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

Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP 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 ATP 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 ²²Na⁺ 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.