The interaction of trichloroethanol with murine recombinant 5‐HT3 receptors
Authors: Downie David L. , Hope Anthony G. , Belelli Delia , Lambert Jeremy J. , Peters John A. , Bentley Kirn R. , Steward Lucinda J. , Chen Chong‐Yang , Barnes Nicholas M. 
Published: July 2012

The effects of ethanol, chloral hydrate and trichloroethanol upon the 5‐HT3 receptor have been investigated by use of electrophysiological techniques applied to recombinant 5‐HT3 receptor subunits (5‐HT3R‐A or 5‐HT3R‐As) expressed in Xenopus laevis oocytes. Additionally, the influence of trichloroethanol upon the specific binding of [3H]‐granisetron to membrane preparations of HEK 293 cells stably transfected with the murine 5‐HT3R‐As subunit and 5‐HT3 receptors endogenous to NG 108‐15 cell membranes was assessed.

Ethanol (30–300 mM), chloral hydrate (1–30 mM) and trichloroethanol (0.3‐10mM), produced a reversible, concentration‐dependent, enhancement of 5‐HT‐mediated currents recorded from oocytes expressing either the 5‐HT3R‐A, or the 5‐HT3R‐As subunit.

Trichloroethanol (5 mM) produced a parallel leftward shift of the 5‐HT concentration‐response curve, reducing the EC50 for 5‐HT from 1 ± 0.04 μm (n = 4) to 0.5 ± 0.01 μm (n = 4) for oocytes expressing the 5‐HT3R‐A. A similar shift, from 2.1 ± 0.05 μm (n = 11) to 1.3 ± 0.1 μm (n = 4), was observed in oocytes expressing the 5‐HT3R‐As subunit. Trichloroethanol (5 mM) had little or no effect upon the maximum current produced by 5‐HT for either recombinant receptor.

Trichloroethanol (5 mM) similarly reduced the EC50 for 2‐methyl‐5‐HT from 13 ± 0.4 μm (n = 4) to 4.6 ± 0.2 μm (n = 4) and from 15 ± 2μm (n = 4) to 5 ± 0.4μm (n = 4) for oocytes expressing the 5‐HT3R‐A and 5‐H3R‐As subunit respectively. Additionally, trichloroethanol (5 mM) produced a clear enhancement of the maximal current to 2‐methyl‐5‐HT (expressed as a percentage of the maximal current to 5‐HT) from 63 ± 0.7% (n = 4) to 101 ± 1.6% (n = 4) and from 9 ± 0.2% (n = 4) to 74 ± 2% (n = 4) for oocytes expressing the 5‐HT3R‐A and 5‐HT3R‐As subunit respectively.

Trichloroethanol (2.5 mM) had no effect upon the Kd, or Bmax, of specific [3H]‐granisetron binding to membrane homogenates of NG 108‐15 cells or HEK 293 cells. Similarly, competition for [3H]‐granisetron binding by the 5‐HT3 receptor antagonists ondansetron and tropisetron was unaffected. However, competition for [3H]‐granisetron binding by the 5‐HT3 receptor agonists, 5‐HT, 2‐methyl‐5‐HT and phenylbiguanide was enhanced by trichloroethanol (2.5 mM).

Unexpectedly, the competition for [3H]‐granisetron binding by the 5‐HT3 receptor antagonist, quipazine, was enhanced by 2.5 mM trichloroethanol. Quipazine (1 nM‐0.3 μm) antagonized 5‐HT‐evoked currents recorded from oocytes expressing the 5‐HT3R‐A subunit with an IC50 of 18 ± 3 nM (n = 4). Additionally, quipazine (30 nM‐0.3 μm) produced a small inward current which was greatly enhanced by 5 mM trichloroethanol and antagonized by 100 nM ondansetron. Collectively, these observations suggest that quipazine may act as a partial agonist.

The demonstration that a recombinant homo‐oligomeric receptor, expressed in a foreign membrane, retains a sensitivity to alcohols, together with the sequencing of alcohol‐insensitive 5‐HT3 receptor subunits, may lead to a better definition of the alcohol binding site(s).