Inhalational Anesthetic-binding Proteins in Rat Neuronal Membranes

Molecular targets of inhaled anesthetics must be represented in the group that specifically bind these drugs, but the paucity of direct binding data has limited the number of candidates for further evaluation. To find candidate targets, we used a combination of photolabeling, two-dimensional gel electrophoresis, and mass spectrometry to identify halothane-binding targets in rat neuronal membranes. Of the 265 spots detected on the two-dimensional gels, 90 were labeled by 14Chalothane, and 34 were identified. Mitochondrial proteins, especially respiratory complex and voltage-dependent anion channels, dominated the labeled group, and there were several examples of subunit- and state-dependent binding. A significant correlation was found between internal protein cavities and binding in a group of proteins with high resolution structures. Therefore, in addition to identifying novel neuronal targets, these data suggest a general molecular feature, the buried cavity, as a dominant attribute of volatile anesthetic-binding sites found in a limited number of neuronal membrane proteins. Molecular targets of inhaled anesthetics must be represented in the group that specifically bind these drugs, but the paucity of direct binding data has limited the number of candidates for further evaluation. To find candidate targets, we used a combination of photolabeling, two-dimensional gel electrophoresis, and mass spectrometry to identify halothane-binding targets in rat neuronal membranes. Of the 265 spots detected on the two-dimensional gels, 90 were labeled by 14Chalothane, and 34 were identified. Mitochondrial proteins, especially respiratory complex and voltage-dependent anion channels, dominated the labeled group, and there were several examples of subunit- and state-dependent binding. A significant correlation was found between internal protein cavities and binding in a group of proteins with high resolution structures. Therefore, in addition to identifying novel neuronal targets, these data suggest a general molecular feature, the buried cavity, as a dominant attribute of volatile anesthetic-binding sites found in a limited number of neuronal membrane proteins. Molecular targets of inhaled anesthetics remain ambiguous, despite electrophysiological evidence for potentially relevant effects at ion channels (1Campagna J. Miller K. Forman S. N. Engl. J. Med. 2003; 348: 2110-2124Crossref PubMed Scopus (620) Google Scholar, 2Franks N.P. Lieb W.R. Nature. 1994; 367: 607-614Crossref PubMed Scopus (1625) Google Scholar, 3Mihic S.J. Ye Q. Wick M.J. Koltchine V.V. Krasowski M.D. Finn S.E. Mascia M.P. Valenzuela C.F. Hanson K.K. Greenblatt E.P. Harris R.A. Harrison N.L. Nature. 1997; 389: 385-389Crossref PubMed Scopus (1096) Google Scholar). Unfortunately, few direct binding data exist for these small hydrophobic of the with volatile Molecular of Scholar). that direct binding of anesthetics to sites in the protein the in ion of with has of these PubMed Scopus Google and has of and binding sites in a of membrane S. PubMed Scopus Google and J. PubMed Scopus Google Scholar, PubMed Scopus Google protein the of these to the we binding J. Google and targets K. J. PubMed Scopus Google for the with at these the for of these targets significant at of to the Therefore, inhaled anesthetics a of binding and to further of the binding novel targets for further and for of we a combination of photolabeling, two-dimensional gel electrophoresis, and spectrometry used voltage-dependent anion protein data used voltage-dependent anion protein data to identify a group of membrane proteins that specifically by there labeled targets, we found a of a number of proteins, and evidence for protein was was were and a was was used for and was used for two-dimensional gel and was and were and were were as by the of and and were in with of were used in was with and the was the of the with was in and at was in and and at for was and at for was and in and was at for and the was and at was as PubMed Scopus Google Scholar). the were in with 14Chalothane, of were used of limited and in at were to at a of for with membrane was and with membrane was with of and were a protein PubMed Scopus Google Scholar). of protein was for the on were with for at to and the was to and was were at used for the were in a and for and in and for were a gel for further in the were with at and with for protein were on at for membrane and were and with to the and of as as the molecular and of data were and and were with and for and on the the labeled and spots were spots were as detected by the on the but detected on the spots were a PubMed Scopus Google and mass and of mass of was to the and was used as the data were the were with the of spots and of gel spots were K. 2003; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). on and were by the of in the of to that in the membrane of protein a of binding. by the molecular in a binding these to labeled proteins of binding and the molecular we were to of binding as the S.E. data were by of by the for data were with A correlation between data was with and the correlation to the of was was and membrane proteins were by two-dimensional electrophoresis, and the spots were with resolution with gel but significant in was of the proteins. detected 265 spots on the but 90 spots