Capsazepine, a synthetic vanilloid that converts the Na,K-ATPase to Na-ATPase

Capsazepine (CH), a synthetic capsaicin analogue, inhibits ATIP hydrolysis by Na,K-ATPase in the presence but not in the absence of K+. Studies with purified membranes revealed that CPZ reduced Na+-dependent phosphorylation by interference with Na+ binding from the intracellular side of the membrane. Kinetic analyses showed that CPZ stabilized an enzyme species that constitutively occluded K+. Low-affinity ATIP interaction with the enzyme was strongly reduced after CPZ treatment; in contrast, indirectly measured interaction with ADP was much increased, which suggests that composite regulatory communication with nucleotides takes place during turnover. Studies with lipid vesicles revealed that CPZ reduced ATP-dependent digitoxigenin-sensitive Na-22(+) influx into K+-loaded vesicles only at saturating ATP concentrations. The drug apparently abolishes the regulatory effect of ATP on the pump. Drawing on previous homology modeling studies of Na,K-ATPase to atomic models of sarcoplasmic reticulum Ca-ATPase and on kinetic data, we propose that CPZ uncouples an Na+ cycle from an Na+/K+ cycle in the pump. The Na+ cycle possibly involves transport through the recently characterized Na+-specific site. A shift to such an uncoupled mode is believed to produce pumps mediating uncoupled Na+ efflux by modifying the transport stoichiometry of single pump units.