Modulation of the smooth-muscle L-type Ca2+ channel alpha 1 subunit (alpha1C-b) by the beta 2a subunit: a peptide which inhibits binding of beta to the I-II linker of alpha 1 induces functional uncoupling
A. Hohaus et al., Modulation of the smooth-muscle L-type Ca2+ channel alpha 1 subunit (alpha1C-b) by the beta 2a subunit: a peptide which inhibits binding of beta to the I-II linker of alpha 1 induces functional uncoupling, BIOCHEM J, 348, 2000, pp. 657-665
Modulation of the smooth-muscle Ca2+ channel alpha 1C-b subunit by the auxi
liary beta 2a subunit was studied in the HEK 293 (cell line from human embr
yonic kidney cells) expression system. In addition, we tested whether the a
lpha 1-beta interaction in functional channels is sensitive to an 18-amino-
acid synthetic peptide that corresponds to the sequence of the defined majo
r interaction domain in the cytoplasmic I-II linker of alpha 1C (AID-peptid
e). Ca2+ channels derived by co-expression of alpha 1C-b and beta 2a subuni
ts exhibited an about 3-fold higher open probability (P-0) than alpha 1C-b
channels. High-P-0 gating of alpha 1C-b.beta 2a channels was associated wit
h the occurrence of long-lasting channel openings [mean open time (tau) > 1
0 ms] which were rarely observed in alpha 1C-b channels. Modulation of fast
gating by the beta 2a subunit persisted in the cell-free, inside-out recor
ding configuration. Biochemical experiments showed that the AID-peptide bin
ds with appreciable affinity to beta 2 subunits of native Ca2+ channels. Bi
nding of the beta 2 protein to immobilized AID-peptide was specifically inh
ibited (K-i of 100 nM) by preincubation with free (uncoupled) AID-peptide,
but not by a corresponding scrambled peptide. Administration of the AID-pep
tide (10 mu M) to the cytoplasmic side of inside-out patches induced a subs
tantial reduction of P-0 of alpha 1C-b.beta 2a channels. The scrambled cont
rol peptide failed to affect alpha 1C-b.beta 2a channels, and the AID-pepti
de (10 mu M) did not modify alpha 1C-b channel function in the absence of e
xpressed beta 2a subunit. Our results demonstrate that the beta 2a subunit
controls fast gating of alpha 1C-b channels, and suggest the alpha 1-beta i
nteraction domain in the cytoplasmic I-II linker of alpha 1C (AID) as a pos
sible target of modulation of the channel. Moreover, our data are consisten
t with a model of alpha 1-beta interaction that is based on multiple intera
ction sites, including AID as a determinant of the affinity of the alpha 1-
beta interaction.