Characterisation of novel human 5-HT3 receptor subunits (HTR3C-E)

Catherine H Gill, Stephen E Moore, Kim Rance, Shahnaz P Yusaf, Nicola Courtenay, Gareth J Sanger, Joanna D Holbrook, Martin J Gunthorpe. Neurology & GI CEDD, GlaxoSmithKline, Third Avenue, Harlow, CM19 5AW. UK.

5-HT3 receptors are members of the superfamily of Cys-loop ligand-gated ion channels. To date, functional channels composed of either a homomeric assembly of HTR3A (5-HT3A) or a heteromeric assembly of HTR3A and HTR3B subunits (5-HT3A/B) have been described. Recently, three novel human genes encoding HTR3C, D and E were reported (Niesler et al., 2003). These subunits show a peripherally restricted pattern of expression, are present in human, dogs and ferret, but are curiously absent in rodents. To gain further insight into their functional properties, and possible physiological roles, we have cloned HTR3C-E from human tissue sources and used a mammalian heterologous expression system to investigate their functional properties using electrophysiology. HTR3C and E were amplified by RT-PCR from human colon and HTR3D from human intestine tissue RNA pools, sequenced in forward and reverse orientation and subcloned into expression vectors. Wild type CHO cells were transiently transfected with HTR3 subunits (either alone or in heteromeric combinations) and the properties of the resultant channels investigated using the whole-cell patch-clamp technique. Cells were routinely voltage-clamped at -60 mV and data are expressed as mean ± s.e. mean from 4-8 cells. The intracellular and extracellular solutions used were (in mM): CsCl 140, MgCl2 4, EGTA 10, HEPES 10 and NaCl 130, KCl 5, CaCl2 2, MgCl2 1, Glucose 30, HEPES 25, respectively (both pH 7.3). Expression of either HTR3A or HTR3A+B resulted in the formation of functional 5-HT gated channels with properties similar to those described previously (Davies et al., 1999; Das & Dillon, 2003). Heteromeric HTR3A+B receptors displayed more rapid activation kinetics, a linear current-voltage relationship and less sensitivity to blockade by picrotoxin compared to homomeric HTR3A receptors (Table 1; Statistical significance was determined using Student’s t-test (*p < 0.05)). In contrast, expression of the novel HTR3 subunits as homomeric complexes did not result in the formation of functional 5-HT sensitive ion channels.

NA – Not Applicable

Further studies will now investigate the propensity for these novel HTR3 subunits to form functional heteromeric receptors with related HTR3 subunits.

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Das & Dillon, (2003). Molecular Brain Research, 119, 207-212.
Niesler et al., (2003).Gene, 310, 101-111.