Photoaffinity Labeling with a Neuroactive Steroid Analogue

Neuroactive steroids modulate the function of γ-aminobutyric acid, type A (GABAA) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3α,5β-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABAA receptors as evidenced by its ability to inhibit binding of [35S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents inXenopus oocytes and human endothelial kidney 293 cells expressing GABAA receptor subunits (α1β2γ2). [3H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of ∼35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [3H]6-AziP concentration of 1.9 μm, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [3H]6-AziP. The β2and β3 subunits of the GABAA receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABAA receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABAA receptor may be mediated by binding to an accessory protein, VDAC-1. Neuroactive steroids modulate the function of γ-aminobutyric acid, type A (GABAA) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3α,5β-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABAA receptors as evidenced by its ability to inhibit binding of [35S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents inXenopus oocytes and human endothelial kidney 293 cells expressing GABAA receptor subunits (α1β2γ2). [3H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of ∼35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [3H]6-AziP concentration of 1.9 μm, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [3H]6-AziP. The β2and β3 subunits of the GABAA receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABAA receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABAA receptor may be mediated by binding to an accessory protein, VDAC-1. γ-aminobutyric acid GABA, type A 3α,5β-6-azi-3-hydroxypregnan-20-one 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid human endothelial kidney matrix-assisted laser desorption ionization time-of-flight t-butylbicyclophosphorothionate voltage-dependent anion channel tetrahydrofuran electrospray ionization Certain endogenous pregnane steroids and their structural analogues are potent anesthetics in vertebrates (1Selye H. Proc. Soc. Exp. Biol. Med. 1941; 46: 116-121Crossref Scopus (342) Google Scholar, 2Atkinson R.M. Davis B. Pratt M.A. Sharpe H.M. Tomich E.G. J. Med. Chem. 1965; 8: 426-432Crossref PubMed Scopus (70) Google Scholar). These neuroactive steroids produce a rapid and reversible depression of the central nervous system indicating that their actions, unlike those of other steroid hormones, are not mediated by transcriptional regulation. In the 1980s it was demonstrated that neuroactive steroids could modulate the function of γ-aminobutyric acid (GABA),1 type A (GABAA) receptors in the central nervous system (3Majewska M.D. Harrison N.L. Schwartz R.D. Barker J.L. Paul S.M. Science. 1986; 232: 1004-1007Crossref PubMed Scopus (2018) Google Scholar, 4Harrison N.L. Simmonds M.A. Brain Res. 1984; 323: 287-292Crossref PubMed Scopus (449) Google Scholar, 5Harrison N.L. Vicini S. Barker J.L. J. Neurosci. 1987; 7: 604-609Crossref PubMed Google Scholar). Low concentrations of the steroids potentiate the actions of GABA whereas higher concentrations directly open the GABAA receptor ion channel (6Cottrell G.A. Lambert J.J. Peters J.A. Br. J. Pharmacol. 1987; 90: 491-500Crossref PubMed Scopus (183) Google Scholar, 7Barker J.L. Harrison N.L. Lange G.D. Owen D.G. J. Physiol. 1987; 386: 485-501Crossref PubMed Scopus (150) Google Scholar). These observations led to the hypothesis that neuroactive steroids produce anesthesia by activating GABAAreceptors and thus enhancing inhibitory synaptic transmission. The strong correlation between the ability of various neuroactive steroid analogues to modulate GABAA receptors and their ability to produce anesthesia strongly supports this hypothesis (8Harrison N.L. Majewska M.D. Harrington J.W. Barker J.L. J. Pharmacol. Exp. Ther. 1987; 241: 346-353PubMed Google Scholar). The mechanism by which neuroactive steroids modulate GABAAreceptor function remains unclear. In previous work we have tested the enantiomers of allopregnanolone (9Wittmer L.L. Hu Y. Kalkbrenner M. Evers A.S. Zorumski C. Covey D.F. Mol. Pharmacol. 