be detected on the of of proteins and of the proteins by of to gel of rat membrane proteins. rat membrane proteins were on a by membrane with the spots of the with spots and in spots were for but be with of these spots to be that labeled spots be on rat protein two-dimensional K. 2003; PubMed Scopus Google Scholar, PubMed Scopus Google in a of 34 proteins the proteins in with the of a a protein spots were as of the protein complex the protein at in of the spots were membrane protein was and of the labeled proteins and of the proteins were in of the respiratory proteins were labeled by with of and the protein of there was significant to the of these proteins. of complex and the of complex was the protein was of complex was the and the were of by the of the with of addition to there were several examples of in a was in in the at were in the voltage-dependent anion and in the as a of to of the To to binding proteins with binding were labeled in and and used for has a halothane-binding of and a of J. PubMed Scopus Google Scholar, K. PubMed Scopus Google Scholar, S. N.P. J. PubMed Scopus Google Scholar). but the and on the and the of protein a of that of the labeled proteins at as of binding that of of used in between and the of in proteins by limited and a limited of must be as of the of the binding of as a of the to the membrane by the molecular the S.E. of data proteins to the complex in between these spots was by of by by of proteins to of the of proteins the in the for the a data correlation of with we 1997; Google and evidence for internal cavities as sites in proteins, we J. S. PubMed Scopus Google Scholar, J. S. PubMed Scopus Google to and and data and these to the data in protein was by the number of cavities as a with with molecular of between and molecular of volatile anesthetics between and and with as of the in and that there a significant between and the number of cavities of and to cavities the that despite the a for binding buried in the to these cavities J. K. S. PubMed Scopus Google and the binding to of of the in and data for 34 proteins and in at with and were found to that the a of a for combination of photolabeling, two-dimensional gel electrophoresis, and in of a of membrane proteins that the inhaled Of the 265 spots detected on the 90 were labeled to be on the Of these 90 evidence of of with with by limited of that of cavities to bind K. J. PubMed Scopus Google Scholar). in of the that membrane and proteins and cavities the small that of the to S.J. 1994; PubMed Scopus Google Scholar). further by the in the of respiratory and and of the small inhaled a proteins with high resolution evidence that molecular the of internal protein cavities of binding to with the used in in that a in and of the protein by the binding of a in molecular in the and the in the of two-dimensional the found at the molecular at a of and a but there in the of but significant in between of the and examples direct evidence for state-dependent binding of inhaled to proteins, a but for a molecular to be of a for as of the and evidence for in R.A. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). we that a and for N. PubMed Scopus Google Scholar). as that the for significant in were that with and that inhaled PubMed Scopus Google Scholar). the for of and with in that these be the we found that at of the respiratory and in a subunit- and state-dependent identify complex in gels, that of the respiratory a halothane-binding in the binding to respiratory complex proteins has a by in was to anesthetics PubMed Scopus Google Scholar). complex in was by several inhaled J. J. J. PubMed Scopus Google Scholar). in complex in 1994; PubMed Scopus Google and in PubMed Scopus Google that effects to and binding to proteins be with effects of as in and in the in of were as to R.A. J. PubMed Scopus Google these effects were a of inhaled in that effects to be PubMed Scopus Google with channels and proteins. the binding targets to these voltage-dependent anion a and of and protein to by a to J. PubMed Scopus Google and evidence that with high W.R. S.J. J. 2003; PubMed Scopus Google Scholar). with the S. PubMed Scopus Google Scholar, N. K. 1997; PubMed Scopus Google in the binding the with in a used to inhaled anesthetics and the for inhaled anesthetics PubMed Scopus Google Scholar). and inhaled anesthetics to with at A the between the complex and with the binding data and suggest a for in for inhaled there was significant in on for in with W.R. S.J. J. PubMed Scopus Google Scholar). there with the these data suggest that the proteins in protein were labeled by in PubMed Scopus Google Scholar). protein and protein to a of inhaled anesthetics the of proteins with the for in the of as proteins of a PubMed Scopus Google of that the proteins, and of and binding of the for of N. J. 1994; PubMed Scopus Google Scholar, K. C.F. J. 2003; PubMed Google a that has there to the of proteins for with the that proteins a significant to the and of these Mitochondrial proteins the for in be of two-dimensional gel PubMed Scopus Google of rat proteins represented of identified. A of the labeled proteins in in but be of of the to membrane proteins we the that proteins, ion channels, binding targets for of these proteins that as and and that proteins labeled by in the of potentially molecular targets for the inhaled direct photolabeling, two-dimensional gel electrophoresis, and mass we find that specifically of rat membrane proteins and that of these proteins the respiratory and the voltage-dependent anion and binding targets for binding with the number of internal protein cavities of we evidence for subunit- and state-dependent binding to the that of these targets to the of Molecular targets of inhaled anesthetics remain ambiguous, despite electrophysiological evidence for potentially relevant effects at ion channels (1Campagna J. Miller K. Forman S. N. Engl. J. Med. 2003; 348: 2110-2124Crossref PubMed Scopus (620) Google Scholar, 2Franks N.P. Lieb W.R. Nature. 