Molecular Determinants of High Affinity Phenylalkylamine Block of L-type Calcium Channels (*)
Abstract
The high affinity phenylalkylamine(-)D888 blocks ion currents through L-type Ca
channels containing the α
subunit with an apparent K
of 50 nM, but N-type Ca
channels in the pheochromocytoma cell line PC12 are blocked with a 100-fold higher K
value of 5 μM. L-type Ca
channels containing α
subunits with the site-directed mutations Y1463A, A1467S, or I1470A in the putative transmembrane segment S6 in domain IV
(IVS6) were 6-12 times less sensitive to block by(-)D888 than control α
. Ca
channels containing paired combinations of these mutations were even less sensitive to block by(-)D888 than the single mutants,
and channels containing all three mutations were >100 times less sensitive to(-)D888 block, similar to N-type Ca
channels. In addition, the Y1463A mutant and all combination mutants including the Y1463A mutation had altered ion selectivity,
suggesting that Tyr-1463 faces the pore and is involved in ion permeation. Since these three critical amino acid residues
are aligned on the same face of the putative IVS6 α-helix, we propose that they contribute to a receptor site in the pore
that confers a high affinity block of L-type channels by(-)D888.
Footnotes
-
↵* This work was supported by National Institutes of Health Grant P01 HL44948 and by a research grant-in-aid from the American Heart Association (to W. A. C.), a postdoctoral research fellowship from the National Institutes of Health (to G. H. H.), and a postdoctoral research fellowship from the Muscular Dystrophy Association (to B. D. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- NGF
-
nerve growth factor
- AI
-
A1467S,I70A
- YA
-
Y1463A,A67S
- YI
-
Y1463A,I70A
- YAI
-
Y1463A, A67S,I70A
- wt
-
wild type.
-
- Received June 12, 1995.
- Revision received July 28, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
