Structure and Function of Ion Pumps Studied by Atomic Force Microscopy and Gene-transfer Experiments Using Chimeric Na⁺/K⁺- and Ca²⁺ ATPases

Abstract

Using novel chimeric Ca²⁺-ATPases modified with portions of Na⁺/K⁺-ATPase, and vice versa, we have identified critical domains that govern ion sensitivity, inhibitor specificity, subunit assembly, and activation by Ca²⁺/calmodulin. Na⁺ and K⁺ sensors have been identified in nonconserved regions of the Na⁺/K⁺-ATPase sequences; the Na⁺ sensor, with 69 amino acids, is localized at the amino-terminal region of the α subunit, whereas the K⁺ sensor, with 161 residues, resides at the carboxy terminus of the same subunit. The assembly domain with the β subunit was identified within the C-terminal 161 amino acids of the Na⁺/K⁺-ATPase a subunit (Figure 1), When this region was incorporated into the corresponding region of the sarcoplasmic reticulum (SR) Ca²⁺-ATPase, the heterodimer of the Na⁺/K⁺-ATPase β subunit and the chimeric SR Ca²⁺-ATPase was formed (25; for details, see Lemas et al. in this volume). The ouabain-sensitive domain was localized within the N-terminal 200 amino acids of the Na⁺/K⁺-ATPase. These variable amino-terminal 200 amino acids, when incorporated into the corresponding regions of the SR Ca²⁺-ATPase, conferred ouabain sensitivity to the SR Ca²⁺-ATPase (15). Addition of the carboxy-terminal 150 amino acids of the plasma membrane (PM) Ca²⁺-ATPase to the Na⁺/K⁺-ATPase a subunit conferred Ca²⁺/ calmodulin sensitivity on the Na⁺/K⁺-ATPase, indicating the existence of potential domains in the Na⁺/K⁺-ATPase that can interact with the carboxy terminus of the PM Ca²⁺-ATPase (for details, see Ishii and Takeyasu in this volume). The successful functional expression of additional chimeric ATPases will allow identification of other critical regions, such as domains responsible for targeting of distinct ATPases to the specific subcellular membranes. This section reviews our recent work on chimeric ATPases. We also address potential abilities of atomic force microscopy (AFM; for details, see Paul et al. in this volume) for analysis of molecular structures of the P-type ATPases.