Bufo marinusbladder H-K-ATPase carries out electroneutral ion transport

Abstract

Bufo marinus bladder H-K-ATPase belongs to the Na-K-ATPase and H-K-ATPase subfamily of oligomeric P-type ATPases and is closely related to rat and human nongastric H-K-ATPases. It has been demonstrated that this ATPase transports K(+) into the cell in exchange for protons and sodium ions, but the stoichiometry of this cation exchange is not yet known. We studied the electrogenic properties of B. marinus bladder H-K-ATPase expressed in Xenopus laevis oocytes. In a HEPES-buffered solution, K(+) activation of the H-K-ATPase induced a slow-onset inward current that reached an amplitude of approximately 20 nA after 1-2 min. When measurements were performed in a solution containing 25 mM HCO at a PCO(2) of 40 Torr, the negative current activated by K(+) was reduced. In noninjected oocytes, intracellular alkalization activated an inward current similar to that due to B. marinus H-K-ATPase. We conclude that the transport activity of the nongastric B. marinus H-K-ATPase is not intrinsically electrogenic but that the inward current observed in oocytes expressing this ion pump is secondary to intracellular alkalization induced by proton transport.

Electrogenicity of Na,K- and H,K-ATPase Activity and Presence of a Positively Charged Amino Acid in the Fifth Transmembrane Segment

“…The wild-type Na,K-ATPase current/voltage curve shows little voltage dependence in the 0 to Ϫ50 mV membrane potential range and a negative slope in the high negative voltage range, similar to what has been described previously for Na,K-pumps expressed in Xenopus oocytes (6,19). Potassium induced a voltage-dependent inward current in the wild-type H,K-ATPase, similar to what we have described previously (8). The K800A and K800E mutants were only weakly voltage-dependent, whereas the K800S mutant displayed stronger voltage dependence along the whole membrane potential range (a 4-fold reduction of the ouabain-sensitive current between 10 and Ϫ110 mV compared with a Ͻ30% decrease for the K800A and K800E mutants over the same voltage range).…”

“…4, A (examples) and C (mean values)). When studied under these conditions, the wild-type Na,K-pump generated outward currents amounting to ϳ120 nA; and as reported previously (8), activation by extracellular K ϩ results only in small inward currents in oocytes expressing the wild-type Bufo bladder H,K-ATPase. An K ϩ -activated and ouabain-sensitive outward current was observed with the K800A mutant H,KATPase and, with a very small amplitude, with the K800E mutant H,K-ATPase, indicating the presence of electrogenic transport activity.…”

“…Fig. 4F also shows the concentration dependence of the inward current induced by K ϩ in the wild-type H,K-ATPase, a current that is related rather to the intracellular alkalization than to an electrogenic transport activity of this H,K-ATPase as shown earlier (8). The maximal K ϩ -induced current was Ϫ25.9 Ϯ 1.8 nA (n ϭ 5), and the apparent affinity for external K ϩ was 0.12 Ϯ 0.08 mM.…”

“…We chose to coexpress these wild-type and mutant ␣-subunits with the B. marinus bladder isoform of the ␤-subunit (␤ bl ), which is the amphibian homolog of the mammalian ␤ 2 -isoform, because the ␣ 1 /␤ bl and ␣ bl /␤ bl dimers are well expressed at a similar level in Xenopus oocytes (8), and it was essential to express the various wild-type and mutant ␣-subunits with the same ␤-subunit to be able to attribute the observed differences to the mutations carried by the ␣-subunit.…”

“…In contrast, the gastric H,KATPase is known to perform an electroneutral exchange of H ϩ for K ϩ , most probably 2 K ϩ ions for 2 protons (7). We have recently shown that the "non-gastric" H,K-ATPase has electroneutral transport activity (8), but this information only indicates that a symmetrical number of cations have to be exchanged. The exact nature of the transported cations has not yet been fully determined, as both H ϩ (9) and Na ϩ (10, 11) seem to be transported in exchange for K ϩ ; and thus, the transport stoichiometry of this ATPase is not known.…”