Biophys J, March 1999, p. 1384-1400, Vol. 76, No. 3

Structural Elements in Domain IV that Influence Biophysical and Pharmacological Properties of Human _1A-Containing High-Voltage-Activated Calcium Channels

SIBIA Neurosciences, Inc., La Jolla, California 92037-4641 USA

We have cloned two splice variants of the human homolog of the _1A subunit of voltage-gated Ca²⁺ channels. The sequences of human _1A-1 and _1A-2 code for proteins of 2510 and 2662 amino acids, respectively. Human _1A-22b1b Ca²⁺ channels expressed in HEK293 cells activate rapidly (_+10mV = 2.2 ms), deactivate rapidly (_-90mV = 148 µs), inactivate slowly (_+10mV = 690 ms), and have peak currents at a potential of +10 mV with 15 mM Ba²⁺ as charge carrier. In HEK293 cells transient expression of Ca²⁺ channels containing _1A/B(f), an _1A subunit containing a 112 amino acid segment of _1B-1 sequence in the IVS3-IVSS1 region, resulted in Ba²⁺ currents that were 30-fold larger compared to wild-type (wt) _1A-2-containing Ca²⁺ channels, and had inactivation kinetics similar to those of _1B-1-containing Ca²⁺ channels. Cells transiently transfected with _1A/B(f)2b1b expressed higher levels of the ₁, _2b, and _1b 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 _1A. _1AE1740R 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 _1ANP shifted steady-state inactivation by 20 mV and increased the fast component of current inactivation twofold. The potency and rate of block by -Aga IVA was increased with _1ANP. These results demonstrate that the IVS3-S4 and IVS5-SS1 linkers play an essential role in determining multiple biophysical and pharmacological properties of _1A-containing Ca²⁺ channels.

Biophys J, March 1999, p. 1384-1400, Vol. 76, No. 3
© 1999 by the Biophysical Society   0006-3495/99/03/1384/17  $2.00

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