Regulation of heterologously expressed 5‐HT_1B receptors coupling to potassium channels in AtT‐20 cells

Abstract

Background and Purpose 5‐HT1B receptors are widely expressed GPCRs and a target of triptans, the most commonly prescribed anti‐migraine drugs. There is very limited information about the acute, agonist‐induced regulation of 5‐HT1B receptor signalling and so we sought to characterize this in a neuron‐like system. Experimental Approach Epitope‐tagged human 5‐HT1B receptors were expressed in mouse AtT20 cells. 5‐HT1B receptor signalling was assessed using whole‐cell patch‐clamp recordings of endogenous G protein‐gated inwardly rectified potassium (GIRK) channels, and receptor localization measured using immunofluorescence. Key Results 5‐HT (EC50 65 nM) and sumatriptan (EC50 165 nM) activated GIRK channels in AtT20 cells expressing 5‐HT1B receptors. Continuous application of both 5‐HT (EC50 120 nM) and sumatriptan (EC50 280 nM) produced profound desensitization of 5‐HT1B receptor signalling within a few minutes. Complete recovery from desensitization was observed after 10 min. Both 5‐HT and sumatriptan induced significant heterologous desensitization of SRIF (somatostatin)‐activated GIRK currents, with the 5‐HT‐induced heterologous desensitization being blocked by the protein kinase inhibitor staurosporine. Both agonists induced modest 5‐HT1B receptor internalization, with a time course much slower than receptor desensitization. Conclusions and Implications In AtT‐20 cells, 5‐HT1B receptors undergo rapid and reversible desensitization at concentrations of agonist similar to those required to activate the receptor. Desensitization is incomplete, and the continued signalling of the receptor in the presence of the agonist may lead to cellular adaptations. Finally, 5‐HT1B receptor activation causes significant heterologous desensitization, which may lead to a reduced effectiveness of unrelated drugs in vivo.

The Effect of Serotonin Receptor 5-HT1B on Lateral Inhibition between Spiny Projection Neurons in the Mouse Striatum

“…This G-protein-coupled receptor acts via G i α subunits leading to a decrease of cellular cAMP and also activates the MAPK pathway ( Masson et al, 2012 ; Wang et al, 2013 ). In addition, several groups linked the receptor to ionic potassium and calcium channels ( Mizutani et al, 2006 ; Heblinski et al, 2019 ). In the substantia nigra and GP 5-HT1B receptors of SPNs are expressed in presynaptic terminals ( Maroteaux et al, 1992 ; Boschert et al, 1994 ; Sari et al, 1999 ; Jolimay et al, 2000 ; Hoyer et al, 2002 ).…”

Brodifacoum does not modulate human cannabinoid receptor-mediated hyperpolarization of AtT20 cells or inhibition of adenylyl cyclase in HEK 293 cells

“…As previously described (Cawston et al, 2013), prolonged application of cannabinoids in AtT20-CB 1 cells produces a response that wanes over time, reflecting desensitization of receptor signaling. The degree to which this desensitization reflects changes in signaling specific to cannabinoid receptors is tested by application of somatostatin, which activates receptors native to AtT20 cells (Günther, Culler & Schulz, 2016; Heblinski, Bladen & Connor, 2019). In these experiments, CP55940 (100 nM) or THC (10 µM) were applied 2 min after addition of brodifacoum (1 µM), and the fluorescence monitored for 30 min before the addition of somatostatin (100 nM) (Fig.…”

“…Activation of GIRK is mediated by the G βγ subunits of G protein heterotrimers, and many Gi/Go coupled receptors effectively signal through this pathway in AtT20 cells (e.g., Mackie et al, 1995; Günther, Culler & Schulz, 2016; Knapman et al, 2013; Heblinski, Bladen & Connor, 2019). We have previously used the fluorescent measurement of membrane potential to study CB 1 and CB 2 agonists, antagonists, and allosteric modulators of CB 1 (Cawston et al, 2013).…”