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Sequence differences between alpha1C and alpha1S Ca2+ channel subunits reveal structural determinants of a guarded
1Institut für Biochemische Pharmakologie, Peter Mayr Strasse 1, A-6020 Innsbruck, Austria.
Abstract:
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.
Insights
Researchers identified key amino acids in calcium channels responsible for (+)-cis-diltiazem block. This reveals a guarded, modulated receptor site for benzothiazepines on L-type calcium channels.
Area of Science:
- Pharmacology
- Molecular Biology
- Biophysics
Background:
- Calcium channels are crucial for cellular function.
- Diltiazem is a calcium channel blocker used clinically.
- Understanding drug-receptor interactions at the molecular level is essential.
Purpose of the Study:
- To elucidate the molecular basis of (+)-cis-diltiazem block in L-type calcium channels.
- To identify specific amino acid residues involved in drug binding and channel modulation.
- To characterize the benzothiazepine receptor on calcium channels.
Main Methods:
- Construction and analysis of alpha1C-a and alpha1S calcium channel chimeras and mutants.
- Functional characterization of channel block by (+)-cis-diltiazem and (-)-gallopamil.
- Site-directed mutagenesis to probe the role of specific amino acids.
Main Results:
- Chimeras containing alpha1C-a sequences were sensitive to (+)-cis-diltiazem, unlike alpha1S.
- Leucine at position 1383 in IVS6 of alpha1C-a is critical for (+)-cis-diltiazem sensitivity.
- Amino acids at positions 1487 and 1504 in IVS6 modulate drug block and channel inactivation.
Conclusions:
- The study identifies specific residues in the calcium channel pore that form a guarded and modulated benzothiazepine receptor.
- These findings provide novel insights into the mechanism of L-type calcium channel block by diltiazem.
- This work advances the understanding of drug interactions with ion channels.