1996; 50: 1581-1586PubMed Google Scholar) and pregnanolone (10Covey D.F. Nathan D. Kalkbrenner M. Nilsson K.R. Hu Y. Zorumski C.F. Evers A.S. J. Pharmacol. Exp. Ther. 2000; 293: 1009-1116PubMed Google Scholar) for their abilities to produce anesthesia and to modulate GABAAreceptor function. These studies showed that both steroid anesthesia and steroid modulation of GABAA receptor function are highly enantioselective, particularly in the case of allopregnanolone. This indicates that neuroactive steroids most likely act via binding to specific recognition sites on the GABAA receptor protein complex, because the enantiomeric pairs have identical physical properties but mirror image shapes. Potentiation of GABA action by neuroactive steroids does not require any specific GABAAsubunit (11Sanna E. Murgia A. Casula A. Biggio G. Mol. Pharmacol. 1997; 51: 484-490PubMed Google Scholar) although the absence of the δ subunit does decrease the sensitivity of the receptor to neuroactive steroids (12Mihalek R.M. Banerjee P.K. Korpi E.R. Quinlan J.J. Firestone L.L. Mi Z.P. Lagenaur C. Tretter V. Sieghart W. Anagnostaras S.G. Sage J.R. Fanselow M.S. Guidotti A. Spigelman I. Li Z. DeLorey T.M. Olsen R.W. Homanics G.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12905-12910Crossref PubMed Scopus (450) Google Scholar). Radioligand binding studies and electrophysiological studies indicate that the putative neuroactive steroid binding sites are not identical to or overlapping with the identified binding sites for benzodiazepines (13Turner D.M. Ransom R.W. Yang J.S. Olsen R.W. J. Pharmacol. Exp. Ther. 1989; 248: 960-966PubMed Google Scholar), GABA (13Turner D.M. Ransom R.W. Yang J.S. Olsen R.W. J. Pharmacol. Exp. Ther. 1989; 248: 960-966PubMed Google Scholar) or picrotoxin (3Majewska M.D. Harrison N.L. Schwartz R.D. Barker J.L. Paul S.M. Science. 1986; 232: 1004-1007Crossref PubMed Scopus (2018) Google Scholar) or to the putative binding site for barbiturates (13Turner D.M. Ransom R.W. Yang J.S. Olsen R.W. J. Pharmacol. Exp. 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Nathan D. Kalkbrenner M. Nilsson K.R. Hu Y. Zorumski C.F. Evers A.S. J. Pharmacol. Exp. Ther. 2000; 293: 1009-1116PubMed Google D. Lambert J.J. Mol. Pharmacol. 1994; 46: Google Scholar) with of protein concentration in were with GABA and of steroid in steroid concentrations from to in a of of binding was as binding in the of and the absence of binding was as binding in the of picrotoxin and from to of were for at A was used for of the was with of and in of to the was by and data were using to the is is steroid is the and is the data was in binding were using a with N.L. Simmonds M.A. Brain Res. 1984; 323: 287-292Crossref PubMed Scopus (449) Google Scholar, M. C. J. Pharmacol. PubMed Scopus Google Scholar). membranes were and in to endogenous of protein concentration in and in a of were for at in the binding was as binding in the of and the absence of binding was as binding in the of were in the or the absence of 6-AziP or were on using a to the was by using data was in oocytes were from with acid to were by for at in in the and at rat GABAA receptor and subunits was using the from receptor were in oocytes were to at in the and at with and The for the rat GABAA receptor subunits were by A. of and C. on and were with a The was with were with and open of were from a via a were at for and at the of was for of and GABA were with of currents were and as G. J. Physiol. PubMed Scopus Google Scholar). In the currents were from 293 cells with subunits in the at of to The receptors were by concentrations of The was on using the A. J. 1996; PubMed Scopus Google Scholar). was using Nature. PubMed Scopus Google Scholar). the were or used for were V. 1999; PubMed Scopus Google Scholar) using or with using a the were in and in using a were with of of and for and the in was by spectrometry. from the were with the receptor β3 antibody the membranes were with for with and with antibody The membranes were for with bands were using the system with the antibody the membranes were with for with and with antibody The membranes were for with bands were using the system with the antibody was as with the that was used as rat brain membranes were in a in at a concentration of of and with [3H]6-AziP for at in the These are the in which 6-AziP binding in rat brain membranes, that the steroid have to its site of The was in a at a of from the The a as the The is by a and the is a This of J.A. PubMed Scopus Google Scholar). The were for and to The concentration and of were on that [3H]6-AziP is and that is these the membranes were by and in and and by on a The were and was in The data were as by and Olsen Olsen R.W. J. PubMed Scopus Google Scholar), the the of and the data as a for on the photolabeled membranes were in and labeled were by The were for in acid at and The were in and to at for from to were in of for 60 at The was at and the was for of GABAA antibody directed against the subunits of the GABAA receptor was was with antibody at and to was