Functional and Structural Analysis of the GABAA Receptor α1 Subunit during Channel Gating and Alcohol Modulation

Abstract

The substituted cysteine accessibility method has proven useful for investigating structural changes of the γ-aminobutyric acid type A (GABAA) receptor during channel gating and allosteric modulation. In the present study, the surface accessibility and reaction rate of propyl- and hexyl-methanethiosulfonate to cysteine residues introduced into the third transmembrane segment of the GABAA receptor α1 subunit were examined. GABA-induced currents in Xenopus oocytes expressing wild type and cysteine mutant GABAA receptors were recorded before and after application of methanethiosulfonate (MTS) reagents in the resting, GABA- or alcohol-bound (ethanol or hexanol) states. Our results indicate that a water-filled cavity exists around the Ala291 and Tyr294 residues of the third transmembrane segment, in agreement indicate that a or hexanol) the cavity around the and residues to the and residues to to the In of the and to reagents in the of GABA-induced that the cavity around and residues to and a structural Our that GABAA receptor a during and channel The substituted cysteine accessibility method has proven useful for investigating structural changes of the γ-aminobutyric acid type A (GABAA) receptor during channel gating and allosteric modulation. In the present study, the surface accessibility and reaction rate of propyl- and hexyl-methanethiosulfonate to cysteine residues introduced into the third transmembrane segment of the GABAA receptor α1 subunit were examined. GABA-induced currents in Xenopus oocytes expressing wild type and cysteine mutant GABAA receptors were recorded before and after application of methanethiosulfonate (MTS) reagents in the resting, GABA- or alcohol-bound (ethanol or hexanol) states. Our results indicate that a water-filled cavity exists around the Ala291 and Tyr294 residues of the third transmembrane segment, in agreement indicate that a or hexanol) the cavity around the and residues to the and residues to to the In of the and to reagents in the of GABA-induced that the cavity around and residues to and a structural Our that GABAA receptor a during and channel receptors and in for The γ-aminobutyric acid type A (GABAA) γ-aminobutyric acid type transmembrane substituted cysteine accessibility γ-aminobutyric acid type transmembrane substituted cysteine accessibility the of in the has a of and that of GABAA receptors GABAA receptor the channel in the subunit has a that in transmembrane a and and a and of GABAA receptor and to the and of GABAA receptors in the to and substituted cysteine accessibility method has to and changes of channel in structural of or of channel during gating and a cysteine introduced into the and a the substituted cysteine the to the of the channel to the cysteine to the surface results that channel gating and allosteric or changes in the of GABAA receptor α1 subunit and that around the that structural or reagents to into the of the and in the and for and GABAA receptors and A cavity for in a the of the or of GABAA and receptors the and of the of GABAA receptor during In the present study, the of the alcohol-bound GABAA receptor the or the to the and changes of the of GABAA receptor α1 subunit during channel gating and modulation. that a water-filled cavity exists around the of and that the cavity to the of during channel gating and results of accessibility of and that the of or of structural of the water-filled around the of during channel gating and in the for and in the of allosteric of the GABAA receptor and wild type and cysteine Ala291 to in of the GABAA α1 subunit were were GABAA receptor subunit and α1 or GABAA receptor a Xenopus oocytes were and the wild type or mutant wild type and in a of introduced in the receptor α1 subunit and a currents were recorded oocytes after a The oocytes were and in for The and were reagents and were of or were in of and were in and in to a The of or reagents were before the surface accessibility of reagents to GABAA currents were to the and currents were to after of in the GABA-induced currents The or of that or of the application of oocytes expressing wild type or mutant GABAA receptors were or in the reagents in or alcohol-bound reagents in or the currents and were The of reagents The of and indicate the of the currents or before and after the application of the the of or the oocytes were for to for before a The were before of reaction the introduced were the of of in the resting, or of or GABA-induced currents or or alcohol-bound were for of the and were before the and GABA-induced currents were the of reaction in alcohol-bound or in were for the the GABA-induced The GABA-induced currents to the were to to the rate the rate the rate the were the of The a of the of application and a of the the of the or to and The were to the in and to the of the the the recorded in a the for A the and the of the mutant and wild type were of the and of receptors of and to or and in the the subunit of GABAA receptor the in the for wild type and mutant GABAA receptors in Xenopus oocytes in cysteine GABAA receptor cysteine mutant α1 were wild type and in the GABA-induced currents and of were and of the GABA-induced currents to wild for the to In cysteine were the of The changes