Structural elements in domain IV that influence biophysical and pharmacological properties of human α1A-containing high-voltage-activated calcium channels

We have cloned two splice variants of the human homolog of the alpha(1A) subunit of voltage-gated Ca2+ channels. The sequences of human alpha(1A-1) and 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 (-90mV) = 148 mu s), inactivate slowly (tau(+10mV) = 690 ms), and have peak currents 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 alpha(1A) subunit containing a 112 amino acid segment of alpha(1A-2)- sequence in the IVS3-IVSS1 region, resulted in Ba2+ currents that were 30-fold larger compared to wild-type (wt) alpha(1A-2)- containing Ca2+ channels, and had inactivation kinetics similar to those of alpha(1B-1)-containing Ca2+ channels. Cells transiently transfected with alpha(1A/B(f))alpha(2b)delta beta(1b), expressed higher levels of the alpha(1), alpha(2b)delta, and beta(1b) subunit polypeptides as detected by immunoblot analysis. By mutation analysis we identified two locations in domain IV within the extracellular loops S3-S4 ((NP1656)-P-1655) and S5-SS1 (E-1740) that influence the biophysical properties of alpha(1A). alpha(1A)E1740R resulted in a threefold increase 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 potency 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 an essential role in determining multiple biophysical and pharmacological properties of alpha(1A)-containing Ca2+ channels.