Tauber M and Ben Chaim Y (2022), The activity of the serotonergic 5-HT1A recepto...

XB-ART-58884

J Biol Chem 2022 Jun 01;2986:101978. doi: 10.1016/j.jbc.2022.101978.

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The activity of the serotonergic 5-HT1A receptor is modulated by voltage and sodium levels.

Tauber M , Ben Chaim Y .

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G protein-coupled receptors are known to play a key role in many cellular signal transduction processes, including those mediating serotonergic signaling in the nervous system. Several factors have been shown to regulate the activity of these receptors, including membrane potential and the concentration of sodium ions. Whether voltage and sodium regulate the activity of serotonergic receptors is unknown. Here, we used Xenopus oocytes as an expression system to examine the effects of voltage and of sodium ions on the potency of one subtype of serotonin (5-hydroxytryptamine [5-HT]) receptor, the 5-HT1A receptor. We found that the potency of 5-HT in activating the receptor is voltage dependent and that it is higher at resting potential than under depolarized conditions. Furthermore, we found that removal of extracellular Na+ resulted in a decrease of 5-HT potency toward the 5-HT1A receptor and that a conserved aspartate in transmembrane domain 2 is crucial for this effect. Our results suggest that this allosteric effect of Na+ does not underlie the voltage dependence of this receptor. We propose that the characterization of modulatory factors that regulate this receptor may contribute to our future understanding of various physiological functions mediated by serotonergic transmission.

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Species referenced: Xenopus laevis
Genes referenced: kcnj3
GO keywords: G-protein coupled receptor binding [+]

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	Figure 1. Voltage dependence of the 5-HT1A receptor. A and B, measurement of the relationship between 5-HT concentration and 5-HT1A receptor–activated GIRK currents at −80 mV and +40 mV, respectively. Basal GIRK current evolved following replacement of the solution to a high K+ solution. Then, three different 5-HT concentrations were applied (1, 10, and 5000 nM, numbered 1–3), and the response for each concentration was measured. C, dose–response curves for −80 mV (black) and +40 mV (red). The responses were normalized to the response evoked by 5000 nM 5-HT at each holding potential. Each point represents the mean (±SEM) from 11 to 50 oocytes. The solid black and red lines were generated by fitting equation 1 to the data (see the Experimental procedures section). The EC50 values obtained for the two graphs were significantly different (p < 0.0001). 5-HT, 5-hydroxytryptamine; G protein–activated inward-rectifying K+.
	Figure 2. Voltage dependence of the activation of the 5-HT1A receptor by buspirone and tandospirone. A, dose–response curves for the activation of the 5-HT1A receptor by buspirone at −80 mV (black) and +40 mV (red). Each point here and in B represents the mean (±SEM) from 12 to 32 oocytes. The solid black and red lines were generated by fitting Equation 1 to the data (see the Experimental procedures section). The EC50 values obtained for the two graphs (301.9 nM at −80 mV and 313.7 at +40 mV) were significantly different (p < 0.0001). B, dose–response curves of tandospirone activated 5-HT1A receptor. The EC50 values obtained for the two graphs (194 nM at −80 mV and 934.1 nM at +40 mV) were significantly different (p < 0.0001). 5-HT, 5-hydroxytryptamine.
	Figure 3. Allosteric effect of sodium ions on the 5-HT1A receptor. A, the maximal amplitude of I5-HT, evoked by 5 μM 5-HT in 72 mM Na+ solution (black) and in Na+-free solution (red). Each data point represents one oocyte, and the mean (±SD) is shown as a horizontal line. The data at the two conditions are not significantly different (unpaired t test, p = 0.79). B, basal IK from experiments in 72 mM Na+ solution (black) described for Figure 1 and in Na+-free solution (red). Each data point represents one oocyte, and the mean (±SD) is shown as horizontal lines. The data at the two conditions are not significantly different (unpaired t test, p = 0.76). C and D, DR curves assembled from various experiments conducted on wt 5-HT1A receptor (C) or Asp92Asn mutant (D) at 72 mM Na+ solution (black circles) and in Na+-free solution (red circles). Each point represents the mean (±SEM) from 8 to 31 oocytes. The solid black and red lines were generated as described previously for Figure 1. The EC50 values obtained for the two graphs at C are significantly different (p = 0.016). The EC50 values obtained for the two graphs at D are not significantly different (p = 0.15). 5-HT, 5-hydroxytryptamine; DR, dose response; I5-HT, 5-HT-induced K+ current.
	Figure 4. Effect of sodium ions on the voltage dependence of the 5-HT1A receptor. A, dose–response curves assembled from various experiments conducted on wt 5-HT1A receptor in Na+-free solution at −80 mV (black circles; taken from Fig. 2B) and at +40 mV (red circles). Each point represents the mean (±SEM) from 7 to 21 oocytes. The solid black and red lines here and in B were generated as described for Figure 1. The dashed lines are the fitting curves of the data obtained in 72 mM Na+ (taken from Fig. 1). The EC50 values obtained for the two graphs (19.4 nM at −80 mV, taken from Figure 3A, and 170.4 nM at +40 mV) are significantly different (p = 0.0018). B, dose–response curves of Asp82Asn mutant receptor in 72 mM Na+ solution at −80 mV (black circles; taken from Fig. 3C) and at +40 mV (red circles). Each point represents the mean (±SEM) from 8 to 31 oocytes. The EC50 values obtained for the two graphs (126 nM at −80 mV and 595.1 nM at +40 mV) are significantly different (p = 0.0018). 5-HT, 5-hydroxytryptamine.
	Figure S1. Effect of 5-HT on GIRK channels. A. A representative recording from an oocyte expressing the GIRK channel. Application of 5 µM 5-HT did not evoke GIRK currents or inhibited basal GIRK currents. B. I-V curve of IK before (black) and after (red) the application of 5-HT. The oocytes were voltage clamped to -100 mV and the currents after depolarizing pulses to various holding potentials from –100 mV to +40 mV at 10 mV increments were measured. Data is mean ± SD from 7 oocytes. The difference between the two conditions is not significant (paired t-test, p>0.2 for all voltages).
	Figure S2. DR curves obtained from oocytes where recordings were conducted from the same oocyte at the two holding potentials (solid symbols and lines). Each point represents the mean (±SEM) from 7-29 oocytes. The EC50 values obtained for the two graphs (2.9 nM at -80 mV and 83.4 nM at +40 mV) were significantly different (p<0.0001). The DR curves constructed from data from all oocytes are shown for comparison (empty symbols and dashed lines; taken from Fig. 1C).
	Figure S3. Buspirone and tandospirone are partial 5-HT1A agonists. Each point represents the ratio between maximal GIRK current evoked by buspirone (A) or tandospirone (B) and the maximal current evoked by 5-HT in the same oocyte. The mean ± SE is shown as horizontal line. The efficacy of both agonists was not affected by the membrane potential (p=0.97 and 0.28 for A and B, respectively).

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Akimova, The serotonin-1A receptor in anxiety disorders. 2009, Pubmed