Glutamate scanning mutagenesis was used to assess the role of the calcicludine binding segment in regulating channel permeation and gating using both Ca2+ and Ba2+ as charge carriers. As expected, wild-type CaV1.2 channels had a Ba2+ conductance ~2× that in Ca2+ (GBa/GCa = 2) and activation was ~10 mV more positive in Ca2+ vs. Ba2+. Of the 11 mutants tested, F1126E was the only one that showed unique permeation and gating properties compared to the wild type. F1126E equalized the CaV1.2 channel conductance (GBa/GCa = 1) and activation voltage dependence between Ca2+ and Ba2+. Ba2+ permeation was reduced because the interactions among multiple Ba2+ ions and the pore were specifically altered for F1126E, which resulted in Ca2+-like ionic conductance and unitary current. However, the high-affinity block of monovalent cation flux was not altered for either Ca2+ or Ba2+. The half-activation voltage of F1126E in Ba2+ was depolarized to match that in Ca2+, which was unchanged from that in the wild type. As a result, the voltages for half-activation and half-inactivation of F1126E in Ba2+ and Ca2+ were similar to those of wild-type in Ca2+. This effect was specific to F1126E since F1126A did not affect the half-activation voltage in either Ca2+ or Ba2+. These results indicate that residues in the outer vestibule of the CaV1.2 channel pore are major determinants of channel gating, selectivity, and permeation.
The Journal of Membrane Biology – Springer Journals
Published: Jan 23, 2010
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