Structural elements in domain IV that influence biophysical and pharmacological properties of human alpha(1A)-containing high-voltage-activated calcium channels
M. Hans et al., Structural elements in domain IV that influence biophysical and pharmacological properties of human alpha(1A)-containing high-voltage-activated calcium channels, BIOPHYS J, 76(3), 1999, pp. 1384-1400
We have cloned two splice variants of the human homolog of the alpha(1A) su
bunit of voltage-gated Ca2+ channels. The sequences of human alpha(1A-1) an
d alpha(1A-2) code for proteins of 2510 and 2662 amino acids, respectively.
Human alpha(1A-2)alpha(2b)delta beta(1b) Ca2+ channels expressed in HEK293
cells activate rapidly (tau(+10mV) =2.2 ms), deactivate rapidly (tau (-90m
V) = 148 mu s), inactivate slowly (tau(+10mV) = 690 ms), and have peak curr
ents ata potential of +10 mV with 15 mM Ba2+, as charge carrier. In HEK293
cells transient expression of Ca2+ channels containing alpha(1A/B(f)), an a
lpha(1A) subunit containing a 112 amino acid segment of alpha(1A-2)- sequen
ce in the IVS3-IVSS1 region, resulted in Ba2+ currents that were 30-fold la
rger compared to wild-type (wt) alpha(1A-2)- containing Ca2+ channels, and
had inactivation kinetics similar to those of alpha(1B-1)-containing Ca2+ c
hannels. Cells transiently transfected with alpha(1A/B(f))alpha(2b)delta be
ta(1b), expressed higher levels of the alpha(1), alpha(2b)delta, and beta(1
b) subunit polypeptides as detected by immunoblot analysis. By mutation ana
lysis we identified two locations in domain IV within the extracellular loo
ps S3-S4 ((NP1656)-P-1655) and S5-SS1 (E-1740) that influence the biophysic
al properties of alpha(1A). alpha(1A)E1740R resulted in a threefold increas
e in current magnitude, a -10 mV shift in steady-state inactivation, and an
altered Ba2+ current inactivation, but did not affect ion selectivity. The
deletion mutant alpha(1A)Delta NP shifted steady-state inactivation by -20
mV and increased the fast component of current inactivation twofold. The p
otency and rate of block by omega-Aga IVA was increased with alpha(1A)Delta
NP. These results demonstrate that the IVS3-S4 and IVS5-SS1 linkers play a
n essential role in determining multiple biophysical and pharmacological pr
operties of alpha(1A)-containing Ca2+ channels.