A single amino acid change in Ca(v)1.2 channels eliminates the permeation and gating differences between Ca(2+) and Ba(2+).
Glutamate scanning mutagenesis was used to assess the role of the calcicludine binding segment in regulating channel permeation and gating using both Ca(2+) and Ba(2+) as charge carriers. As expected, wild-type Ca(V)1.2 channels had a Ba(2+) conductance ~2x that in Ca(2+) (G(Ba)/G(Ca) = 2) and activation was ~10 mV more positive in Ca(2+) vs. Ba(2+). 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 Ca(V)1.2 channel conductance (G(Ba)/G(Ca) = 1) and activation voltage dependence between Ca(2+) and Ba(2+). Ba(2+) permeation was reduced because the interactions among multiple Ba(2+) ions and the pore were specifically altered for F1126E, which resulted in Ca(2+)-like ionic conductance and unitary current. However, the high-affinity block of monovalent cation flux was not altered for either Ca(2+) or Ba(2+). The half-activation voltage of F1126E in Ba(2+) was depolarized to match that in Ca(2+), which was unchanged from that in the wild type. As a result, the voltages for half-activation and half-inactivation of F1126E in Ba(2+) and Ca(2+) were similar to those of wild-type in Ca(2+). This effect was specific to F1126E since F1126A did not affect the half-activation voltage in either Ca(2+) or Ba(2+). These results indicate that residues in the outer vestibule of the Ca(V)1.2 channel pore are major determinants of channel gating, selectivity, and permeation.
Li
Zhe
Z
Department of Cardiology, Renmin Hospital and Cardiovascular Research Institute, Wuhan University, China.
Wang
Xianming
X
Gao
Guofeng
G
Qu
Dongmei
D
Yu
Buwei
B
Huang
Congxin
C
Elmslie
Keith S
KS
Peterson
Blaise Z
BZ
D001464:Barium / Q000378:metabolism*; D001692:Biological Transport / Q000235:genetics / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004594:Electrophysiology; D005971:Glutamates / Q000235:genetics / Q000502:physiology; D006801:Humans; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques
D020746:Calcium Channels, L-Type; D005971:Glutamates; D001464:Barium; D002118:Calcium