in the and the wild type wild type or currents and to the in and currents of cysteine to and to and currents for the wild type and mutant GABAA receptors in The the to oocytes receptor of to in a of currents or for wild type and mutant GABAA receptors The the to oocytes receptor of to in a of in the the surface accessibility of propyl- and hexyl-methanethiosulfonate and reagents to introduced into the The wild type or mutant α1 were wild type and in Xenopus and GABA-induced currents in Xenopus oocytes expressing wild type GABAA receptors before and after of wild type GABAA receptors to GABA-induced currents and application of in the of or GABA-induced currents in wild type GABAA receptor cysteine residues were for reaction the or reaction the in wild type GABAA receptors GABA- or alcohol-bound changes or (ethanol or hexanol) in the mutant receptors the accessibility of and to introduced cysteine in GABA-induced currents were recorded before and after reagents or The GABA-induced currents and receptors were after reagents to mutant receptors in the and the mutant results and GABA-induced GABA-induced currents The results and were that a exists around of reagents in the the of receptors and that mutant receptors in the to or reaction of the reagents mutant of GABA-induced currents of wild type and GABAA receptors in the or GABA-induced the and were wild type and mutant GABAA indicate that the wild type in a of the The of and indicate the of the GABA-induced currents before and after the application of the for of the the to receptor of to of reagents wild type and mutant GABAA oocytes expressing wild type and and GABAA receptors the of of currents or of in the of in the of in the of in the of in the application of reagents in the of GABA-induced currents of the mutant GABAA of reagents the or currents of the and and or of in the of in the and The and were reagents in the or in the of GABA-induced currents and of the mutant were to results in the GABA-induced GABA-induced currents mutant and The were reagents in of in the GABA-induced currents in oocytes expressing mutant GABAA receptors were before and after of reagents or to changes of accessibility of reagents in the alcohol-bound GABA-induced currents of and mutant receptors were after reagents and or of in the or of in the and and reagents and and were to reagents in the and states. or in the of or GABA-induced currents and the or GABA-induced and or the currents and of in the or reaction of reagents introduced cysteine the to the cysteine and the of of the substituted cysteine the of substituted reaction of the were and to in the and the around or residues in the in the alcohol-bound or states. the rate of of to and in the and of and of mutant were for resting, alcohol-bound and in the in the or alcohol-bound that GABA-induced changes around and The rate of mutant in of to in the of or the and accessibility of reagents in the of and of reaction expressing The GABA-induced currents application of in the for of the GABA-induced currents to GABA-induced application to and The rate in rate of of cysteine in the resting, and for and for the the to in a of and that the cysteine residues that reagents a surface to cysteine reagents results that the of cysteine that reagents for the In that of the GABAA receptor α1 subunit changes during channel gating and allosteric or to allosteric of GABAA changes and changes and In study, to changes in of the GABAA receptor α1 subunit during and In study, that the mutant to the of or currents after that the and in the were to the of the surface or a water-filled cavity has that water-filled cavity to the and reagents to and that residues the cavity reagents in the reagents the and in the of The results that the water-filled cavity around and to the and residues to to reagents that the of a water-filled cavity around or that to the to or to In the cavity around to the that in the alcohol-bound around the of of the and to reagents in the of GABA-induced that the surface around and residues to and of a structural that of in the of Tyr294 in that to reagents of the cavity and reagents in the the that structural of to the to the The and to reagents in the and in the alcohol-bound and in the states. of of reagents in the or of or in that reagents to the that the of the that or in the to to of wild type and and that the of or the in the of or that the around the of the GABAA receptor subunit in the of that the of of and of in structural that the allosteric of GABAA the of GABA-induced currents before and after of reagents mutant of in the and of or The and the in A and in states. receptor of to of to that the reagents The and were to to in the of Our that reagents were and in that and or the cysteine or reagents to the accessibility or in and or were in the and the that for changes in the of GABAA receptor α1 subunit of or of the in the and to the introduced the rate of reaction reagents The rate reagents a cysteine the the and the The the of the in the of the in the and the of the The to a cysteine that the in and and the the of the introduced of of the GABAA receptor α1 the rate of reaction of to of the cysteine The rate of the mutant that the around the in the alcohol-bound in the or states. The rate of the mutant in the the rate in the or alcohol-bound states. that the around the