Capsaicin binds to the intracellular domain of the capsaicin-activated ionchannel

Capsaicin (CAP) excites small sensory neurons, causing pain, neurogenic inf lammation, and other visceral reflexes. These effects have been proposed to be the result of CAP activation of a nonselective cation current. It is ge nerally assumed that CAP binds to an extracellular domain of the membrane r eceptor. However,the exact binding site is not known because of the lipophi lic nature of CAP. To determine whether the binding domain is extracellular or intracellular, we tested the effect of a synthetic water-soluble CAP an alog, DA-5018.HCl, on current activation. CAP activated the 45 pS (at -60 m V) nonselective cation channel from either side of the membrane. However, D A-5018.HCl, which had a greater potency and efficacy than CAP, activated th e channels only from the cytosolic side of the patch membrane in a capsazep ine, a CAP receptor antagonist, reversible manner. When applied extracellul arly, DA-5018.HCl did not, but CAP did, activate whole-cell currents in sen sory neurons, as well as in oocytes expressing vanilloid receptor 1, a rece ntly cloned CAP receptor. Hydrogen ions, reported as a possible endogenous activator of cation current, failed to elicit any current when acidic mediu m (pH 5.0-6.0) was applied intracellularly, indicating that H+ does not med iate the CAP effect. These results indicate that CAP and its analog bind to the cytosolic domain of the CAP receptor and suggest that an endogenous CA P-like substance other than H+ may be present in the cell.