calcium channels can be divided into two subgroups based on their activation threshold, low-voltage-activated (LVA) and high-voltage-activated (HVA). Auxiliary subunits of the HVA calcium channels contribute significantly to biophysical properties of the channels. We have cloned and characterized members of two families of auxiliary subunits: alpha2delta and gamma. Two new alpha2delta subunits, alpha2delta-2 and alpha2delta-3, regulate all classes of HVA calcium channels. While the ubiquitous alpha2delta-2 modulates both neuronal and non-neuronal channels with similar efficiency, the alpha2delta-3 subunit regulates Ca(v)2.3 channels more effectively. Furthermore, alpha2delta-2 may modulate the LVA Ca(v)3.1 channel. Four new gamma subunits, gamma-2, gamma-3, gamma-4 and gamma-5, were characterized. The gamma-2 subunit modulated both the non-neuronal Ca(v)1.2 channel and the neuronal Ca(v)2.1 channel. The gamma-4 subunit affected only the Ca(v)2.1 channel. The gamma-5 subunit may be a regulatory subunit of the LVA Ca(v)3.1 channel. The Ca(v)1.2 channel is a major target for treatment of cardiovascular diseases. We have mapped the interaction site for clinically important channel blockers - dihydropyridines (DHPs) - and analysed the underlying inhibition mechanism. High-affinity inhibition is characterized by interaction with inactivated state of the channel. Its structural determinants are amino acids of the IVS6 segment, with smaller contribution of the IS6 segment, which contributes to voltage-dependence of DHP inhibition. Removal of amino acids responsible for the high-affinity inhibition revealed a low-affinity open channel block, in which amino acids of the IIIS5 and IIIS6 segments take part. Experiments with a permanently charged DHP suggested that there is another low-affinity interaction site on the alpha(1) subunit. We have cloned and characterized murine neuronal LVA Ca(v)3.1 channel. The channel has high sensitivity to the organic blocker mibefradil, moderate sensitivity to phenytoin, and low sensitivity to ethosuximide, amiloride and valproat. The channel is insensitive to tetrodotoxin and DHPs. The inorganic blockers Ni2+ and Cd2+ are moderately effective compared to La3+. The current through the Ca(v)3.1 channel inactivates faster with Ba2+ compared to Ca2+. Molecular determinants of fast inactivation are located in amino side of the intracellular carboxy terminus. The voltage dependence of charge movement is very shallow compared to the voltage dependence of current activation. Transfer of 30 % of charge correlates with activation of 70 % of measurable macroscopic current. Prolonged depolarization does not immobilize charge movement of the Ca(v)3.1 channel.     

Molecular determinants of voltage-dependent slow inactivation of the Ca2+ channel.

Ba(2+) current through the L-type Ca(2+) channel inactivates essentially by voltage-dependent mechanisms with fast and slow kinetics. Here we found that slow inactivation is mediated by an annular determinant composed of hydrophobic amino acids located near the cytoplasmic ends of transmembrane segments S6 of each repeat of the alpha(1C) subunit. We have determined the molecular requirements that completely obstruct slow inactivation. Critical interventions include simultaneous substitution of A752T in IIS6, V1165T in IIIS6, and I1475T in IVS6, each preventing in additive manner a considerable fraction of Ba(2+) current from inactivation. In addition, it requires the S405I mutation in segment IS6. The fractional inhibition of slow inactivation in tested mutants caused an acceleration of fast inactivation, suggesting that fast and slow inactivation mechanisms are linked. The channel lacking slow inactivation showed approximately 45% of the sustained Ba(2+) or Ca(2+) current with no indication of decay. The remaining fraction of the current was inactivated with a single-exponential decay (pi(f) approximately 10 ms), completely recovered from inactivation within 100 ms and did not exhibit Ca(2+)-dependent inactivation properties. No voltage-dependent characteristics were significantly changed, consistent with the C-type inactivation model suggesting constriction of the pore as the main mechanism possibly targeted by Ca(2+) sensors of inactivation.     

Opposite effects of a single IIIS5 mutation on phenylalkylamine and dihydropyridine interaction with L-type Ca2+ channels

Replacement of L-type Ca(2+) channel alpha(1) subunit residue Thr-1066 in segment IIIS5 by a tyrosine residue conserved in the corresponding positions of non-L-type Ca(2+) channels eliminates high dihydropyridine sensitivity through a steric mechanism. To determine the effects of this mutation on phenylalkylamine interaction, we exploited the availability of Ca(v)1.2DHP(-/-) mice containing the T1066Y mutation. In contrast to dihydropyridines, increased protein-dependent binding of the phenylalkylamine (-)-[(3)H]devapamil occurred to Ca(v)1.2DHP(-/-) mouse brain microsomes. This effect could be attributed to an at least 2-fold increase in affinity as determined by saturation analysis and binding inhibition experiments. The latter also revealed a higher affinity for (-)-verapamil but not for (-)-gallopamil. The mutation caused a pronounced slowing of (-)-[(3)H]devapamil dissociation, indicating a stabilization of the drug-channel complex. The increased affinity of mutant channels was also evident in functional studies after heterologous expression of wild type and T1066Y channels in Xenopus laevis oocytes. 100 mum (-)-verapamil inhibited a significantly larger fraction of Ba(2+) inward current through mutant than through WT channels. Our results provide evidence that phenylalkylamines also interact with the IIIS5 helix and that the geometry of the IIIS5 helix affects the access and/or binding of different chemical classes of Ca(2+) channel blockers to their overlapping binding domains. Mutation of Thr-1066 to a non-L-type tyrosine residue can be exploited to differentially affect phenylalkylamine and dihydropyridine binding to L-type Ca(2+) channels.     

