Abstract

Homopentameric complexes of either the A or As subunit of the 5-hydroxytryptamine3 receptor form Ca(2+)-permeable channels that can be activated by the selective agonist 1-(m-chlorophenyl)-biguanide (mCPBG). In both N1E-115 neuroblastoma cells and human embryonic kidney 293 cells stably expressing the 5-HT3 receptor As subunit, (+)-verapamil, (-)-verapamil, diltiazem, and nimodipine caused reversible and concentration-dependent (IC50 = 2.5-6.5 microM) inhibition of the increases in cytosolic [Ca2+] evoked by mCPBG. In voltage-clamped human embryonic kidney 293 cells stably expressing the 5-HT3 receptor As subunit, similar concentrations of the Ca2+ channel antagonists (IC50 = 3.0-6.8 microM) accelerated the rate at which 5-HT-evoked currents decayed without affecting the amplitude of the peak current. In equilibrium competition binding assays to membranes from Sf9 cells infected with the 5-HT3 receptor As subunit, [3H]mCPBG and [3H]granisetron were displaced by (+)-verapamil, (-)-verapamil, and diltiazem; (+)-verapamil was approximately 10-fold more potent than (-)-verapamil and approximately-30-fold more potent than diltiazem. Nimodipine neither displaced [3H]granisetron binding nor affected its displacement by diltiazem and (+)-verapamil. The stereoselectivity of verapamil binding, which contrasts with the similar potency of each isomer in functional assays, was maintained when the incubations were performed at 20 degrees or when an antagonist of the 5-HT3 receptor, [3H]granisetron, was used as the radioligand. The interaction between verapamil and either [3H]mCPBG or [3H]granisetron binding was not competitive. We conclude that the inhibition of [3H]mCPBG binding by diltiazem and verapamil is mediated by a site that is distinct from both the agonist-binding site and from the site through which nimodipine inhibits 5-HT3 receptor function. Our results provide evidence for allosteric regulation of agonist binding to 5-HT3 receptors and the first example of a ligandgated ion channel whose function is directly inhibited by members of all three major classes of L-type Ca2+ channel antagonists.