Mutational analysis of Glu-327 of Na+-K+-ATPase reveals stimulation of 86Rb+ uptake by external K+

A competition assay of⁸⁶Rb⁺uptake in HeLa cells transfected with ouabain-resistantNa⁺-K⁺-ATPasemutants revealed a stimulation of⁸⁶Rb⁺uptake at low external concentrations (1 mM) of competitor(K⁺). Of the models that weretested, those that require that two K⁺ be bound before transportoccurs gave the worst fits. Random and ordered binding schemesdescribed the data equally well. General models in which both bindingand transport were allowed to be cooperative yielded parameter errorslarger than the parameters themselves and could not be utilized. Modelsthat assumed noncooperative transport always showed positivecooperativity in binding. E327Q and E327L mutated forms of rat₂ had lower apparent affinities for the first K⁺ bound than didwild-type rat ₂ modified to beouabain resistant. The mutations did not affect the apparent affinityof the second K⁺ bound. Modelsthat assumed noncooperativity in binding always showed positivelycooperative transport, i.e., enzymes with two K⁺ bound had a higher flux thanthose with one K⁺ bound. Increasesin external Na⁺ decreased theapparent affinity for K⁺ for allmodels and decreased the ratio of the apparent influx rate constantsfor E327L.