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.     

L-type Ca2+ channel function and expression in neonatal rabbit ventricular myocytes

L-type Ca(2+) channel-mediated, Ca(2+)-induced Ca(2+) release (CICR) is the dominant mode of excitation-contraction (E-C) coupling in the mature mammalian myocardium but is thought to be absent in the fetal and newborn mammalian myocardium. Furthermore, the characteristics and contributors of E-C coupling at the earliest developmental stages are poorly understood. In this study, we measured [(3)H](+)PN200-110 dihydropyridine binding capacity, functionality and expression of the L-type Ca(2+) channel, and cytosolic [Ca(2+)] ([Ca(2+)](i)) at various developmental stages (3, 6, 10, 20, and 56 days old) to characterize ontogenetic changes in E-C coupling. We found that 1) the whole cell L-type Ca(2+) channel peak current (I(Ca)) density increased slightly in parallel with cell growth, but the current-voltage relationship, the steady-state activation, and the maximum DHP binding and binding affinity did not exhibit significant developmental changes; 2) sarcoplasmic reticulum Ca(2+) dependence of inactivation rates of L-type Ca(2+) channel and peak of I(Ca) density were only observed after 10 days of age, which temporally coincides with transverse (T)-tubule formation; 3) the relationship between [Ca(2+)](i) and voltage changed from a linear relationship at the earliest developmental stages to a "bell-shaped" relationship at the later developmental stages, presumably corresponding to a switch from reverse-mode Na/Ca exchange-dependent to I(Ca)-dependent E-C coupling; and 4) the expression of two different splice variants of Ca(V)1.2, IVS3A and IVS3B, switched from predominantly IVS3A at the earliest stages to IVS3B at the later developmental stages. Our data suggest that whereas the density of functional dihydropyridine receptors (DHPRs) increases only slightly during ontogeny, the enhancement of functional coupling between DHPR and ryanodine receptor is dramatic between the second and third weeks after birth. Furthermore, we found that the differential expression of splice variants during development temporally correlated with the appearance of I(Ca)-dependent E-C coupling and T-tubule formation.     

Regulation of Ca-sensitive inactivation of a 1-type Ca2+ channel by specific domains of beta subunits.

Ca2+ channel auxiliary beta subunits have been shown to modulate voltage-dependent inactivation of various types of Ca2+ channels. The beta1 and beta2 subunits, that are differentially expressed with the L-type alpha1 Ca2+ channel subunit in heart, muscle and brain, can specifically modulate the Ca2+-dependent inactivation kinetics. Their expression in Xenopus oocytes with the alpha1C subunit leads, in both cases, to biphasic Ca2+ current decays, the second phase being markedly slowed by expression of the beta2 subunit. Using a series of beta subunit deletion mutants and chimeric constructs of beta1 and beta2 subunits, we show that the inhibitory site located on the amino-terminal region of the beta2a subunit is the major element of this regulation. These results thus suggest that different splice variants of the beta2 subunit can modulate, in a specific way, the Ca2+ entry through L-type Ca2+ channels in different brain or heart regions.     

Acid pairs increase the N-terminal Ca2+ affinity of CaM by increasing the rate of Ca2+ association

A series of N-terminal calmodulin (CaM) mutants was generated to probe the relationship between the N-terminal Ca(2+) affinity and the number of paired, negatively charged Ca(2+) chelating residues in the N-terminal Ca(2+)-binding sites of CaM. When the number of acid pairs [negatively charged residues at positions +x and -x (X-axis), +y and -y (Y-axis), and +z and -z (Z-axis)] was increased from zero to one and then to two, a progressive increase was seen in the N-terminal Ca(2+) affinities. The maximal ranges of the increases observed in the N-terminal Ca(2+) affinity were approximately 8-8.5-fold for site I, approximately 4.5-5-fold for site II, and approximately 11-fold for both sites, in comparison to the mutants containing no acid pairs. The maximal values of N-terminal Ca(2+) affinity were bestowed by the presence of five acidic chelating residues in site I or II, individually. Addition of the sixth acidic chelating residue (third acid pair) to both N-terminal Ca(2+)-binding sites reduced the N-terminal Ca(2+) affinity. The increases in Ca(2+) affinity observed were caused by an increase in the Ca(2+) association rates for the Y- and Z-axis acid pairs, while the X-axis acid pair caused a reduction in the Ca(2+) dissociation rates.