Diltiazem causes open channel block of recombinant 5-HT3 receptors.

1. To extend our knowledge of the site and mechanism of action of L-type Ca2+ channel antagonists on 5-HT3 receptors, whole-cell voltage clamp electrophysiology was used to investigate the action of one of these compounds, diltiazem, on the recombinant receptor expressed in human embryonic kidney (HEK) 293 cells. 2. Application of diltiazem with 5-HT (30 microM) caused an increase in the rate of receptor current decay, but did not significantly affect peak current (Ip), the EC50 or the Hill coefficient, indicating a non-competitive mechanism of action. Pre-application of the antagonist had no effect indicating that diltiazem mediates its effects by binding preferentially to the open state of the 5-HT3 receptor. 3. To examine the effects of diltiazem on the open state of the receptor in more detail we used 10 mM 5-hydroxyindole (5-OHi) to reduce receptor desensitisation. These experiments showed that diltiazem causes a rapid, reversible, block in the presence of agonist but can become trapped in the unliganded state of the receptor by prior washout of agonist. Dose-inhibition data yielded an IC50 of 5.5 microM and a Hill coefficient of 0.96; inhibition was slightly voltage dependent as the degree of blockade at +60 mV was reduced. 4. The Hill coefficient of near unity suggests a single molecule of diltiazem mediates inhibition and, indeed, kinetic analysis verified that the interaction of diltiazem with the 5-HT3 receptor was well described by a bimolecular reaction scheme. The results suggest that diltiazem acts by causing open-channel block of the 5-HT3 receptor.