Mechanism of Action of Salt Adaptation Mutations in Artemia franciscana
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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. The ion-coordinating residues in the α subunit of the pump are usually conserved in most species. However, the brine shrimp (Artemia franciscana) that lives in extreme saline conditions express a pump with two asparagine to lysine substitutions within the ion binding site region (Jorgensen and Amat, 2008). We used two-electrode voltage clamp on Na+-loaded Xenopus oocytes to evaluate the effect of the equivalent substitutions (N333K and N785K) individually and concurrently on the function of Xenopus Na+/K+ pumps. In particular, we studied the effect of these mutations on activation of pump currents by eternal K+ and on voltage-dependent conformational changes related to external Na+ binding (charge voltage (Q-V) curves). The apparent affinity for K+ in the absence of Na+ was reduced (approximately ten-fold) by the N785K, mutation while N333K and the double mutant had similar affinity to the wild-type. The centers of the Q-V curves were displaced approximately -80 mV by both individual mutations suggesting a reduced (greater than ten-fold) external Na+ affinity. Surprisingly, the double mutant showed a slightly smaller shift in the Q-V, indicating non-additive effects on external Na+ affinity and energetic coupling between the residues. 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 the ion-coordinating N785 in the Na+ bound conformation. Therefore, 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.