MOLECULAR DISSECTION OF FUNCTIONAL DOMAINS OF THE E1E2-ATPASE USING SODIUM AND CALCIUM-PUMP CHIMERIC MOLECULES

Proposed models for the catalytic subunit of the E1E2-ATPases (ion pumps) predict that the first four transmembrane domains (M1 - M4) reside in the NH2 terminal one-third of the molecule, and the remainder (M5 - M10) in the COOH terminal one-third. The amino-acid sequences for the 5'-(p-fluorosulfonyl)-benzoyl-adenosine (FSBA) binding region residing just before M5 segment are very well conserved among distinct ion pumps. Taking advantage of these models, we have constructed a set of chicken chimeric ion pumps between the (Na+ + K+)-ATPase-alpha-subunit and the Ca2+-ATPase using the FSBA-binding site as an exchange junction, thereby preserving overall topological structure as E1E2 ATPases. From various functional assays on these chimeric ion pumps, including ouabain-inhibitable ATPase activity, Ca2+ binding, Ca2+ uptake, and subunit assembly based on immuno-coprecipitation, the following conclusions were obtained: (a) A (Na+ + K+)-ATPase inhibitor, ouabain, binds to the regions before M4 in the alpha-subunit and exerts its inhibitory effect. (b) The regions after M5 of the (Na+ + K+)-ATPase alpha-subunit bind the beta-subunit, even when these regions are incorporated into the corresponding domains in the Ca2+-ATPase. (c) The corresponding domains of the Ca2+-ATPase, the regions after M5 bind Ca-45 even when it is incorporated into the corresponding position of the (Na+ + K+)-ATPase-alpha-subunit.