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.

Three new familial hemiplegic migraine mutants affect P/Q-type Ca(2+) channel kinetics.

Missense mutations in the pore-forming human alpha(1A) subunit of neuronal P/Q-type Ca(2+) channels are associated with familial hemiplegic migraine. We studied the functional consequences on P/Q-type Ca(2+) channel function of three recently identified mutations, R583Q, D715E, and V1457L after introduction into rabbit alpha(1A) and expression in Xenopus laevis oocytes. The potential for half-maximal channel activation of Ba(2+) inward currents was shifted by > 9 mV to more negative potentials in all three mutants. The potential for half-maximal channel inactivation was shifted by > 7 mV in the same direction in R583Q and D715E. Biexponential current inactivation during 3-s test pulses was significantly faster in D715E and slower in V1457L than in wild type. Mutations R583Q and V1457L delayed the time course of recovery from channel inactivation. The decrease of peak current through R583Q (30.2%) and D715E (30. 1%) but not V1457L (18.7%) was more pronounced during 1-Hz trains of 15 100-ms pulses than in wild type (18.2%). Our data demonstrate that the mutations R583Q, D715E, and V1457L, like the previously reported mutations T666M, V714A, and I1819L, affect P/Q-type Ca(2+) channel gating. We therefore propose that altered channel gating represents a common pathophysiological mechanism in familial hemiplegic migraine.

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.

Structural basis of drug binding to L Ca2+ channels.

At least five different types of voltage-gated Ca2+ channels exist in electrically excitable mammalian cells. Only one type, the family of L-type Ca2+ channels (L channels), contains high-affinity binding domains within their alpha 1-subunits for different chemical classes of drugs (Ca2+ channel antagonists; exemplified by isradipine, verapamil and diltiazem). Their stereoselective, high-affinity binding induces block of channel-mediated Ca2+ inward currents in heart and smooth muscle, resulting in antihypertensive, cardiodepressive and antiarrhythmic effects. Amino acids involved in drug binding have recently been identified using photoaffinity labelling, chimeric alpha 1-subunits and site-directed mutagenesis. Insertion of the drug-binding amino acids enabled the transfer of drug-sensitivity into Ca2+ channels that are insensitive to Ca2+ channel antagonists ('gain-of-function' approach). In this review, Jörg Striessing and colleagues summarize the present knowledge about the molecular architecture of L channel drug-binding domains and the implications for Ca2+ channel pharmacology and drug development.

Familial hemiplegic migraine mutations change alpha1A Ca2+ channel kinetics.

Missense mutations in the pore-forming human alpha1A subunit of neuronal P/Q-type Ca2+ channels are associated with familial hemiplegic migraine (FHM). The pathophysiological consequences of these mutations are unknown. We have introduced the four single mutations reported for the human alpha1A subunit into the conserved rabbit alpha1A (R192Q, T666M, V714A, and I1819L) and investigated possible changes in channel function after functional expression of mutant subunits in Xenopus laevis oocytes. Changes in channel gating were observed for mutants T666M, V714A, and I1819L but not for R192Q. Ba2+ current (IBa) inactivation was slightly faster in mutants T666M and V714A than in wild type. The time course of recovery from channel inactivation was slower than in wild type in T666M and accelerated in V714A and I1819L. As a consequence, accumulation of channel inactivation during a train of 1-Hz pulses was more pronounced for mutant T666M and less pronounced for V714A and I1819A. Our data demonstrate that three of the four FHM mutations, located at the putative channel pore, alter inactivation gating and provide a pathophysiological basis for the postulated neuronal instability in patients with FHM.

Nine L-type amino acid residues confer full 1,4-dihydropyridine sensitivity to the neuronal calcium channel alpha1A subunit. Role of L-type Met1188.

