Jansen M , Bali M , Akabas MH .

GM077660 NIGMS NIH HHS , K99-NS059841 NINDS NIH HHS , NS030808 NINDS NIH HHS , R01 NS030808-14 NINDS NIH HHS , K99 NS059841-01 NINDS NIH HHS , K99 NS059841-02 NINDS NIH HHS , R01 NS030808 NINDS NIH HHS , K99 NS059841 NINDS NIH HHS , R01 GM077660 NIGMS NIH HHS

	Figure 1. Constructs used in this study. (A) Schematic depiction of constructs. The N-terminal ligand binding domain is followed by transmembrane segments (black boxes). M1, M2, and M3 are connected by short loops. The cytoplasmic domain is mainly formed by a large loop (gray box) between M3 and M4. The amino acid sequence of the α-helical end of M3, the M3M4 loop (shaded gray) and the α-helical beginning of M4(Unwin, 2005), is shown. Amino acids that were removed/introduced are shaded gray. Arginines mutated in the 5-HT3A-QDA mutant are indicated by asterisks. (B) Homology models of the 5-HT3A wild type (left) and 5-HT3A-glvM3M4 (right) based on nAChR model (Unwin, 2005). Arginines in the 5-HT3A MA helices and in the truncated M3M4 loop of 5-HT3A-glvM3M4 are shown in spacefilling representation. The only part of the intracellular domain that is shown (left) are the MA helices because the rest of this domain is disordered in the nAChR structure. (C) SDS-PAGE/Western blot analysis of total and plasma membrane protein fractions from oocytes. 5-HT3A-V5-wt protein (53 kD) and 5-HT3A-V5-glvM3M4 protein (41 kD) bands are observed.
	Figure 2. The pharmacological characteristics of 5-HT3A-glvM3M4 are comparable to 5-HT3A. (A) Application of increasing 5-HT concentrations elicits inward currents with increasing amplitudes in 5-HT3A-glvM3M4–expressing oocyte. 5-HT concentrations (μM) are above corresponding trace. (B) Concentration–response relationship for 5-HT activation of 5-HT3A-glvM3M4 (closed squares) and 5-HT3A (open squares). Currents were normalized to the maximum response for individual oocytes (n = 8). (C) Coapplication of increasing picrotoxin concentrations with the 5-HT EC30 concentration to 5-HT3A-glvM3M4 inhibits 5-HT–activated currents. Picrotoxin concentrations (μM) are above corresponding trace. (D) Picrotoxin concentration–inhibition relationship for 5-HT3A-glvM3M4 (closed squares) and 5-HT3A (open squares) (n ≥ 3). (E) Diltiazem concentration–inhibition relationship. Experiments performed as in C except with diltiazem in place of picrotoxin (n ≥ 3). (F) GABA concentration–response curves for activation of GABA ρ1-glvM3M4 (closed squares) and GABA ρ1 (open squares) (n ≥ 3). In all panels, data points are mean ± SEM.
	Figure 3. 5-HT3A-glvM3M4 is cation selective with increased single channel conductance. (A) Representative single channel recordings from an outside-out patch containing 5-HT3A-glvM3M4 channels at different holding voltages in the presence of 1 μM 5-HT. The closed state level is indicated by c and dotted line; openings are downward deflections. Records filtered at 1 kHz for display purpose. (B) All-points histograms fitted with two or three Gaussian functions representing closed level and one or two open channels. (C) Mean current and standard deviation obtained from the fit in B plotted as a function of voltage. Chord conductance estimated by linear regression. (D) 100 nM ondansetron inhibits whole cell current induced by 1 μM 5-HT. (E) Change in reversal potential induced by reduction in external NaCl concentration (E2, E3, and E4 contain 145, 72.5, and 36.25 mM NaCl, respectively) for 5-HT3A and (F) for 5-HT3A-glvM3M4.
	Figure 4. Coexpression of hRIC-3 inhibits 5-HT3A but not 5-HT3A-glvM3M4 expression. Average peak currents elicited by a saturating 5-HT concentration recorded in oocytes injected with 10 ng mRNA for 5-HT3A or 5-HT3A-glvM3M4 either alone or together with 5 ng mRNA for hRIC-3.

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