Gee, V.J.; (2005) Molecular and functional characterization of neuronal nicotinic and 5-HT/subscript3 receptors. Doctoral thesis , University of London.

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Abstract

Unlike many nicotinic subunits, the al nicotinic subunit and the 5-HT3A subunit are able to form functional homomeric receptors. Despite sharing this ability, they have differences in cell-surface expression in human embryonic kidney (HEK) cells, with 5HT3A subunits forming a large number of functional receptors, whereas the al subunit forms few or no correctly folded receptors as assayed by radioligand binding with 125I a-BTX. A series of chimeras between al and 5HT3A were constructed to investigate which domains of the subunits were important for folding and cell surface expression. Only chimeras that contained the region from the beginning of Ml to the end of M3, and M4 domain of the 5HT3A subunit were able to form correctly folded receptors. The chimeras that gave high levels of radioligand binding were also found to be functional using whole-cell patch-clamp recording. Functional characteristics were examined, and differences were found in single channel conductance and desensitization. Chimeras with the large cytoplasmic loop and the extracellular N-terminal region of al had larger single channel conductances than the 5HT3A receptor, with the inclusion of the al large cytoplasmic loop and the N-terminal domain increasing the conductance by approximately 10 and 2 fold respectively Co-expression of al with RIC3 (a protein originally identified in C. elegans) in HEK cells, results in high levels of cell surface expression. These receptors were shown to be functional, with whole cell responses from 20-300 pA. showing fast desensitization (time constant of 66 13 ms) and strong inward rectification. RIC3 was also shown to increase the functional expression of the a8 and the rat a3p2 nAChRs, which rarely formed detectable functional receptors when expressed alone. On co-expression with RIC3 almost all cells expressing a8 responded giving an average response of 240 pA, and all cells expressing a3 32 responded giving an average response of 99 pA. C. elegans RIC3 has been shown to increase the functional expression of the human 5HT3A receptor by 168 %. Human RIC3 had no effect on human 5HT3A and in fact decreased the functional expression of the murine 5HT3A by 59 %.

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