| pA2 online © Copyright 2004 The British Pharmacological Society |
073P
University of Bath Summer Meeting July 2004 |
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A determinant of single channel conductance within the large intracellular loop of a nicotinic acetylcholine receptor 1T.G. Hales, 2J.I. Dunlop, 2S.P. Kelley, 1T. Deeb, 2J.J. Lambert & 2J.A. Peters, 1Department of Pharmacology & Physiology, The George Washington University, 2300 Eye Street NW, Washington DC 20037, USA & 2Division of Pathology and Neuroscience, Ninewells Hospital and Medical School, The University of Dundee, Dundee, DD1 9SY, UK |
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Print abstractThree arginine residues, particularly R436, within a putative 
-helix (the HA-stretch) of the large cytoplasmic loop of the human 5-HT3Areceptor influence single channel conductance (
) profoundly (Kelley et al., 2003). Such residues are postulated to frame narrow openings in the cytoplasmic vestibule of the channel within the ion permeation pathway (Miyazawa et al., 1999; Kelley et al., 2003). This study examines whether residues homologous to R436 within the HA-stretch of a neuronal nicotinic ACh receptor similarly affect .
Rat wild-type (WT), or mutant, nicotinic ACh 4 and ß2 subunits were co-expressed from cDNAs introduced into HEK 293 cells by transient transfection. Mutant 4 (F588R) and ß2 (Q443R) subunits were constructed by standard molecular biological techniques. Fluctuation analysis of macroscopic current responses, or single channel events recorded from outside-out membrane patches, in response to nicotine (100 nM - 100 µM) were used to estimate for WT and mutant receptors (Davies et al., 1999; Kelley et al., 2003).
Fluctuation analysis revealed a for WT 4 ß2 receptors of 27.3 ± 1.2 pS (n = 10). A similar value of 30.3 ± 0.8 pS (n = 7) was provided by direct recording of single channel events. The for receptors carrying the double mutation 4(F588R)/ ß2(Q443R) was significantly depressed as assessed both by fluctuation analysis (12.8 ± 0.4 pS; n = 10) and single channel recording (16.1 ± 0.5 pS; n = 7) (p < 0.001 t-test). Receptors assembled from the WT 4 subunit and ß2(Q443R) subunit, or the 4(F588R) and WT ß2 subunit, yielded single channel events with values of 26.8 ± 0.7 pS (n = 4) and 28.3 ± 0.9 pS (n = 4), respectively, only the former of which was significantly lower than the WT receptor (p < 0.05 ANOVA and posthoc Tukey test).
The results indicate that the introduction of 2 arginine residues at a crucial location within the HA-stretch impacts significantly upon the of nicotinic ACh receptors assembled from 4 and ß2 subunits. Cytoplasmic residues may thus prove to be important determinants of across additional members of the Cys-loop family.
Davies, P.A. et al. (1999). Nature, 397, 359-363
Kelley, S.P. et al. (2003). Nature, 424, 321-324
Miyazawa, A. et al. (1999). J. Mol. Biol., 288, 765-786
Supported by a Grant from the Wellcome Trust to J.A.P. and J.J.L