Photolabile Amiloride Derivatives as Cation Site Probes of the Na,K-ATPase

Abstract

Treatment of purified canine renal Na,K-ATPase with a range of photoactivatable amiloride derivatives results in inhibition of ATPase activity prior to illumination. Inhibition by amiloride derivatives substituted on a guanidium N could not be prevented by the presence of either K or Na; however, these cations could protect the enzyme against inhibition by derivatives substituted on the 5-position of the pyrazine ring. In the case of 5-(N-ethyl-[2'-methoxy-4'-nitrobenzyl])amiloride (NENMBA), the presence of monovalent cations (Na, K, and Rb) protected the enzyme effectively against inhibition, with concentrations in the millimolar range. ATP did not prevent inhibition; furthermore, native and NENMBA-treated enzyme exhibited normal levels of high affinity [3H]ADP (and hence ATP) binding. The rate of inhibition increased with increasing concentrations of NENMBA. Extensive washing of NENMBA-inhibited enzyme did not restore ATPase activity, showing that NENMBA has an extremely slow off-rate for dissociation from its inhibitory site. Partially inhibited enzyme could be rapidly pelleted and resuspended in NENMBA-free buffer and inhibition was observed to continue, albeit at a somewhat diminished rate, suggesting that NENMBA gains access to its inhibitory site after partitioning into the lipid phase rather than directly from the aqueous solution. Photolysis of NENMBA-inhibited enzyme resulted in covalent incorporation of the reagent into the alpha-subunit of the Na,K-ATPase, as observed by separation of labeled protein on a Laemmli gel and Western analysis using a polyclonal amiloride antibody. Almost all of the covalent labeling could be prevented by the presence of Rb in the incubation and labeling medium. These results suggest that NENMBA inhibits the Na, K-ATPase by disruption of the cation transport domain rather than the catalytic domain of the enzyme and that it promises to be a useful tool for cation site localization.