Mechanism of Action of Salt Adaptation Mutations in Artemia Franciscana

Abstract

Nearly all animals maintain a large electrochemical gradient for Na+ across the plasma membrane. This gradient is generated by the Na-K pump, which exports 3 Na+ and imports 2 K+ per ATP molecule hydrolyzed. Ion-coordinating residues in the α subunit are usually conserved, but the brine shrimp (Artemia franciscana) living in extreme saline conditions express a pump with two asparagine to lysine substitutions within the ion binding site region (Jorgensen and Amat (2008) J. Memb Biol. 221:39-49). We used two-electrode voltage clamp on Na+-loaded Xenopus oocytes to evaluate the effect of the substitutions (N333K and N785K) individually and concurrently on the function of Xenopus Na/K pumps. We studied their effect on activation of pump currents by eternal K+ and on voltage-dependent conformational changes related to external Na+ binding (charge movement). The center of the Q-V curves are displaced by ∼-80 mV by both individual mutations suggesting a reduced (>10 fold) external Na+ affinity. Surprisingly the double mutant showed a nearly identical shift in the Q-V, indicating non-additive effects on external Na+ affinity. Apparent affinity for K+ in the absence of Na+ was reduced (∼10-fold) by the N785K mutation while N333K and the double mutant had similar affinity to the wild type. These results can be explained with recent structures of the Na/K pump with Na+ or K+ bound. N333, outside the ion-binding pocket, forms a hydrogen bond with ion-coordinating N785 in the Na+ bound conformation. Once the disruption of normal Na+ coordination by N785K is in place the mutation N333K does not affect Na+ binding. This contrasts with previous findings regarding internal Na+ binding. We are investigating intracellular ion dependence in these mutants by measuring currents in patch clamp and enzymatic activity in membrane preparations. Supported by NSF-MCB-1243842 & NIH-NS081570-01.