Abstract
PurposeIt was shown by several experimental studies that some G protein coupled receptors (GPCR) are sensitive to sodium ions. Furthermore, mutagenesis studies or the determination of crystal structures of the adenosine A2A or δ-opioid receptor revealed an allosteric Na+ binding pocket near to the highly conserved Asp2.50. Within a previous study, the influence of NaCl concentration onto the steady-state GTPase activity at the human histamine H3 receptor (hH3R) in presence of the endogenous histamine or the inverse agonist thioperamide was analyzed. The purpose of the present study was to examine and quantify the Na+-sensitivity of hH3R on a molecular level.MethodsTo achieve this, we developed a set of equations, describing constitutive activity and the different ligand-receptor equilibria in absence or presence of sodium ions. Furthermore, in order to gain a better understanding of the ligand- and Na+-binding to hH3R on molecular level, we performed molecular dynamic (MD) simulations.ResultsThe analysis of the previously determined experimental steady-state GTPase data with the set of equations presented within this study, reveals that thioperamide binds into the orthosteric binding pocket of the hH3R in absence or presence of a Na+ in its allosteric binding site. However, the data suggest that thioperamide binds preferentially into the hH3R in absence of a sodium ion in its allosteric site. These experimental results were supported by MD simulations of thioperamide in the binding pocket of the inactive hH3R. Furthermore, the MD simulations revealed two different binding modes for thioperamide in presence or absence of a Na+ in its allosteric site.ConclusionThe mathematical model presented within this study describes the experimental data regarding the Na+-sensitivity of hH3R in an excellent manner. Although the present study is focused onto the Na+-sensitivity of the hH3R, the resulting equations, describing Na+- and ligand-binding to a GPCR, can be used for all other ion-sensitive GPCRs.
Highlights
G protein coupled receptors (GPCRs), one of the largest protein families within the human genome, play an important role in several physiological and pathophysiological processes (Wise et al 2002; Foord et al 2005; Jacoby et al 2006)
The data suggest that thioperamide binds preferentially into the histamine H3 receptor (hH3R) in absence of a sodium ion in its allosteric site
The mathematical model presented within this study describes the experimental data regarding the Na+-sensitivity of hH3R in an excellent manner
Summary
G protein coupled receptors (GPCRs), one of the largest protein families within the human genome, play an important role in several physiological and pathophysiological processes (Wise et al 2002; Foord et al 2005; Jacoby et al 2006). The location of the allosteric sodium binding site was supported by mutagenesis of the highly conserved Asp2.50 into the neutral alanine (Neve et al 1991; Schetz and Sibley 2001) or asparagine (Ceresa and Limbird 1994; Schnell and Seifert 2010) This is supported by experimental results at hH3R, where the Asp2.50Asn mutant was found to partially mimic the effect of high sodium chloride concentrations by suppressing constitutive activity (Schnell and Seifert 2010). With MD studies the binding pathway of a sodium ion into the allosteric sodium binding site of the D2 receptor (Selent et al 2010) and the μ-opioid receptor (Yuan et al 2013) were observed
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