Split Na+-Ca2+ exchangers. Implications for function and expression.
The Na(+)-Ca(2+) exchanger has nine transmembrane segments, with a large cytoplasmic loop between the fifth and sixth transmembrane segments. The protein was split within the cytoplasmic loop into two domains consisting of the first five transmembrane segments and the last four transmembrane segments, respectively. The two domains were either expressed individually or coexpressed. Each of the two domains with different lengths of the cytoplasmic loop was fused to green fluorescent protein. We show that coexpression of both domains is required for proper membrane targeting and for expression of functional exchange activity. Fusion to green fluorescent protein does not alter biophysical properties of the exchange process. In addition, truncation of a large portion of the cytoplasmic loop does not alter important properties of the exchanger such as Na(+)-dependent inactivation, activation by chymotrypsin, or exchanger inhibitory peptide (XIP) sensitivity.
Departments of Physiology and Medicine and the Cardiovascular Research Laboratories, University of California, School of Medicine, Los Angeles, California 90095-1760, USA.
M
M
Ottolia
S
S
John
Z
Z
Qiu
K D
KD
Philipson
D000818:Animals; D002118:Calcium / Q000493:pharmacokinetics; D002462:Cell Membrane / Q000378:metabolism; D002918:Chymotrypsin / Q000378:metabolism / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D004594:Electrophysiology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000096:biosynthesis* / Q000737:chemistry* / Q000502:physiology*; D014981:Xenopus
D008164:Luminescent Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D019831:Sodium-Calcium Exchanger; C066745:exchanger inhibitory peptide; D049452:Green Fluorescent Proteins; D012964:Sodium; D002918:Chymotrypsin; D002118:Calcium