Elsevier

European Journal of Pharmacology

Volume 815, 15 November 2017, Pages 233-240
European Journal of Pharmacology

Neuropharmacology and analgesia
N-(2-methoxyphenyl) benzenesulfonamide, a novel regulator of neuronal G protein-gated inward rectifier K+ channels

https://doi.org/10.1016/j.ejphar.2017.09.020Get rights and content

Abstract

G protein-gated inward rectifier K+ (GIRK) channels are members of the super-family of proteins known as inward rectifier K+ (Kir) channels and are expressed throughout the peripheral and central nervous systems. Neuronal GIRK channels are the downstream targets of a number of neuromodulators including opioids, somatostatin, dopamine and cannabinoids. Previous studies have demonstrated that the ATP-sensitive K+ channel, another member of the Kir channel family, is regulated by sulfonamide drugs. Therefore, to determine if sulfonamides also modulate GIRK channels, we screened a library of arylsulfonamide compounds using a GIRK channel fluorescent assay that utilized pituitary AtT20 cells expressing GIRK channels along with the somatostatin type-2 and -5 receptors. Enhancement of the GIRK channel fluorescent signal by one compound, N-(2-methoxyphenyl) benzenesulfonamide (MPBS), was dependent on the activation of the channel by somatostatin. In whole-cell patch clamp experiments, application of MPBS both shifted the somatostatin concentration-response curve (EC50 = 3.5 nM [control] vs.1.0 nM [MPBS]) for GIRK channel activation and increased the maximum GIRK current measured with 100 nM somatostatin. However, GIRK channel activation was not observed when MPBS was applied to the cells in the absence of somatostatin. While the MPBS structural analog 4-fluoro-N-(2-methoxyphenyl) benzenesulfonamide also augmented the somatostatin-induced GIRK fluorescent signal, no increase in the signal was observed with the sulfonamides tolbutamide, sulfapyridine and celecoxib. In conclusion, MPBS represents a novel prototypic GPCR-dependent regulator of neuronal GIRK channels.

Introduction

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue (Hibino et al., 2010, Lusscher and Slesinger, 2010). GIRK channels are members of the super-family of proteins known as inward rectifier K+ (Kir) channels (Hibino et al., 2010, Lusscher and Slesinger, 2010) that function to stabilize the cell resting membrane potential near the K+ equilibrium potential. Four GIRK channel subunits are expressed in mammals: GIRK1 (Kir3.1), GIRK2 (Kir3.2), GIRK3 (Kir3.3) and GIRK4 (Kir3.4) (Hibino et al., 2010, Lusscher and Slesinger, 2010, Luján et al., 2014). The GIRK1, GIRK2 and GIRK3 subunits are expressed in a number of regions of the brain including the hippocampus, cerebellum, substantia nigra, locus coeruleus and ventral tegmental area (Hibino et al., 2010, Lusscher and Slesinger, 2010, Luján et al., 2014). In addition, GIRK1 and GIRK2 subunits are expressed in the superficial layers of the spinal dorsal horn and are enriched in the postsynaptic membranes of substantia gelatinosa neurons (Lusscher and Slesinger, 2010).

In the CNS a large number of neuromodulators including opioids, somatostatin, dopamine and cannabinoids activate GIRK channels (Hibino et al., 2010, Lusscher and Slesinger, 2010). Binding of these agents to their cognate G protein-coupled receptors (GPCRs) causes the dissociation of the βγ subunits of pertussis toxin-sensitive G proteins (Gβγ) which subsequently bind to and activate the GIRK channel (Logothetis et al., 1987, Kurachi et al., 1989). This activation by Gβγ is dependent on the interaction of phosphatidylinositol 4,5-bisphosphate (PIP2) with the GIRK channel (Huang et al., 1998, Zhang et al., 1999a); most likely occurring at the interface of the transmembrane and cytoplasmic domains of the channel (Whorton and MacKinnon, 2011). Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters.

Sulfonamides represent a large and diverse group of chemicals used as antibiotic (sulfapyridine) anti-inflammatory (celexcoxib) and diuretic (furosemide) agents. In addition, binding of sulfonamide drugs such as tolbutamide and diazoxide to the sulfonylurea receptor (SUR) in target cells leads to an inhibition or activation of ATP-sensitive K+ (KATP) channels (Miller et al., 1999, Whiteaker et al., 2007); members of the Kir channel family (Kir6.x). In this study a small library of arylsulfonamide compounds was screened using a GIRK channel fluorescent assay that utilized pituitary AtT20 cells (Walsh, 2011, Günther et al., 2016). Enhancement of the GIRK channel fluorescent signal by one compound, N-(2-methoxyphenyl) benzenesulfonamide (MPBS) was dependent on the activation of the channel by somatostatin. In whole-cell patch clamp experiments of the AtT20 cells, MPBS caused both a leftward shift in the somatostatin concentration-response curve for channel activation and increased the maximum GIRK current. Our results suggest that MPBS represents a novel GPCR-dependent regulator of neuronal GIRK channels.

Section snippets

AtT20 cell culture and plating

The AtT20 pituitary cell line was obtained from ATCC (AtT-20/D16y-F2, CRL-1795) and grown in DMEM media with 10% horse serum + Pen-Strep. Cells were plated on poly-l-lysine-coated glass coverslips (5000 cells per coverslip) (patch clamp recording) and in black 96-well plates (Corning) (30,000 cells per well) (fluorescent measurements). AtT20 cells stably transfected with the cannabinoid type 1 (CB1) receptor were a gift from Dr. Ken Mackie (Indiana University, Bloomington, IN) (Mackie et al.,

Regulation of GIRK channels by MPBS

Fig. 1 displays the MP-sensitive fluorescent dye signal measured over time in cultured AtT20 cells using a microplate reader. The AtT20 cells were chosen in these experiments for two reasons. First, the AtT20 cell line endogenously expresses the somatostatin type 2 and 5 receptors (SSTR2 & SSTR5), voltage-gated Ca2+ channels and GIRK1/GIRK2 channels (Mackie et al., 1995, Kuzhikandathil et al., 1998, Theodoropoulou and Stalla, 2013). Second, expression of GIRK/GIRK2 channels in heterologous

Discussion

The goal of this study was to determine if sulfonamide compounds modulate neuronal GIRK channels. For this purpose a small library of arylsulfonamide compounds was screened using a GIRK channel fluorescent assay that utilized AtT20 cells. Pretreatment of the cells with the compound MPBS enhanced the somatostatin-activated GIRK channel fluorescent signal. The potency of MPBS in the assay was greater during stimulation with a low concentration of somatostatin (5 nM) versus a maximal concentration

Acknowledgements

The authors thank Ms. Aisha Shabaan and Maribel Vazquez for their excellent technical assistance. This work was supported by US Public Health Service award NS-071530 (K.B.W.) and a Magellan fellowship from the University of South Carolina (D.W.G.) 18080-17-43697.

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