the and alcohol-bound states. results to reagents in the and states. The reaction rate of the mutant in the reaction rate in the alcohol-bound states. the of the to to that changes or the surface of the segment to the around and to the or results of accessibility and reaction rate of that the of or during channel gating or and the of a or of that in the water-filled cavity in that the of cavity or a receptor in the cavity of of and of the of channel In a of to that the in the has to the of GABAA receptors in a water-filled cavity that the for of in the present for of the water-filled and of the cavity that in the of the receptors and in for The γ-aminobutyric acid type A (GABAA) γ-aminobutyric acid type transmembrane substituted cysteine accessibility γ-aminobutyric acid type transmembrane substituted cysteine accessibility the of in the has a of and that of GABAA receptors The GABAA receptor the channel in the subunit has a that in transmembrane a and and a and of GABAA receptor and to the and of GABAA receptors in the to and The substituted cysteine accessibility method has to and changes of channel in structural of or of channel during gating and a cysteine introduced into the and a the substituted cysteine the to the of the channel to the cysteine to the surface results that channel gating and allosteric or changes in the of GABAA receptor α1 subunit and that around the that structural or reagents to into the of the and in the and for and GABAA receptors and A cavity for in a the of the or of GABAA and receptors the and of the of GABAA receptor during In the present study, the of the alcohol-bound GABAA receptor the or states. the to the and changes of the of GABAA receptor α1 subunit during channel gating and modulation. that a water-filled cavity exists around the of and that the cavity to the of during channel gating and results of accessibility of and that the of or of structural of the water-filled around the of during channel gating and in the for and in the of allosteric of the GABAA receptor and wild type and cysteine Ala291 to in of the GABAA α1 subunit were were GABAA receptor subunit and α1 or GABAA receptor a Xenopus oocytes were and the wild type or mutant wild type and in a of introduced in the receptor α1 subunit and a currents were recorded oocytes after a The oocytes were and in for The and were reagents and were of or were in of and were in and in to a The of or reagents were before the surface accessibility of reagents to GABAA currents were to the and currents were to after of in the GABA-induced currents The or of that or of the application of oocytes expressing wild type or mutant GABAA receptors were or in the reagents in or alcohol-bound reagents in or the currents and were The of reagents The of and indicate the of the currents or before and after the application of the the of or the oocytes were for to for before a The were before of reaction the introduced were the of of in the resting, or of or GABA-induced currents or or alcohol-bound were for of the and were before the and GABA-induced currents were the of reaction in alcohol-bound or in were for the the GABA-induced The GABA-induced currents to the were to to the rate the rate the rate the were the of The a of the of application and a of the the of the or to and The were to the in and to the of the the the recorded in a the for A the and the of the mutant and wild type were of the and wild type and cysteine Ala291 to in of the GABAA α1 subunit were were GABAA receptor subunit and α1 or GABAA receptor a Xenopus oocytes were and the wild type or mutant wild type and in a of introduced in the receptor α1 subunit and a currents were recorded oocytes after a The oocytes were and in for The and were reagents and were of or were in of and were in and in to a The of or reagents were before the surface accessibility of reagents to GABAA currents were to the and currents were to after of in the GABA-induced currents The or of that or of the application of oocytes expressing wild type or mutant GABAA receptors were or in the reagents in or alcohol-bound reagents in or the currents and were The of reagents The of and indicate the of the currents or before and after the application of the the of or the oocytes were for to for before a The were before of reaction the introduced were the of of in the resting, or of or GABA-induced currents or or alcohol-bound were for of the and were before the and GABA-induced currents were the of reaction in alcohol-bound or in were for the the GABA-induced The GABA-induced currents to the were to to the rate the rate the rate the were the of The a of the of application and a of the the of the or to and The were to the in and to the of the the the recorded in a the for A the and the of the mutant and wild type were of the and of receptors of and to or and in the the subunit of GABAA receptor the in the for wild type and mutant GABAA receptors in Xenopus oocytes in cysteine GABAA receptor cysteine mutant α1 were wild type and in the GABA-induced currents and of were and of the GABA-induced currents to wild for the to In cysteine were the of The changes in the and the wild type wild type or currents and to the in and currents of cysteine to and to and currents for the wild type and mutant GABAA receptors in The the to oocytes receptor of to in a of currents