1994; 367: 607-614Crossref PubMed Scopus (1625) Google Scholar, 3Mihic S.J. Ye Q. Wick M.J. Koltchine V.V. Krasowski M.D. Finn S.E. Mascia M.P. Valenzuela C.F. Hanson K.K. Greenblatt E.P. Harris R.A. Harrison N.L. Nature. 1997; 389: 385-389Crossref PubMed Scopus (1096) Google Scholar). Unfortunately, few direct binding data exist for these small hydrophobic of the with volatile Molecular of Scholar). that direct binding of anesthetics to sites in the protein the in ion of with has of these PubMed Scopus Google and has of and binding sites in a of membrane S. PubMed Scopus Google and J. PubMed Scopus Google Scholar, PubMed Scopus Google protein the of these to the we binding J. Google and targets K. J. PubMed Scopus Google for the with at these the for of these targets significant at of to the Therefore, inhaled anesthetics a of binding and to further of the binding novel targets for further and for of we a combination of photolabeling, two-dimensional gel electrophoresis, and spectrometry used voltage-dependent anion protein data used voltage-dependent anion protein data to identify a group of membrane proteins that specifically by there labeled targets, we found a of a number of proteins, and evidence for protein was was were and a was was used for and was used for two-dimensional gel and was and were and were were as by the of and and were in with of were used in was with and the was the of the with was in and at was in and and at for was and at for was and in and was at for and the was and at was as PubMed Scopus Google Scholar). the were in with 14Chalothane, of were used of limited and in at were to at a of for with membrane was and with membrane was with of and were a protein PubMed Scopus Google Scholar). of protein was for the on were with for at to and the was to and was were at used for the were in a and for and in and for were a gel for further in the were with at and with for protein were on at for membrane and were and with to the and of as as the molecular and of data were and and were with and for and on the the labeled and spots were spots were as detected by the on the but detected on the spots were a PubMed Scopus Google and mass and of mass of was to the and was used as the data were the were with the of spots and of gel spots were K. 2003; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). on and were by the of in the of to that in the membrane of protein a of binding. by the molecular in a binding these to labeled proteins of binding and the molecular we were to of binding as the S.E. data were by of by the for data were with A correlation between data was with and the correlation to the of was was was was were and a was was used for and was used for two-dimensional gel and was and were and were were as by the of and and were in with of were used in was with and the was the of the with was in and at was in and and at for was and at for was and in and was at for and the was and at was as PubMed Scopus Google Scholar). the were in with 14Chalothane, of were used of limited and in at were to at a of for with membrane was and with membrane was with of and were a protein PubMed Scopus Google Scholar). of protein was for the on were with for at to and the was to and was were at used for the were in a and for and in and for were a gel for further in the were with at and with for protein were on at for membrane and were and with to the and of as as the molecular and of data were and and were with and for and on the the labeled and spots were spots were as detected by the on the but detected on the spots were a PubMed Scopus Google and mass and of mass of was to the and was used as the data were the were with the of spots and of gel spots were K. 2003; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). on and were by the of in the of to that in the membrane of protein a of binding. by the molecular in a binding these to labeled proteins of binding and the molecular we were to of binding as the S.E. data were by of by the for data were with A correlation between data was with and the correlation to the of was was and membrane proteins were by two-dimensional electrophoresis, and the spots were with resolution with gel but significant in was of the proteins. detected 265 spots on the but 90 spots be detected on the of of proteins and of the proteins by of to spots were for but be with of these spots to be that labeled spots be on rat protein two-dimensional K. 2003; PubMed Scopus Google Scholar, PubMed Scopus Google in a of 34 proteins the proteins in with the of a a protein spots were as of the protein complex the protein at in of the spots were membrane protein was and of the labeled proteins and of the proteins were in of the respiratory proteins were labeled by with of and