used as antibody R.W. G.E. 7: Scopus Google Scholar). VDAC-1 was immunoprecipitated using antibody directed against the N terminus of VDAC-1 was with VDAC-1 antibody at and protein A and to was used as protein was from the by in for membranes were with and to the of protein for two-dimensional The membranes were in and of and at a concentration of M. E. R. G. 1999; PubMed Scopus Google Scholar). The was and at for at in the lysate was with a protein The lysate was with and used to a in a for at The was on an system using the for for and for the the were in and on of a that was for at V. The were for or with or In the photolabeled were identified by In two were in in which membranes were photolabeled with 6-AziP and in which membranes were photolabeled with [3H]6-AziP. The were and to in the on a The was with The with was and in the identified in the were and by spectrometry. In this protein labeled by [3H]6-AziP could be The on the to were The were for mass by Y. in Science. Scholar) and by and was at and at of The were with The were by mass on an mass data and data was with was at by mass on a ion mass were to the of [3H]6-AziP. The that of the in of [3H]6-AziP as a band with the as 6-AziP of the in the with 6-AziP of the at a higher as an for the of a of 6-AziP was an of The band at as a function of with a of 6-AziP the of a neuroactive electrophysiological and binding studies were the 6-AziP on the by in oocytes expressing In the absence of GABA, 6-AziP not any 6-AziP the of the GABA the as evidenced with the the by 6-AziP to by GABA in an expressing the subunit at 6-AziP GABA-elicited currents with an of In the pregnanolone an of the of the GABA with an of also at the of 6-AziP on currents by GABA in from the 293 cells with in the absence and of are shown in The open in the absence and of 6-AziP are in The data that the of steroid to an in the channel open The open is and and in the of or of open with the of currents that the of currents in oocytes be for by an in the channel open Neuroactive steroids are to inhibit binding of the of to the picrotoxin binding site of GABAA receptors (3Majewska M.D. Harrison N.L. Schwartz R.D. Barker J.L. Paul S.M. Science. 1986; 232: 1004-1007Crossref PubMed Scopus (2018) Google Scholar). that 6-AziP binding in rat brain membranes with an of and a of pregnanolone binding with an of Neuroactive steroids are also of binding to GABAAreceptors N.L. Simmonds M.A. Brain Res. 1984; 323: 287-292Crossref PubMed Scopus (449) Google Scholar, M. C. J. Pharmacol. PubMed Scopus Google Scholar). 6-AziP effects to those of other neuroactive steroids and to that 6-AziP at the putative steroid binding the effects of 6-AziP and pregnanolone on binding were in rat brain membranes. 6-AziP produced an of specific binding to rat brain membranes. This is to the produced by pregnanolone the electrophysiological and binding studies indicate that 6-AziP the of a neuroactive although it the electrophysiological and a in to were to the effects of 6-AziP as an in anesthetic because of of the the for photolabeling membranes, membranes were with [3H]6-AziP for of and of in the membranes was These showed that of [3H]6-AziP rat brain was at of a was used in the protein sites of [3H]6-AziP rat brain membranes were photolabeled with concentrations of [3H]6-AziP and by with shown in was two protein at ∼35 kDa and at were labeled in a concentration-dependent The 35-kDa was half-maximally labeled at a [3H]6-AziP concentration of 1.9 μm, whereas the 60-kDa was labeled at higher concentrations the 35-kDa protein was labeled at 6-AziP concentrations at which the steroid is have on and of this that [3H]6-AziP labeled a specific binding site on the 35-kDa protein, we photolabeling could be by with other of the 35-kDa protein by [3H]6-AziP was not by with concentrations of or indicating that it was not binding a steroid receptor or a 1994; PubMed Scopus Google Scholar, D. M. E. M. M. 2000; PubMed Scopus Google Scholar) not In photolabeling was in a concentration-dependent by pregnanolone The of and the concentration of pregnanolone to inhibit photolabeling as a function of 6-AziP with a be that the of the between [3H]6-AziP and pregnanolone of binding sites and the 6-AziP concentration as a function of the it is not to that pregnanolone photolabeling via a mechanism. for the photolabeling data is that the 60-kDa protein is a GABAAreceptor subunit and that the 35-kDa protein is a of a GABAA receptor this we membranes from cells with GABAA subunits and from cells that 6-AziP GABAA receptors in we the ability of 6-AziP to inhibit binding in the membranes. shown in 6-AziP binding with an of and a of