or for wild type and mutant GABAA receptors The the to oocytes receptor of to in a of in the the surface accessibility of propyl- and hexyl-methanethiosulfonate and reagents to introduced into the The wild type or mutant α1 were wild type and in Xenopus and GABA-induced currents in Xenopus oocytes expressing wild type GABAA receptors before and after of wild type GABAA receptors to GABA-induced currents and application of in the of or GABA-induced currents in wild type GABAA receptor cysteine residues were for reaction the or reaction the in wild type GABAA receptors GABA- or alcohol-bound changes or (ethanol or hexanol) in the mutant receptors the accessibility of and to introduced cysteine in GABA-induced currents were recorded before and after reagents or The GABA-induced currents and receptors were after reagents to mutant receptors in the and the mutant results and GABA-induced GABA-induced currents The results and were that a exists around of reagents in the the of receptors and that mutant receptors in the to or reaction of the reagents mutant of reagents wild type and mutant GABAA oocytes expressing wild type and and GABAA receptors the of of currents or of in the of in the of in the of in the of in the application of reagents in the of GABA-induced currents of the mutant GABAA of reagents the or currents of the and and or of in the of in the and The and were reagents in the or in the of GABA-induced currents and of the mutant were to results in the GABA-induced GABA-induced currents mutant and The were reagents in of in the GABA-induced currents in oocytes expressing mutant GABAA receptors were before and after of reagents or to changes of accessibility of reagents in the alcohol-bound GABA-induced currents of and mutant receptors were after reagents and or of in the or of in the and and reagents and and were to reagents in the and states. or in the of or GABA-induced currents and the or GABA-induced and or the currents and of in the or reaction of reagents introduced cysteine the to the cysteine and the of of the substituted cysteine the of substituted reaction of the were and to in the and the around or residues in the in the alcohol-bound or states. the rate of of to and in the and of and of mutant were for resting, alcohol-bound and in the in the or alcohol-bound that GABA-induced changes around and The rate of mutant in of to in the of or the and accessibility of reagents in the of and of reaction expressing The GABA-induced currents application of in the for of the GABA-induced currents to GABA-induced application to and The rate in rate of of cysteine in the resting, and for and for the the to in a and of receptors of and to or and in the the subunit of GABAA receptor the in the for wild type and mutant GABAA receptors in Xenopus oocytes in cysteine GABAA receptor cysteine mutant α1 were wild type and in the GABA-induced currents and of were and of the GABA-induced currents to wild for the to In cysteine were the of The changes in the and the wild type wild type or currents and to the in and currents of cysteine to and to of in the the surface accessibility of propyl- and hexyl-methanethiosulfonate and reagents to introduced into the The wild type or mutant α1 were wild type and in Xenopus and GABA-induced currents in Xenopus oocytes expressing wild type GABAA receptors before and after of wild type GABAA receptors to GABA-induced currents and application of in the of or GABA-induced currents in wild type GABAA receptor cysteine residues were for reaction the or reaction the in wild type GABAA receptors GABA- or alcohol-bound changes or (ethanol or hexanol) in the mutant receptors the accessibility of and to introduced cysteine in GABA-induced currents were recorded before and after reagents or The GABA-induced currents and receptors were after reagents to mutant receptors in the and the mutant results and GABA-induced GABA-induced currents The results and were that a exists around of reagents in the the of receptors and that mutant receptors in the to or reaction of the reagents mutant of in the application of reagents in the of GABA-induced currents of the mutant GABAA of reagents the or currents of the and and or of in the of in the and The and were reagents in the or in the of GABA-induced currents and of the mutant were to results in the GABA-induced GABA-induced currents mutant and The were reagents in of in the GABA-induced currents in oocytes expressing mutant GABAA receptors were before and after of reagents or to changes of accessibility of reagents in the alcohol-bound GABA-induced currents of and mutant receptors were after reagents and or of in the or of in the and and reagents and and were to reagents in the and states. or in the of or GABA-induced currents and the or GABA-induced and or the currents and of in the or reaction of reagents introduced cysteine the to the cysteine and the of of the substituted cysteine the of substituted reaction of the were and to in the and the around or residues in the in the alcohol-bound or states. the rate of of to and in the and of and of mutant were for resting, alcohol-bound and in the in the or alcohol-bound that GABA-induced changes around and The rate of mutant in of to in the of or the and accessibility of reagents in the of and of and that the cysteine residues that reagents a surface