the protein of there was significant to the of these proteins. of complex and the of complex was the protein was of complex was the and the were of by the of the with of addition to there were several examples of in a was in in the at were in the voltage-dependent anion and in the as a of to of the To to binding proteins with binding were labeled in and and used for has a halothane-binding of and a of J. PubMed Scopus Google Scholar, K. PubMed Scopus Google Scholar, S. N.P. J. PubMed Scopus Google Scholar). but the and on the and the of protein a of that of the labeled proteins at as of binding that of of used in between and the of in proteins by limited and a limited of must be as of the of the binding of as a of the to the membrane by the molecular the S.E. of data proteins to the complex in between these spots was by of by by of proteins to of the of proteins the in the for the a data correlation of with we 1997; Google and evidence for internal cavities as sites in proteins, we J. S. PubMed Scopus Google Scholar, J. S. PubMed Scopus Google to and and data and these to the data in protein was by the number of cavities as a with with molecular of between and molecular of volatile anesthetics between and and with as of the in and that there a significant between and the number of cavities of and to cavities the that despite the a for binding buried in the to these cavities J. K. S. PubMed Scopus Google and the binding to of of the in and data for 34 proteins and in at with and were found to that the a of a for and membrane proteins were by two-dimensional electrophoresis, and the spots were with resolution with gel but significant in was of the proteins. detected 265 spots on the but 90 spots be detected on the of of proteins and of the proteins by of to spots were for but be with of these spots to be that labeled spots be on rat protein two-dimensional K. 2003; PubMed Scopus Google Scholar, PubMed Scopus Google in a of 34 proteins the proteins in with the of a a protein spots were as of the protein complex the protein at in of the spots were membrane protein was and of the labeled proteins and of the proteins were in of the respiratory proteins were labeled by with of and the protein of there was significant to the of these proteins. of complex and the of complex was the protein was of complex was the and the were of by the of the with of addition to there were several examples of in a was in in the at were in the voltage-dependent anion and in the as a of to of the To to binding proteins with binding were labeled in and and used for has a halothane-binding of and a of J. PubMed Scopus Google Scholar, K. PubMed Scopus Google Scholar, S. N.P. J. PubMed Scopus Google Scholar). but the and on the and the of protein a of that of the labeled proteins at as of binding that of of used in between and the of in proteins by limited and a limited of must be as of the of the binding of proteins the in the for the a data correlation of with we 1997; Google and evidence for internal cavities as sites in proteins, we J. S. PubMed Scopus Google Scholar, J. S. PubMed Scopus Google to and and data and these to the data in protein was by the number of cavities as a with with molecular of between and molecular of volatile anesthetics between and and with as of the in and that there a significant between and the number of cavities of and to cavities the that despite the a for binding buried in the to these cavities J. K. S. PubMed Scopus Google and the binding to of of the in and data for 34 proteins and in at with and were found to that the a of a for combination of photolabeling, two-dimensional gel electrophoresis, and in of a of membrane proteins that the inhaled Of the 265 spots detected on the 90 were labeled to be on the Of these 90 evidence of of with with by limited of that of cavities to bind K. J. PubMed Scopus Google Scholar). in of the that membrane and proteins and cavities the small that of the to S.J. 1994; PubMed Scopus Google Scholar). further by the in the of respiratory and and of the small inhaled a proteins with high resolution evidence that molecular the of internal protein cavities of binding to with the used in in that a in and of the protein by the binding of a in molecular in the and the in the of two-dimensional the found at the molecular at a of and a but there in the of but significant in between of the and examples direct evidence for state-dependent binding of inhaled to proteins, a but for a molecular to be of a for as of the and evidence for in R.A. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). we that a and for N. PubMed Scopus Google Scholar). as that the for significant in were that with and that inhaled PubMed Scopus Google Scholar). the for of and with in that these be the we found that at of the respiratory and in a subunit- and state-dependent identify complex in gels, that of the respiratory a halothane-binding in the binding to respiratory complex proteins has a by in was to anesthetics PubMed Scopus Google Scholar). complex in was by several inhaled J. J. J. PubMed Scopus Google Scholar). in complex in 1994; PubMed Scopus Google and in PubMed Scopus Google that effects to and binding to proteins be with effects of as in and in the in of were as to R.A. J. PubMed Scopus Google these effects were a of inhaled in that effects to be PubMed Scopus Google with channels and