Membranes and Bioenergetics
Mutation of Amino Acid Residues in the Putative Transmembrane Segments of the Cardiac Sarcolemmal Na+-Ca2+ Exchanger*

https://doi.org/10.1074/jbc.271.23.13385Get rights and content
Under a Creative Commons license
open access

We have examined the role of conserved regions and acidic or basic residues located in the putative transmembrane segments of the cardiac sarcolemmal Na+-Ca2+ exchanger by site-directed mutagenesis. The α-1 and α-2 repeats are transmembrane regions of internal similarity, which are highly conserved among Na+-Ca2+ exchangers. We find that Na+-Ca2+ exchange activity is highly sensitive to mutagenesis in the α-repeats. Mutation at residues Ser-109, Ser-110, Glu-113, Ser-139, Asn-143, Thr-810, Ser-811, Asp-814, Ser-818, or Ser-838 resulted in loss of exchanger activity. Mutation at residues Thr-103, Gly-108, Pro-112, Glu-120, Gly-138, Gly-809, Gly-837, and Asn-842 resulted in reduced exchanger activity, and altered current-voltage relationships were observed with mutations at residues Gly-138 and Gly-837. Only mutation at residue Ser-117 appeared to leave exchanger activity unaffected. Thus, the α-repeats appear to be important components for ion binding and translocation. Another region implicated in exchanger function is a region of similarity to the Na+,K+ pump (Nicoll, D. A., Longoni, S., Philipson, K. D. (1990) Science 250, 562-565). Mutations at two residues in the pump-like region, Glu-199 and Thr-203, resulted in nonfunctional exchangers, while mutation at two other residues, Glu-196 and Gly-200, had no effect. The role of acidic and basic residues in the transmembrane segments was also examined. Mutation of several basic residues (Arg-42, His-744, Lys-751, Lys-797, and His-858) did not affect exchange activity. Of the acidic residues located outside of the α-repeat and pump-like regions (Asp-740, Asp-785, and Asp-798), only mutation at Asp-785 resulted in reduction of exchanger activity.

Cited by (0)

*

This work was supported by the National Institutes of Health Grant HL49101, the American Heart Association, Greater Los Angeles Affiliate, and the Laubisch Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Current address: Division of Cardiovascular Sciences, St. Boniface Hospital Research Centre, Winnipeg, Manitoba R2G 2A6, Canada.

Current address: Dept. of Physiology, Faculty of Medicine, Kyoto University, Kyoto 606-01, Japan.