Single amino acid substitutions within the ion permeation pathway alter single-channel conductance of the human L-type cardiac Ca2+ channel.
Voltage-dependent L-type Ca2+ channels select for Ca2+ and other divalent cations by high-affinity Ca2+ binding and ion-ion interactions in the permeation pathway. We have recently identified a series of highly conserved glutamate residues, located within the SS2 segments of each of the four repeats of the human heart Ca2+ channel alpha 1 subunit, as major determinants of ion selectivity of the channel. To further investigate the functional role of these glutamate residues in ion permeation, we have individually neutralized the glutamic acids in repeats II and IV by substitution with alanine or glutamine. Wild-type and mutant Ca2+ channels were expressed in Xenopus oocytes. Apparent affinity for external Ca2+ was assessed by measuring the block by Ca2+ of inward Li+ currents through the Ca2+ channels. Mutations reducing net negative charge at these positions resulted in a 10-fold reduction in the affinity of the channel for external Ca2+. Single-channel conductance was measured with either divalent or monovalent cations as the charge carriers. Substitution of glutamic acid at position 677 with either alanine or glutamine increased single-channel conductance, whereas the same substitutions at position 1387 resulted in a decrease in single-channel conductance with Ba2+ as the charge carrier. In contrast, the unitary current amplitude carried by Na+, in the absence of external divalent cations, was not altered by these mutations. The results suggest that these conserved glutamate residues participate in high-affinity Ca2+ binding within the pore. The different effects on Ba2+ permeation indicate an asymmetric arrangement of the glutamate residues between repeats II and IV.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, OH 45267-0575.
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Yatani
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Bahinski
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Mikala
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Yamamoto
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Schwartz
D000595:Amino Acid Sequence; D000818:Animals; D015220:Calcium Channels / Q000145:classification / Q000235:genetics* / Q000502:physiology*; D004553:Electric Conductivity; D006801:Humans; D007477:Ions; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D010539:Permeability; D014981:Xenopus
D015220:Calcium Channels; D007477:Ions