Mutation of amino acid residues in the putative transmembrane segments of the cardiac sarcolemmal Na+-Ca2+ exchanger.
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 alpha-1 and alpha-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 alpha-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 alpha-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 alpha-repeat and pump-like regions (Asp-740, Asp-785, and Asp-798), only mutation at Asp-785 resulted in reduction of exchanger activity.
Nicoll
D A
DA
Department of Physiology, UCLA, School of Medicine, Los Angeles, California 90095-1760, USA.
Hryshko
L V
LV
Matsuoka
S
S
Frank
J S
JS
Philipson
K D
KD
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D017124:Conserved Sequence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D017354:Point Mutation; D012091:Repetitive Sequences, Nucleic Acid; D012508:Sarcolemma / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium