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 α1A subunit of voltage-gated Ca2+ channels. The sequences of human α1A-1 and α1A-2 code for proteins of 2510 and 2662 amino acids, respectively. Human α1A-2α2bδβ1b Ca2+ channels expressed in HEK293 cells activate rapidly (τ+10mV=2.2ms), deactivate rapidly (τ–90mV=148μs), inactivate slowly (τ+10mV=690ms), and have peak currents at a potential of +10mV with 15mM Ba2+ as charge carrier. In HEK293 cells transient expression of Ca2+ channels containing α1A/B(f), an α1A subunit containing a 112 amino acid segment of α1B-1 sequence in the IVS3-IVSS1 region, resulted in Ba2+ currents that were 30-fold larger compared to wild-type (wt) α1A-2-containing Ca2+ channels, and had inactivation kinetics similar to those of α1B-1-containing Ca2+ channels. Cells transiently transfected with α1A/B(f)α2bδβ1b expressed higher levels of the α1, α2bδ, and β1b subunit polypeptides as detected by immunoblot analysis. By mutation analysis we identified two locations in domain IV within the extracellular loops S3-S4 (N1655P1656) and S5-SS1 (E1740) that influence the biophysical properties of α1A. α1AE1740R resulted in a threefold increase in current magnitude, a −10mV shift in steady-state inactivation, and an altered Ba2+ current inactivation, but did not affect ion selectivity. The deletion mutant α1AΔNP shifted steady-state inactivation by −20mV and increased the fast component of current inactivation twofold. The potency and rate of block by ω-Aga IVA was increased with α1AΔNP. These results demonstrate that the IVS3-S4 and IVS5-SS1 linkers play an essential role in determining multiple biophysical and pharmacological properties of α1A-containing Ca2+ channels.