McKinnon NK , Bali M , Akabas MH .

NS030808 NINDS NIH HHS , R01 NS030808 NINDS NIH HHS , T32 GM007288 NIGMS NIH HHS

	Figure 1. Construct design and initial characterization of 5-HT3A M3M4 loop truncation constructs.(A) Schematic depiction of 5-HT3A truncation constructs. The N-terminal ligand binding domain is connected to the 4 transmembrane segments (open rectangles). M1, M2 and M3 are connected by short loops. The M3M4 cytoplasmic domain including the MA α-helix have been removed and replaced with a short loop (red) corresponding to inserted amino acid linkers. LVHK and VLDR represent the terminal and initial amino acids of the M3 and M4 transmembrane domains, respectively. (B) View of the M3 transmembrane segment thru the M4 segment (including the cytoplasmic M3M4 loop) of a single subunit of the 2.9 à high resolution crystal structure Gloeobacter violaceus GLIC protein (PDB file: 3EAM). The SQPARAA residues that were predicted to form the M3M4 loop are highlighted (green) and the side chains are shown in stick format. Note that the final three residues are part of the M4 α helix on the left. (C) Average peak current recorded from oocyctes expressing 5-HT3A-WT and 5-HT3A-M3M4 loop truncations at a saturating 5-HT (10 µM) concentration. Bars represent the mean ± SEM. The shortened cytoplasmic loops are depicted by the amino acid peptides listed. * (p<0.05) and ** (p<0.0001) indicates peak current significantly different from WT by one way ANOVA using Dunnett's multiple comparison post hoc text. (D) SDS-PAGE/Western blot analysis of total and plasma membrane protein fractions from oocyctes expressing(GSA)n = 1–3 insertion constructs. 5-HT3A-V5-wild type protein (53 kDa) and 5-HT3A-V5-(GSA)n = 1–3 (41 kDa) bands are observed.
	Figure 2. 5-HT concentration-response relationships of the 5-HT3A-An = 1–7 constructs are comparable to WT.(A) Peak current recorded from oocytes expressing 5-HT3A wild type and 5-HT3A-A n = 1–7 loop constructs at a saturating 5-HT (10 µM) concentration. The shortened cytoplasmic loops are depicted by the number of alanines [A] present between the M3 and M4 transmembrane domains. (B) Concentration-response relationship for 5-HT activation of the 5-HT3A-An = 1–7 constructs (A7 □, A6 ▿, A5 ⧫, A4 ⋄, A3 ▴, A2 ○ and WT <$>\raster="rg1"<$>). Currents were normalized to the maximum response for individual oocytes (n = 3–6). Mean ± SEM shown. ** indicates peak current significantly different from WT by one way ANOVA using Dunnett's multiple comparison post hoc test (p<0.0001).
	Figure 3. 5-HT3A-An = 1,3,5 receptors desensitize more rapidly that 5-HT3A-An = 2,4,6,7.Representative traces for oocytes expressing 5-HT3A- (A) WT (B) A7, A6, A4, A2 (C) and A5, A3, A1 receptors during prolonged exposure to 10 µM 5-HT. (A) The desensitizing component of the current from an oocyte expressing WT 5-HT3A receptors is fit by a single exponential decay function. The dots represent the fitted line which overlays the current trace. (A). Note the difference in the time scale between panels (B) and (C). The A5, A3, and A1 receptors desensitized an order of magnitude faster than A7, A6, A4 and A2 receptors which were similar to WT.
	Figure 4. Characterization of 5-HT3A-QDA and 5-HT3A-An receptors in transfected HEK 293 cells using patch clamp recording.(A) Current/voltage relationship of 5-HT3A-QDA receptors. Representative traces from outside-out patch held at various negative voltages used for the construction of the I/V relationship in panel B. (B) Mean current amplitudes, as determined from all points histograms fitted with a sum of Gaussian functions, are plotted as a function of holding voltage. The line is the linear regression fit to the data points and its slope gives the slope conductance of the channel, 67±7 pS (n = 5). (C, E, G) Single channel recordings from a cell-attached patches containing 5-HT3A-QDA (C) channels, 5-HT3A-A5 (E) and 5-HT3A-A1 (G) at −60 mV in the presence of 50 µM 5-HT. (C) The mean open time for the main conductance is 103±7 ms (n = 5). Note that occasional subconductance levels are visible but rare. Channel openings are represented as downward deflections. Records filtered at 1 kHz for display purposes. (E) The recordings from 5-HT3A-A5 showed a larger conductance with shorter openings, mean open time = 8±2 ms (n = 4), and a smaller sub-conducting state with a mean open time of 48±5 ms. (D, F, H) All-points histograms of the recording on the left were fitted with three Gaussian functions representing the closed and open channel levels and are shown for 5-HT3A-QDA (D) channels, 5-HT3A-A5 (F) and 5-HT3A-A1 (H).
	Figure 5. Characterization of the 5-HT3A-A1 insertion construct.(A) Currents elicited by repeated applications of 10 µM 5-HT are significantly smaller than the current following the initial 5-HT application in the 5-HT3A-A1 receptor. The wash time denoted by the//was 6 min. Insert shows an expanded time scale of the indicated region. The second current response amplitude was 11.6% of the initial response. (B) Radioactive 5-HT binding to oocytes expressing 5-HT3A-WT and 5-HT3A-An insertion constructs was determined by subtracting 5-HT binding to water-injected oocytes from 5-HT binding to channel expressing oocytes. Each bar represents the mean 5-HT binding ± SEM for at least 10 channel expressing oocytes from at least three separate oocyte isolations. ** indicates radioactive 5-HT binding significantly different from WT by one way ANOVA using Dunnett's multiple comparison post hoc test (p<0.0001).
	Figure 6. Sequence alignment GluClcrys and 5-HT3A and cartoon illustrating transmembrane region of GluClcrys.(A) Sequence alignment of the C-terminal region of mouse 5-HT3A receptor and GluClcrys. The GluClcrys M3 and M4 α helical membrane-spanning segments are indicated by the gray rectangles. The M3M4 loop region replaced by two alanines in the 5-HT3A-A2 construct is shown underlined in bold. The 5-HT3A-A2 construct yielded functional receptors with channel kinetics similar to wild type 5-HT3A receptors. Note that the 5-HT3A-A2 construct lacks 16 residues aligned with the cytoplasmic end of the GluCl M4 segment. (B) Cartoon representation of the transmembrane domain of one subunit of GluCl (PDB: 3RHW). The cyan region at the cytoplasmic end of the M4 segment indicates the residues that were replaced by two alanine residues in the5-HT3A-A2 construct.

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