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, transport s 1 Ca2+ and 1 K+ in exchange for 4 Na+. The present work describes the pro perties of chimeric constructs of the two exchanger types. Ca2+ gel overlay experiments have identified a high affinity (Kd in the 1 mu M 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 retina l Ca2+-binding domain in the cytosolic loop of the cardiac exchanger confer red K+-dependence to the Ca2+ uptake activity of the chimeric constructs ex pressed in HeLa cells. The apparent K-m of the K+ effect was about 1 mM. Ex periments with C-terminally truncated versions of the retinal insert indica ted that the sequence between Leu643 and Asp733 was critical in mediating K + sensitivity of the recombinant chimeras. Thus, the high affinity Ca2+-bin ding domain in the main cytosolic loop of the retinal exchanger may regulat e the activity of the retinal protein by binding Ca2+, and by conferring to it K+ sensitivity.