Martina J. Sinnegger

Familial Hemiplegic Migraine Mutations Change α1ACa2+ Channel Kinetics

Missense mutations in the pore-forming human α1A 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 α1A subunit into the conserved rabbit α1A(R192Q, T666M, V714A, and I1819L) and investigated possible changes in channel function after functional expression of mutant subunits inXenopus laevis oocytes. Changes in channel gating were observed for mutants T666M, V714A, and I1819L but not for R192Q. Ba2+ current (I Ba) 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.

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 (α1S or α1C) to a weakly sensitive, nonstereoselective class A (α1A) calcium channel. Transfer of three amino acids of transmembrane segment IVS6 of L-type α1 into the α1A 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.

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 α1 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 α1 subunits with α2δ and β1a 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 α1C-a), which also further increased the sensitivity to DHP antagonists. Asp-955, which is found at the corresponding position in the calcium channel α1S 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 α-helix of transmembrane segment IIIS5, form a crucial DHP binding motif.

Nine L-type Amino Acid Residues Confer Full 1,4-Dihydropyridine Sensitivity to the Neuronal Calcium Channel α1ASubunit 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 α1subunit 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; α1C-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 α1A subunit using site-directed mutagenesis. 1,4-Dihydropyridine agonist and antagonist modulation of barium inward currents mediated by the mutant α1A subunits, coexpressed with α2δ and β1a subunits inXenopus laevis oocytes, was investigated with the two-microelectrode voltage clamp technique. The resulting mutant α1A-DHPi displayed low sensitivity for 1,4-dihydropyridines. Analysis of the 1,4-dihydropyridine binding region of an ancestral L-type α1 subunit previously cloned from Musca domestica body wall muscle led to the identification of Met1188 (α1C-a numbering) as an additional critical constituent of the L-type 1,4-dihydropyridine binding domain. The introduction of this residue into α1A-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.

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 Ca2+ channel block by the 1,4-dihydropyridine (DHP) (+)-isradipine was analyzed in L-type channel constructs (α1Lc; 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 Ca2+ channel mutant (α1A-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. Ca2+ channel inactivation was modulated by coexpressing the α1A-DHP- or α1Lc-subunits in Xenopus oocytes with either the β2a- or the β1a-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 Ca2+ channel block by (+)-isradipine: (i) a 3-fold larger fraction of α1A-DHP/β1achannels in steady state inactivation at −80 mV (compared with α1A-DHP/β2a) did not enhance the block by (+)-isradipine; (ii) different steady state inactivation of α1Lc 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 α1Lc/β1a 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.

Sequence Differences between α1C and α1S 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 α1C-aCa2+ channels. Chimeras consisted of either rabbit heart (α1C-a) or carp skeletal muscle (α1S) sequence in transmembrane segments IIIS6, IVS6, and adjacent S5-S6 linkers. Only chimeras containing sequences from α1C-awere efficiently blocked by (+)-cis-diltiazem, whereas the phenylalkylamine (−)-gallopamil efficiently blocked both constructs. Carp skeletal muscle and rabbit heart Ca2+ channel α1 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 α1C-a to IVS6 of α1Ssignificantly 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 α1subunit 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.