to cysteine reagents results that the of cysteine that reagents for the In that of the GABAA receptor α1 subunit changes during channel gating and allosteric or to allosteric of GABAA changes and changes and In study, to changes in of the GABAA receptor α1 subunit during and In study, that the mutant to the of or currents after that the and in the were to the of the surface or a water-filled cavity has that water-filled cavity to the and reagents to and that residues the cavity reagents in the reagents the and in the of The results that the water-filled cavity around and to the and residues to to reagents that the of a water-filled cavity around or that to the to or to In the cavity around to the that in the alcohol-bound around the of of the and to reagents in the of GABA-induced that the surface around and residues to and of a structural that of in the of Tyr294 in that to reagents of the cavity and reagents in the the that structural of to the to the The and to reagents in the and in the alcohol-bound and in the states. of of reagents in the or of or in that reagents to the that the of the that or in the to to of wild type and and that the of or the in the of or that the around the of the GABAA receptor subunit in the of that the of of and of in structural that the allosteric of GABAA of to that the reagents The and were to to in the of Our that reagents were and in that and or the cysteine or reagents to the accessibility or in and or were in the and the that for changes in the of GABAA receptor α1 subunit of or of the in the and to the introduced the rate of reaction reagents The rate reagents a cysteine the the and the The the of the in the of the in the and the of the The to a cysteine that the in and and the the of the introduced of of the GABAA receptor α1 the rate of reaction of to of the cysteine The rate of the mutant that the around the in the alcohol-bound in the or states. The rate of the mutant in the the rate in the or alcohol-bound states. that the around the the and alcohol-bound states. results to reagents in the and states. The reaction rate of the mutant in the reaction rate in the alcohol-bound states. the of the to to that changes or the surface of the segment to the around and to the or results of accessibility and reaction rate of that the of or during channel gating or and the of a or of that in the water-filled cavity in that the of cavity or a receptor in the cavity of of and of the of channel In a of to that the in the has to the of GABAA receptors in a water-filled cavity that the for of in the present for of the water-filled and of the cavity that in the of the of and that the cysteine residues that reagents a surface to cysteine reagents results that the of cysteine that reagents for the In that of the GABAA receptor α1 subunit changes during channel gating and allosteric or to allosteric of GABAA changes and changes and In study, to changes in of the GABAA receptor α1 subunit during and In study, that the mutant to the of or currents after that the and in the were to the of the surface or a water-filled cavity has that water-filled cavity to the and reagents to and that residues the cavity reagents in the reagents the and in the of The results that the water-filled cavity around and to the and residues to to reagents that the of a water-filled cavity around or that to the to or to In the cavity around to the that in the alcohol-bound around the of of the and to reagents in the of GABA-induced that the surface around and residues to and of a structural that of in the of Tyr294 in that to reagents of the cavity and reagents in the the that structural of to the to the The and to reagents in the and in the alcohol-bound and in the states. of of reagents in the or of or in that reagents to the that the of the that or in the to to of wild type and and that the of or the in the of or that the around the of the GABAA receptor subunit in the of that the of of and of in structural that the allosteric of GABAA of to that the reagents The and were to to in the of Our that reagents were and in that and or the cysteine or reagents to the accessibility or in and or were in the and the that for changes in the of GABAA receptor α1 subunit of or of the in the and to the introduced the rate of reaction reagents The rate reagents a cysteine the the and the The the of the in the of the in the and the of the The to a cysteine that the in and and the the of the introduced of of the GABAA receptor α1 the rate of reaction of to of the cysteine The rate of the mutant that the around the in the alcohol-bound in the or states. The rate of the mutant in the the rate in the or alcohol-bound states. that the around the the and alcohol-bound states. results to reagents in the and states. The reaction rate of the mutant in the reaction rate in the alcohol-bound states. the of the to to that changes or the surface of the segment to the around and to the or results of accessibility and reaction rate of that the of or during channel gating or and the of a or of that in the water-filled cavity in that the of cavity or a receptor in the cavity of of and of the of channel In a of to that the in the has to the of GABAA receptors in a water-filled cavity that the for of in the present for of the water-filled and of the cavity that in the of the for for and and for of