Abstract
To elucidate the mechanism underlying the interaction between the L-type Ca2+ channel and the dihydropyridines (DHPs), contribution of the repeat III was studied by constructing chimeras between the DHP-sensitive α1C and DHP-insensitive α1E subunits. The chimeras were transiently expressed in human embryonic kidney 293 cells and the whole-cell Ba2+ current (IBa) was recorded. Mutating Thr1061 to Tyr in IIIS5 of the α1C sequence completely abolished the inhibition and stimulation of IBaby the antagonist (+)-isradipine and agonist (−)-Bay K 8644, whereas mutating Gln1065 to Met in IIIS5 decreased the affinity for isradipine 100-fold without affecting the stimulating effect of Bay K 8644. The conserved amino acid residue Tyr1174 in IIIS6 of the α1C subunit was necessary for the high affinity DHP block. The DHP-dependent block and stimulation of IBa were transferred to the α1E channel by the mutation of two amino acid residues in IIIS5 (Y1295T, M1299Q), three residues in IIIS6 (F1406I, F1409I, V1414M) and three residues in IVS6 (I1706Y, F1707M, L1714I). The mutated α1E channel was stimulated 2.8-fold by 1 μm Bay K 8644 and blocked by isradipine with an IC50 value of 60 nm. These results show that mutation of Thr1061 in the α1C sequence results in a DHP-insensitive L-type channel and that transfer of the high affinity DHP sensitivity requires mutation of eight amino acid residues in the α1E sequence.
Footnotes
- Received April 23, 1997.
- Accepted June 20, 1997.
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Send reprint requests to: Hiroyuki Ito, Institut für Pharmakologie und Toxikologie der TU München, Biedersteiner Strasse 29, 80802 München, Germany. E-mail:pharma{at}ipt.med.tu-muenchen.de
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This work was supported by grants from Deutsche Forschungsgemeinschaft, Bundesministerium für Bildung und Forschung, and Fond der Chemie.
- The American Society for Pharmacology and Experimental Therapeutics
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