Testing K+-like occlusion of cations in the Na+/K+-ATPase.

ROLANDO C. ROSSI; RODOLFO M. GONZÁLEZ-LEBRERO; SERGIO B. KAUFMAN; PATRICIO J. GARRAHAN

Woods Hole, MA, USA.

Congreso; 11th International ATPase Conference & 59th Annual Meeting and Symposium of the Society of General Physiologists.; 2005

Society of General Physiologists.

The K+-congener Rb+ can become spontaneously occluded in the Na+/K+-ATPase, leading to a state that slowly exchanges cations with de medium. Therefore, the rate of 86Rb+ occlusion can be used to test whether any other cation (X+) can lead the enzyme to a state that slows down 86Rb+ occlusion (perhaps with X+ being occluded). With this aim, we measured the time course of 86Rb+ occlusion at 25ºC in a purified preparation of Na+/K+-ATPase, after addition of 86Rb+ (20 µM, final concentration) under two conditions: (i) X+ was preincubated with the enzyme, or (ii) X+ was added together with 86Rb+. Media contained 25 mM Imidazole-HCl, pH 7.4. We found that, when the cation was K+, Rb+, Cs+, Tl+, NH4+, or Na+ + oligomycin, the rate of 86Rb+ occlusion for condition (i) was significantly slower than that of condition (ii). When Na+, Li+, or Mg2+ were used, no significant difference between the time courses for both conditions was observed. To discriminate if those cations that showed differences between conditions (i) and (ii) were acting as K+, we tested the ability of X+ to block Rb+ deocclusion (Forbush, 1987, J. Biol. Chem. 262:11116-11127). We therefore equilibrated the enzyme with 20 µM 86Rb+ and followed the time courses of 86Rb+ deocclusion after a 1/20 dilution in media with or without (control) X+. We found that, when X+ was K+, Rb+, Tl+, or Cs+, the rate of 86Rb+ deocclusion was slower than that of the control, whereas when Na+, Li+, NH4+, Mg2+, or oligomycin + Na+ were used, no significant differences with the control were observed. These results agree with the rule that those cations that become occluded like K+ should show differences between the rates of occlusion for conditions (i) and (ii), and be good blockers of Rb+ deocclusion. NH4+ seems to be an exception to this rule.   [Supported by CONICET, ANPCyT and UBA]