Structural elements in domain IV that influence biophysical and pharmacological properties of human alpha(1A)-containing high-voltage-activated calcium channels

Citation
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
Citations number
62
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOPHYSICAL JOURNAL
ISSN journal
00063495 → ACNP
Volume
76
Issue
3
Year of publication
1999
Pages
1384 - 1400
Database
ISI
SICI code
0006-3495(199903)76:3<1384:SEIDIT>2.0.ZU;2-0
Abstract
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.