Electrogenic partial reactions of the gastric H,K-ATPase

¹ University of Konstanz
² University of California, Los Angeles
³ University of California, los Angeles

^* To whom correspondence should be addressed. E-mail: h-j.apell{at}uni-konstanz.de.

Submitted on November 24, 2004
Revised on January 10, 2005
Accepted on 23 February 2005

	Abstract

The fluorescent styryl dye RH421 was used to identify and investigate the electrogenic reaction steps of the H,K-ATPase pump cycle. Equilibrium titration experiments were performed with membrane vesicles isolated from hog gastric mucosa, and cytoplasmic and luminal binding of K⁺ and H⁺ ions was studied. It was found that the binding and release steps of both ion species in both principal conformations of the ion pump, E₁ and P-E₂, are electrogenic, while the conformation transitions do not contribute significantly to a charge movement within the membrane dielectric. This behavior is in agreement with the transport mechanism found for the Na,K-ATPase and the SR Ca-ATPase. The data were analyzed on the basis of an Post-Albers reaction cycle. For proton binding two pK values were found in both conformations: 6.7 and 4.5 in the E₁ conformation, 6.7 and 2 in the P-E₂ conformation. The equilibrium dissociation constants for K⁺ binding on the cytoplasmic side were 11 mM and 16 mM. The respective equilibrium dissociation constants on the luminal side were obtained via K⁺ concentration dependence of the enzyme activity and determined to be 0.11 mM for both luminal binding sites.

Key Words: cation binding, electrochromic dye, fluorescence measurements, gastric proton pump