The N-terminal portion of the main cytosolic loop mediates K+ sensitivity in the retinal rod Na+/Ca2+-K+-exchanger.
Two types of Na+/Ca2+-exchangers have been characterized in the literature: The first is the cardiac, skeletal muscle and brain type, which exchanges 1 Ca2+ for 3 Na+, the second, found in retinal photosensor cells, transports 1 Ca2+ and 1 K+ in exchange for 4 Na+. The present work describes the properties of chimeric constructs of the two exchanger types. Ca2+ gel overlay experiments have identified a high affinity (Kd in the 1 microM range) Ca2+-binding domain between Glu601 and Asp733 in the main cytosolic loop of the retinal protein, just after transmembrane domain 5. Insertion of the retinal Ca2+-binding domain in the cytosolic loop of the cardiac exchanger conferred K+-dependence to the Ca2+ uptake activity of the chimeric constructs expressed in HeLa cells. The apparent Km of the K+ effect was about 1 mM. Experiments with C-terminally truncated versions of the retinal insert indicated that the sequence between Leu643 and Asp733 was critical in mediating K+ sensitivity of the recombinant chimeras. Thus, the high affinity Ca2+-binding domain in the main cytosolic loop of the retinal exchanger may regulate the activity of the retinal protein by binding Ca2+, and by conferring to it K+ sensitivity.
Seiler
E P
EP
Department of Biochemistry III, Swiss Federal Institute of Technology (ETH), Zurich, Switzerland.
Guerini
D
D
Guidi
F
F
Carafoli
E
E
D000818:Animals; D016470:Bacteremia; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004285:Dogs; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D011188:Potassium / Q000737:chemistry / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger; D013347:Subcellular Fractions / Q000378:metabolism
D002352:Carrier Proteins; D017931:DNA Primers; D019831:Sodium-Calcium Exchanger; C074530:potassium-dependent sodium-calcium exchanger; D011188:Potassium; D002118:Calcium