cells binding indicating the absence of GABAA the of photolabeling and membranes with [3H]6-AziP. of of these that the and 60-kDa were photolabeled in both of membranes. This indicates that the 60-kDa protein is to be a GABAA receptor and the 35-kDa protein is to be a GABAA receptor subunit identify the 35-kDa protein, we isolated the photolabeled protein using two-dimensional of were on of membranes photolabeled with [3H]6-AziP. These of and were at the most likely of the protein in on to the identified on the were and identified by The identified protein in both was VDAC-1 The protein were also by The identified protein in both was voltage-dependent anion In A voltage-dependent anion was also and in B voltage-dependent anion was also that the identified by mass were the that were studies were A antibody directed against the N terminus of VDAC-1 was used for The VDAC-1 antibody 35-kDa protein, confirming that VDAC-1 is photolabeled by [3H]6-AziP. in which antibody was not of any the 60-kDa protein was also by the VDAC-1 suggesting that the two labeled and are In some labeling and in some VDAC-1 a band of kDa was also This band was and was thus to of of the labeled be that the VDAC-1 antibody immunoprecipitated a of the in the 35-kDa This is at in to the that the was not using a antibody to VDAC-1 showed that the a of VDAC-1 is immunoprecipitated by the is also that 35-kDa other VDAC-1 and are photolabeled by [3H]6-AziP. for a between VDAC-1 and the GABAA VDAC-1 was immunoprecipitated from a lysate of rat brain The protein was on to a and by using antibody directed against the and β3 subunits of the GABAA The VDAC-1 antibody co-immunoprecipitated GABAA as evidenced by of to the and β3 subunits of the GABAA in which antibody was not in of any GABAA These indicate that VDAC-1 is with the GABAA receptor of the neuroactive steroid binding sites for anesthesia remains an the GABAA receptor is an and approaches have to identify or binding sites on the GABAA receptor are for the of A binding site may of the protein, may be binding sites but The used to receptors may thus The binding may be on a protein other the In this study we have used photoaffinity a to neuroactive steroid binding sites that of the The photoaffinity labeling reagent we have including the that the is at a that is not to and of with any acid J. G. PubMed Scopus Google Scholar). 6-AziP the neuroactive steroid effects on GABAA receptor function as evidenced by and and by binding A of 6-AziP is that it is and potent as a modulator of GABAA receptor function as In this the that 6-AziP a of the neuroactive steroid binding sites that modulate GABAA receptor function. The in this study is that 6-AziP VDAC-1 in rat brain in a and specific The that VDAC-1 is labeled with with the that pregnanolone indicates that 6-AziP with a binding site on VDAC-1 and is not to an and were also identified in protein from two-dimensional remains to be of these is also photolabeled by VDAC-1 as is a protein, in the M. E. M. 1996; PubMed Scopus Google Scholar). is a of a of that to the M. J. Biol. Chem. 1996; PubMed Scopus Google Scholar). is a channel that is to be in various of including M. J. 1997; Scopus Google Scholar, S. A. Y. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, S. Y. Y. Y. Y. J. Biol. 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S. 1999; PubMed Scopus Google Scholar). it is that VDAC-1 as a ion channel in the its and be with VDAC-1 could be of a with the GABAA receptor in which neuroactive steroid binding to VDAC-1 GABAA receptor function. This be to the mechanism which anesthetics to the protein, and thus modulate receptor function Y. PubMed Scopus Google Scholar). is also that endogenous binding to could be an mechanism for of function. be to the effects of neuroactive steroid binding to in of both anesthetic effects and regulation. The of of the of neuroactive steroid binding sites on VDAC-1 J. Biol. Chem. PubMed Scopus Google Scholar). is neuroactive steroids to the GABAA receptor protein indicate that 6-AziP does not the GABAA 6-AziP does a 60-kDa protein, this protein is labeled in 293 cells expressing and not expressing GABA of [3H]6-AziP GABAAreceptors from 293 cells to any protein not it is that 6-AziP does not neuroactive steroid binding and a could to a GABAA receptor are in the of the 60-kDa protein and its to the GABAA 6-AziP is the effective neuroactive steroid photoaffinity labeling The of VDAC-1 as a neuroactive protein the that neuroactive steroids may modulate GABAA receptor function by binding to an accessory of the between VDAC-1 and the GABAA as as of other photolabeled by on the of neuroactive steroid