Single amino acid substitutions within the ion permeation pathway alter single-channel conductance of the human L-type cardiac Ca2+ channel

Atsuko Yatani, Anthony Bahinski, G. Mikala, Satoshi Yamamoto, Arnold Schwartz

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

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 a, 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. The glutamic acid at position 1387 may be only peripherally involved in the formation of a high-affinity Ca2+-binding site. We suggest that disruption of this site results in an undisturbed stabilization of inner sites, thereby causing a decrease in divalent cation conductance. Substitution at position 677 disrupts coordination of both inner and outer sites, resulting in weaker divalent cation binding and increased conductance.

Original languageEnglish
Pages (from-to)315-323
Number of pages9
JournalCirculation Research
Volume75
Issue number2
Publication statusPublished - Aug 1994

Fingerprint

Amino Acid Substitution
Divalent Cations
Glutamic Acid
Ions
Glutamine
Alanine
Glutamates
Monovalent Cations
Mutation
Xenopus
Ion Channels
Oocytes
Binding Sites

Keywords

  • Ca channels
  • High-affinity Ca binding
  • Pore structure
  • Single-channel conductance
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

Cite this

Single amino acid substitutions within the ion permeation pathway alter single-channel conductance of the human L-type cardiac Ca2+ channel. / Yatani, Atsuko; Bahinski, Anthony; Mikala, G.; Yamamoto, Satoshi; Schwartz, Arnold.

In: Circulation Research, Vol. 75, No. 2, 08.1994, p. 315-323.

Research output: Contribution to journalArticle

Yatani, Atsuko ; Bahinski, Anthony ; Mikala, G. ; Yamamoto, Satoshi ; Schwartz, Arnold. / Single amino acid substitutions within the ion permeation pathway alter single-channel conductance of the human L-type cardiac Ca2+ channel. In: Circulation Research. 1994 ; Vol. 75, No. 2. pp. 315-323.
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N2 - 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 a, 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. The glutamic acid at position 1387 may be only peripherally involved in the formation of a high-affinity Ca2+-binding site. We suggest that disruption of this site results in an undisturbed stabilization of inner sites, thereby causing a decrease in divalent cation conductance. Substitution at position 677 disrupts coordination of both inner and outer sites, resulting in weaker divalent cation binding and increased conductance.

AB - 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 a, 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. The glutamic acid at position 1387 may be only peripherally involved in the formation of a high-affinity Ca2+-binding site. We suggest that disruption of this site results in an undisturbed stabilization of inner sites, thereby causing a decrease in divalent cation conductance. Substitution at position 677 disrupts coordination of both inner and outer sites, resulting in weaker divalent cation binding and increased conductance.

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KW - Site-directed mutagenesis

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