DIRECT INHIBITION OF 5-HYDROXYTRYPTAMINE(3) RECEPTORS BY ANTAGONISTS OF L-TYPE CA2+ CHANNELS

Homopentameric complexes of either the A or As subunit of the 5-hydrox ytryptamine(3) receptor form Ca2+-permeable channels that can be activ ated 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, (-)-v erapamil, diltiazem, and nimodipine caused reversible and concentratio n-dependent (IC50=2.5-6.5 mu M) inhibition of the increases in cytosol ic [Ca2+] evoked by mCPBG. In voltage-clamped human embryonic kidney 2 93 cells stably expressing the 5-HT3 receptor As subunit, similar conc entrations of the Ca2+ channel antagonists (IC50=3.0-6.8 mu M) acceler ated 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, [H-3]mCPBG and [H-3]granisetron were displaced by (+)-verapa mil, (-)-verapamil, and diltiazem; (+)-verapamil was similar to 10-fol d more potent than (-)-verapamil and similar to 30-fold more potent th an diltiazem, Nimodipine neither displaced [H-3]granisetron binding no r affected its displacement by diltiazem and (+)-verapamil. The stereo selectivity of verapamil binding, which contrasts with the similar pot ency of each isomer in functional assays, was maintained when the incu bations were performed at 20 degrees or when an antagonist of the 5-HT 3 receptor, [H-3]granisetron, was used as the radioligand. The interac tion between verapamil and either [H-3]mCPBG or [H-3]granisetron bindi ng was not competitive. We conclude that the inhibition of [H-3]mCPBG binding by diltiazem and verapamil is mediated by a site that is disti nct from both the agonist-binding site and from the site through which nimodipine inhibits 5-HT3 receptor function. Our results provide evid ence for allosteric regulation of agonist binding to 5-HT3 receptors a nd the first example of a ligand-gated ion channel whose function is d irectly inhibited by members of all three major classes of L-type Ca2 channel antagonists.