proteins. the binding targets to these voltage-dependent anion a and of and protein to by a to J. PubMed Scopus Google and evidence that with high W.R. S.J. J. 2003; PubMed Scopus Google Scholar). with the S. PubMed Scopus Google Scholar, N. K. 1997; PubMed Scopus Google in the binding the with in a used to inhaled anesthetics and the for inhaled anesthetics PubMed Scopus Google Scholar). and inhaled anesthetics to with at A the between the complex and with the binding data and suggest a for in for inhaled there was significant in on for in with W.R. S.J. J. PubMed Scopus Google Scholar). there with the these data suggest that the proteins in protein were labeled by in PubMed Scopus Google Scholar). protein and protein to a of inhaled anesthetics the of proteins with the for in the of as proteins of a PubMed Scopus Google of that the proteins, and of and binding of the for of N. J. 1994; PubMed Scopus Google Scholar, K. C.F. J. 2003; PubMed Google a that has there to the of proteins for with the that proteins a significant to the and of these Mitochondrial proteins the for in be of two-dimensional gel PubMed Scopus Google of rat proteins represented of identified. A of the labeled proteins in in but be of of the to membrane proteins we the that proteins, ion channels, binding targets for of these proteins that as and and that proteins labeled by in the of potentially molecular targets for the inhaled direct photolabeling, two-dimensional gel electrophoresis, and mass we find that specifically of rat membrane proteins and that of these proteins the respiratory and the voltage-dependent anion and binding targets for binding with the number of internal protein cavities of we evidence for subunit- and state-dependent binding to the that of these targets to the of combination of photolabeling, two-dimensional gel electrophoresis, and in of a of membrane proteins that the inhaled Of the 265 spots detected on the 90 were labeled to be on the Of these 90 evidence of of with with by limited of that of cavities to bind K. J. PubMed Scopus Google Scholar). in of the that membrane and proteins and cavities the small that of the to S.J. 1994; PubMed Scopus Google Scholar). further by the in the of respiratory and and of the small inhaled a proteins with high resolution evidence that molecular the of internal protein cavities of binding to with the used in in that a in and of the protein by the binding of a in molecular in the and the in the of two-dimensional the found at the molecular at a of and a but there in the of but significant in between of the and examples direct evidence for state-dependent binding of inhaled to proteins, a but for a molecular to be of a for as of the and evidence for in R.A. J. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). we that a and for N. PubMed Scopus Google Scholar). as that the for significant in were that with and that inhaled PubMed Scopus Google Scholar). the for of and with in that these be the we found that at of the respiratory and in a subunit- and state-dependent identify complex in gels, that of the respiratory a halothane-binding in the binding to respiratory complex proteins has a by in was to anesthetics PubMed Scopus Google Scholar). complex in was by several inhaled J. J. J. PubMed Scopus Google Scholar). in complex in 1994; PubMed Scopus Google and in PubMed Scopus Google that effects to and binding to proteins be with effects of as in and in the in of were as to R.A. J. PubMed Scopus Google these effects were a of inhaled in that effects to be PubMed Scopus Google with channels and proteins. the binding targets to these voltage-dependent anion a and of and protein to by a to J. PubMed Scopus Google and evidence that with high W.R. S.J. J. 2003; PubMed Scopus Google Scholar). with the S. PubMed Scopus Google Scholar, N. K. 1997; PubMed Scopus Google in the binding the with in a used to inhaled anesthetics and the for inhaled anesthetics PubMed Scopus Google Scholar). and inhaled anesthetics to with at A the between the complex and with the binding data and suggest a for in for inhaled there was significant in on for in with W.R. S.J. J. PubMed Scopus Google Scholar). there with the these data suggest that the proteins in protein were labeled by in PubMed Scopus Google Scholar). protein and protein to a of inhaled anesthetics the of proteins with the for in the of as proteins of a PubMed Scopus Google of that the proteins, and of and binding of the for of N. J. 1994; PubMed Scopus Google Scholar, K. C.F. J. 2003; PubMed Google a that has there to the of proteins for with the that proteins a significant to the and of these Mitochondrial proteins the for in be of two-dimensional gel PubMed Scopus Google of rat proteins represented of identified. A of the labeled proteins in in but be of of the to membrane proteins we the that proteins, ion channels, binding targets for of these proteins that as and and that proteins labeled by in the of potentially molecular targets for the inhaled direct photolabeling, two-dimensional gel electrophoresis, and mass we find that specifically of rat membrane proteins and that of these proteins the respiratory and the voltage-dependent anion and binding targets for binding with the number of internal protein cavities of we evidence for subunit- and state-dependent binding to the that of these targets to the of for and S. for a of the