The Na/K pump actively exports 3 Na ؉ in exchange for 2 K ؉ across the plasmalemma of animal cells. As in other P-type ATPases, pump function is more effective when the relative affinity for transported ions is altered as the ion binding sites alternate between opposite sides of the membrane. Deletion of the five C-terminal residues from the ␣-subunit diminishes internal Na ؉ (Na duced an inward flow of Na ؉ through Na/K pumps at negative potentials. Guanidinium ؉ can also permeate truncated pumps, whereas N-methyl-D-glucamine cannot. Because guanidinium o ؉ can also traverse normal Na/K pumps in the absence of both Na o ؉ and K o ؉ and can also inhibit Na/K pump currents in a Na ؉ -like voltagedependent manner, we conclude that the normal pathway transited by the first externally released Na ؉ is large enough to accommodate guanidinium ؉ .C-terminal truncation ͉ ion access channel ͉ ion binding ͉ Na,K-ATPase ͉ voltage dependence
Truncation of the α‐subunit's last 5 residues renders Na/K pumps with a reduced affinity for intracellular Na (Nai), without effects on affinity for external K (Ko) (Morth et al, 2007). We used two electrode voltage clamp, and cut‐open oocyte to investigate the effect of the ΔKESYY truncation on electrogenic transport by Xenopus Na/K pumps (Q120RN131D‐α1+β3). Pump currents (IP) were measured 2‐3 days after cRNA injection in Nai‐loaded oocytes. The K0.5 for IP activation by Ko in Na‐free NMDG external solutions was ≈0.2 mM, both in eggs expressing truncated (TP) or normal (NP) pumps; TP expressing eggs showed larger IP (IPmax≈300 nA) than NP expressing eggs (IPmax≈150 nA). Western blots suggest better expression of TP at the plasma membrane. Surprisingly, in Ko‐free solutions, TP but not NP, presented a voltage‐ and Nao‐dependent inward current of ≈ ‐250 nA at ‐160 mV with 135 mM Nao, that is reduced by ≈50% at 67 mM Nao. Consistent with a low Nao affinity in TP, the voltage‐dependent distribution of transient charge movement (which describes the E1‐E2 occupancy in the Na branch of transport) is shifted by ≈‐100 mV compared with NP. The effect of combining ΔKESYY with other mutations that modify external Na affinity will also be presented.
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