Corradi J et al. (2015), 5-HT3 Receptor Brain-Type B-Subunits are Differ...

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ACS Chem Neurosci 2015 Jul 15;67:1158-64. doi: 10.1021/acschemneuro.5b00080.

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5-HT3 Receptor Brain-Type B-Subunits are Differentially Expressed in Heterologous Systems.

Corradi J , Thompson AJ , McGonigle I , Price KL , Bouzat C , Lummis SC .

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Genes for five different 5-HT3 receptor subunits have been identified. Most of the subunits have multiple isoforms, but two isoforms of the B subunits, brain-type 1 (Br1) and brain-type 2 (Br2) are of particular interest as they appear to be abundantly expressed in human brain, where 5-HT3B subunit RNA consists of approximately 75% 5-HT3Br2, 24% 5-HT3Br1, and <1% 5-HT3B. Here we use two-electrode voltage-clamp, radioligand binding, fluorescence, whole cell, and single channel patch-clamp studies to characterize the roles of 5-HT3Br1 and 5-HT3Br2 subunits on function and pharmacology in heterologously expressed 5-HT3 receptors. The data show that the 5-HT3Br1 transcriptional variant, when coexpressed with 5-HT3A subunits, alters the EC50, nH, and single channel conductance of the 5-HT3 receptor, but has no effect on the potency of competitive antagonists; thus, 5-HT3ABr1 receptors have the same characteristics as 5-HT3AB receptors. There were some differences in the shapes of 5-HT3AB and 5-HT3ABr1 receptor responses, which were likely due to a greater proportion of homomeric 5-HT3A versus heteromeric 5-HT3ABr1 receptors in the latter, as expression of the 5-HT3Br1 compared to the 5-HT3B subunit is less efficient. Conversely, the 5-HT3Br2 subunit does not appear to form functional channels with the 5-HT3A subunit in either oocytes or HEK293 cells, and the role of this subunit is yet to be determined.

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	Figure 1. Alignment of the N-terminal region of 5-HT3B, 5-HT3Br1, and 5-HT3Br2 subunits. 5HT3Br1 subunits differ from 5-HT3B subunits only in the extreme N-terminus, while 5HT3Br2 has ∼100 fewer amino acids and is missing the β1 and β2/loop D regions. Differences in the subunits are due to alternative splicing so the remaining sequences are identical.
	Figure 2. Traces of macroscopic currents from 5-HT3 receptors expressed in oocytes measured using voltage clamp electrophysiology. Typical examples of current traces over a range of 5-HT concentrations for 5-HT3A, 5-HT3AB, 5-HT3ABr1, and 5-HT3ABr2 receptors from the same batch of oocytes are shown. There are distinct differences in the shapes of all the currents.
	Figure 3. Responses from 5-HT3 receptors expressed in HEK293 cells measured using a fluorescent membrane potential sensitive dye. Typical examples of 5-HT-induced responses over a range of 5-HT concentrations for 5-HT3A, 5-HT3AB, 5-HT3ABr1, and 5-HT3ABr2 receptors are shown. The response profiles of 5-HT3A and 5-HT3AB receptors are different, with 5-HT3ABr1 receptor responses similar to 5-HT3AB receptor responses, and 5HT3Br2 receptor responses similar to 5-HT3A receptor responses. F = arbitrary fluorescent units.
	Figure 4. Appearance of 5-HT3AB receptor characteristics differ following transfection with 5-HT3B or 5-HT3Br1 subunits. Cells were transfected with 5-HT3A subunit cDNA (20 ng per well) and various amounts of 5-HT3B or 5-HT3Br1 subunit DNA, and incubated for 2 (A) or 3 (B) days. Higher EC50 and lower nH values (i.e., 5-HT3AB receptor characteristics) were observed in cells incubated for 2 days with 2 and 20 ng of 5-HT3B subunit cDNA, but those transfected with 0.2 ng of cDNA had responses with characteristics consistent with homomeric 5-HT3A receptors. Cells transfected with 200 ng of 5-HT3BR1 subunit cDNA had 5-HT3AB receptor characteristics after 3 days of incubation. Responses with characteristics consistent with homomeric 5-HT3A receptors were observed for cells transfected with 2 or 20 ng of cDNA, and for cells incubated for 2 days (data not shown). Data = mean ± SEM, n = 3–6;*significantly different from 5-HT3A receptor responses.
	Figure 5. Potencies of ligands at different 5-HT3 receptors expressed in HEK293 cells. The Ki values of a range of competitive 5-HT3 receptor ligands were not significantly different for all the different subtypes. Data = mean ± SEM, n = 3–6.
	Figure 6. Single-channel currents of 5-HT3AB and 5-HT3ABr1 receptors expressed in HEK293 cells. Single channels activated by 10 μM 5-HT were recorded from cells transfected with 5-HT3A together with 5-HT3B or 5-HT3Br1 subunits (1:3 A:B or Br1 ratio; total DNA, 4 μg/dish). Recordings were made 3 days after transfection. Channels are shown as upward deflections at different membrane potentials and two different temporal scales for each receptor. Filter: 10 kHz. Representative amplitude histograms at different membrane potentials are shown. At the bottom, representative open- and burst-duration histograms for each receptor at −100 mV are shown.
	Figure 7. Current–voltage (IV) relationships for 5-HT3AB and 5-HT3ABr1 receptors expressed in HEK293 cells. Data corresponds to the mean amplitude (I) ± SD for at least 160 opening events from three different cells, transfected as in Figure 6, for each condition. The mean amplitude was obtained from the corresponding amplitude histogram. The conductance was obtained from the slope of the curve. Data are not significantly different (p > 0.05).
	Figure 8. Macroscopic and single-channel recordings from HEK293 cells cotransfected with 5-HT3A or in combination with 5-HT3B, 5-HT3Br1, or 5-HT3Br2 subunits. Representative traces of macroscopic (top of each panel) and single-channel currents (bottom of each panel) from cells transfected with only 5-HT3A or together with 5-HT3Br2 or 5-HT3B subunits are shown (subunit ratio 1:3 for A:B and A:Br1, and 1:9 for A:Br2, total DNA was 4 μg/dish). Macroscopic currents were recorded in the whole cell configuration at a holding potential of −50 mV and were elicited by a pulse of 100 μM 5-HT (gray bar). Single-channel currents were recorded from cell-attached patches at −100 mV in the presence of 10 μM 5-HT. Channel openings are shown as upward deflections.

References [+] :

Andersen, Stoichiometry for activation of neuronal α7 nicotinic receptors. 2013, Pubmed