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Mathematical analysis of the sodium sensitivity of the human histamine H 3 receptor

View Article: PubMed Central - PubMed

ABSTRACT

Purpose: It 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.

Methods: To 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.

Results: The 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.

Conclusion: The 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.

No MeSH data available.


Sodium binding channel of an aminergic GPCR. A, Na+-binding channel of an aminergic GPCR with highly conserved amino acids. B, Distribution of amino acids being in direct contact to the sodium binding channel of all human aminergic GPCRs.
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Fig9: Sodium binding channel of an aminergic GPCR. A, Na+-binding channel of an aminergic GPCR with highly conserved amino acids. B, Distribution of amino acids being in direct contact to the sodium binding channel of all human aminergic GPCRs.

Mentions: In general, functional data, e.g. obtained within the steady-state GTPase assay are analyzed by determination of the pEC50 value of a ligand. However, pEC50 values represent a complex quantity, consisting of distinct ligand- and/or receptor specific contributions, as shown above. Thus, a comprehensive explanation of pEC50 values on a molecular level with the help of computational methods may be a challenge. In contrast, if functional data will be analyzed using the equations mentioned above, distinct equilibrium constants, which can be related to results of molecular modelling studies, can be obtained. For example, using the present equations, it is possible to determine the binding constant for the sodium ion from the extracellular side, via the orthosteric binding site into its allosteric binding site. It may be suggested that there are no significant differences for the binding constant of the sodium ion from the extracellular side into the orthosteric binding pocket, because this process is mainly driven by an electrostatic attraction of the positively charged sodium ion and negatively charged amino acids in the orthosteric binding pocket, like the highly conserved Asp3.32. Thus, significant differences in the related constants K1 or K2 between sodium sensitive and sodium insensitive GPCRs are not expected. Of course, the binding of the sodium ion from the orthosteric to the allosteric binding site is also suggested to be driven by an electrostatic attraction between the sodium ion and the highly conserved Asp2.50 in the allosteric binding site. However, a comparison of all amino acids, forming the binding channel for the sodium ion from the orthosteric to the allosteric binding site, between the human aminergic GPCRs reveals distinct differences (Figure 9). Thus, it can be suggested that differences in amino acids between the human aminergic GPCRs within this channel may have a large influence onto sodium sensitivity, and consequently may have influence onto the constant K3, which corresponds to the transition of the sodium ion from its orthosteric to its allosteric binding site. Due to the differences in amino acids in direct neighbourhood to the sodium binding channel (Figure 9), it will be interesting to perform similar studies, as presented within this work, at other human aminergic GPCRs and to compare the resulting constants K3. This may give a more detailed insight onto the sodium sensitivity of GPCRs on a molecular level. Furthermore, the constants K6 (here describing the binding of thioperamide to the receptor with a sodium ion being in its allosteric binding pocket) and K9 (here describing the binding of histamine to the receptor with a sodium ion being in its allosteric binding pocket) are suggested to have an influence onto the sodium sensitivity of a GPCR. In general, if a sodium ion is bound in its allosteric binding site it has to be taken into account that this may have an influence onto the orthosteric ligand binding pocket, e.g. amino side chains being located in near neighbourhood to the allosteric and orthosteric binding site may change its conformation in dependence of absence or presence of a sodium ion in its allosteric site. Consequently this may have influence onto the binding properties of a ligand to its binding pocket. This hypothesis is supported by the MD simulations of thioperamide in the inactive hH3R (Figure 6). The results suggest that the binding mode of thioperamide is dependent of the absence or presence of a sodium ion in the allosteric pocket.Figure 9


Mathematical analysis of the sodium sensitivity of the human histamine H 3 receptor
Sodium binding channel of an aminergic GPCR. A, Na+-binding channel of an aminergic GPCR with highly conserved amino acids. B, Distribution of amino acids being in direct contact to the sodium binding channel of all human aminergic GPCRs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4644138&req=5

Fig9: Sodium binding channel of an aminergic GPCR. A, Na+-binding channel of an aminergic GPCR with highly conserved amino acids. B, Distribution of amino acids being in direct contact to the sodium binding channel of all human aminergic GPCRs.
Mentions: In general, functional data, e.g. obtained within the steady-state GTPase assay are analyzed by determination of the pEC50 value of a ligand. However, pEC50 values represent a complex quantity, consisting of distinct ligand- and/or receptor specific contributions, as shown above. Thus, a comprehensive explanation of pEC50 values on a molecular level with the help of computational methods may be a challenge. In contrast, if functional data will be analyzed using the equations mentioned above, distinct equilibrium constants, which can be related to results of molecular modelling studies, can be obtained. For example, using the present equations, it is possible to determine the binding constant for the sodium ion from the extracellular side, via the orthosteric binding site into its allosteric binding site. It may be suggested that there are no significant differences for the binding constant of the sodium ion from the extracellular side into the orthosteric binding pocket, because this process is mainly driven by an electrostatic attraction of the positively charged sodium ion and negatively charged amino acids in the orthosteric binding pocket, like the highly conserved Asp3.32. Thus, significant differences in the related constants K1 or K2 between sodium sensitive and sodium insensitive GPCRs are not expected. Of course, the binding of the sodium ion from the orthosteric to the allosteric binding site is also suggested to be driven by an electrostatic attraction between the sodium ion and the highly conserved Asp2.50 in the allosteric binding site. However, a comparison of all amino acids, forming the binding channel for the sodium ion from the orthosteric to the allosteric binding site, between the human aminergic GPCRs reveals distinct differences (Figure 9). Thus, it can be suggested that differences in amino acids between the human aminergic GPCRs within this channel may have a large influence onto sodium sensitivity, and consequently may have influence onto the constant K3, which corresponds to the transition of the sodium ion from its orthosteric to its allosteric binding site. Due to the differences in amino acids in direct neighbourhood to the sodium binding channel (Figure 9), it will be interesting to perform similar studies, as presented within this work, at other human aminergic GPCRs and to compare the resulting constants K3. This may give a more detailed insight onto the sodium sensitivity of GPCRs on a molecular level. Furthermore, the constants K6 (here describing the binding of thioperamide to the receptor with a sodium ion being in its allosteric binding pocket) and K9 (here describing the binding of histamine to the receptor with a sodium ion being in its allosteric binding pocket) are suggested to have an influence onto the sodium sensitivity of a GPCR. In general, if a sodium ion is bound in its allosteric binding site it has to be taken into account that this may have an influence onto the orthosteric ligand binding pocket, e.g. amino side chains being located in near neighbourhood to the allosteric and orthosteric binding site may change its conformation in dependence of absence or presence of a sodium ion in its allosteric site. Consequently this may have influence onto the binding properties of a ligand to its binding pocket. This hypothesis is supported by the MD simulations of thioperamide in the inactive hH3R (Figure 6). The results suggest that the binding mode of thioperamide is dependent of the absence or presence of a sodium ion in the allosteric pocket.Figure 9

View Article: PubMed Central - PubMed

ABSTRACT

Purpose: It 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.

Methods: To 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.

Results: The 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.

Conclusion: The 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.

No MeSH data available.