Pharmacological modulation by 1,4-dihydropyridines is a central feature of L-type calcium channels. Recently, eight L-type amino acid residues in transmembrane segments IIIS5, IIIS6, and IVS6 of the calcium channel alpha1 subunit were identified to substantially contribute to 1,4-dihydropyridine sensitivity. To determine whether these eight L-type residues (Thr1066, Gln1070, Ile1180, Ile1183, Tyr1490, Met1491, Ile1497, and Ile1498; alpha1C-a numbering) are sufficient to form a high affinity 1,4-dihydropyridine binding site in a non-L-type calcium channel, we transferred them to the 1, 4-dihydropyridine-insensitive alpha1A subunit using site-directed mutagenesis. 1,4-Dihydropyridine agonist and antagonist modulation of barium inward currents mediated by the mutant alpha1A subunits, coexpressed with alpha2delta and beta1a subunits in Xenopus laevis oocytes, was investigated with the two-microelectrode voltage clamp technique. The resulting mutant alpha1A-DHPi displayed low sensitivity for 1,4-dihydropyridines. Analysis of the 1,4-dihydropyridine binding region of an ancestral L-type alpha1 subunit previously cloned from Musca domestica body wall muscle led to the identification of Met1188 (alpha1C-a numbering) as an additional critical constituent of the L-type 1,4-dihydropyridine binding domain. The introduction of this residue into alpha1A-DHPi restored full sensitivity for 1,4-dihydropyridines. It also transferred functional properties considered hallmarks of 1, 4-dihydropyridine agonist and antagonist effects (i.e. stereoselectivity, voltage dependence of drug modulation, and agonist-induced shift in the voltage-dependence of activation). Our gain-of-function mutants provide an excellent model for future studies of the structure-activity relationship of 1, 4-dihydropyridines to obtain critical structural information for the development of drugs for neuronal, non-L-type calcium channels.

Two amino acid residues in the IIIS5 segment of L-type calcium channels differentially contribute to 1,4-dihydropyridine sensitivity.

The transmembrane segment IIIS5 of the L-type calcium channel alpha1 subunit participates in the formation of the 1,4-dihydropyridine (DHP) interaction domain (Grabner, M., Wang, Z., Hering, S., Striessnig, J., and Glossmann, H. (1996) Neuron 16, 207-218). We applied mutational analysis to identify amino acid residues within this segment that contribute to DHP sensitivity. DHP agonist and antagonist modulation of Ba2+ inward currents was assessed after coexpression of chimeric and mutant calcium channel alpha1 subunits with alpha2delta and beta1a subunits in Xenopus oocytes. Whereas DHP antagonists required Thr-1066, DHP agonist modulation crucially depended on the additional presence of Gln-1070 (numbering according to alpha1C-a), which also further increased the sensitivity to DHP antagonists. Asp-955, which is found at the corresponding position in the calcium channel alpha1S subunit from carp skeletal muscle, displayed functional similarity to Gln-1070 with respect to DHP interaction. We conclude that these residues (Thr-1066 plus Gln-1070 or Asp-955), which are located in close vicinity on the same side of the putative alpha-helix of transmembrane segment IIIS5, form a crucial DHP binding motif.

Transfer of high sensitivity for benzothiazepines from L-type to class A (BI) calcium channels.

To investigate the molecular basis of the calcium channel block by diltiazem, we transferred amino acids of the highly sensitive and stereoselective L-type (alpha1S or alpha1C) to a weakly sensitive, nonstereoselective class A (alpha1A) calcium channel. Transfer of three amino acids of transmembrane segment IVS6 of L-type alpha1 into the alpha1A subunit (I1804Y, S1808A, and M1811I) was sufficient to support a use-dependent block by diltiazem and by the phenylalkylamine (-)-gallopamil after expression in Xenopus oocytes. An additional mutation F1805M increased the sensitivity for (-)-gallopamil but not for diltiazem. Our data suggest that the receptor domains for diltiazem and gallopamil have common but not identical molecular determinants in transmembrane segment IVS6. These mutations also identified single amino acid residues in segment IVS6 that are important for class A channel inactivation.

Coordination of Ca2+ by the pore region glutamates is essential for high-affinity dihydropyridine binding to the cardiac Ca2+ channel alpha 1 subunit.

The molecular determinants for Ca2+ modulation of dihydropyridine (DHP) binding to cardiac Ca2+ channels were identified by mutational neutralization of the glutamate residues that comprise the Ca2+ channel selectivity filter. The binding activity of the DHP (+)-[3H]isradipine, monitored after expression of wild-type and mutant alpha 1 subunits in COS-7 cells, was markedly reduced in four single mutants and a double mutant. Evidence for decreased Ca2+ affinity was obtained for two single mutants in kinetic and equilibrium binding studies. Mutational destabilization of Ca2+ binding resulted in a concomitant decrease of (+)-[3H]isradipine binding affinity. Recovery of (+)-[3H]isradipine binding activity by the allosteric modulator (+)-tetrandrine in two single mutants was associated with a recovery of Ca2+ and DHP binding kinetics to wild-type values. Our findings demonstrate that high-affinity DHP binding is dependent on Ca2+ coordination by glutamate residues which form the selectivity filter of the channel pore.