Distinct Structural Elements in the First Membrane-spanning Segment of the Epithelial Sodium Channel

Abstract

Epithelial Na+ channels (ENaCs) comprise three subunits that have been proposed to be arranged in either an α2βγ or a higher ordered configuration. Each subunit has two putative membrane-spanning segments (M1 and M2), intracellular amino and carboxyl termini, and a large extracellular loop. We have used the TOXCAT assay (a reporter assay for transmembrane segment homodimerization) to identify residues within the transmembrane segments of ENaC that may participate in important structural interactions within ENaC, with which we identified a candidate within We and the ENaC the the of in the of ENaC, we We that within the of and with a with a We that within the of and with a in which the of and structural that to the ENaC that the of may be or a and may be in interactions to or that two Epithelial Na+ channels (ENaCs) comprise three subunits that have been proposed to be arranged in either an α2βγ or a higher ordered configuration. Each subunit has two putative membrane-spanning segments (M1 and M2), intracellular amino and carboxyl termini, and a large extracellular loop. We have used the TOXCAT assay (a reporter assay for transmembrane segment homodimerization) to identify residues within the transmembrane segments of ENaC that may participate in important structural interactions within ENaC, with which we identified a candidate within We and the ENaC the the of in the of ENaC, we We that within the of and with a with a We that within the of and with a in which the of and structural that to the ENaC that the of may be or a and may be in interactions to or that two Na+ used ENaC, Na+ membrane-spanning membrane-spanning of used ENaC, Na+ membrane-spanning membrane-spanning of in the of that the and and and the of the in ENaC a in Na+ and and ENaC and subunits arranged in either a α2βγ or higher ordered Each subunit has two membrane-spanning and M2), intracellular amino and carboxyl termini, and large extracellular of the of channels that have in and of the ENaC has been the of has been to be of the segments with the residues extracellular to segments three subunits the α2βγ the two subunits to be of the that the of three subunits the to have that three subunits and to the that residues with segments that have the of within of the that of may be proposed for the of the ENaC the a with the the and a that the of the the of has been to be and to the to the of in channels we have used the TOXCAT assay to identify residues within the ENaC transmembrane that may participate in important structural interactions within the assay the of to of a to the of a transmembrane we identified a candidate within We and the ENaC the an to the of in the of ENaC, we with the of we to We that within the of and with a with a in to in in the of the we a in the putative that the of the of and large a in which the of and structural that to the ENaC that the of may be either or a and may with structural with the of and the and subunits of the of the for the of the a of the membrane-spanning and for the transmembrane in which the transmembrane segment an to the for the membrane-spanning segments of the and subunits the and and to the and of of in the to and of two in the of to and ENaC to to of of the and of the for in the of and with a of with and and and to in for and of the to that an for of membrane-spanning of the to within the of the a within the the membrane-spanning of that for and ENaC ENaC used for ENaC subunits a the of for and ENaC subunits with and of with of for ENaC and for in and a and a and a with a and a of with and the in a with of and with a of either or and the in to the in for to ENaC the of ENaC with of or or subunits with and for in with and for with with for in and with with for and to in of and for in a of of the in with to a with and to for and with of in an an and a to a a in the and the with a for and of channels of with and of ENaC the of of the ENaC membrane-spanning segments to within the of a we a TOXCAT assay the transmembrane and of transmembrane and the putative membrane-spanning to the the to with the of a reporter be the of the to a for the TOXCAT assay for of the transmembrane segments (M1 and for ENaC subunits and the to that ENaC has the to of the a which residues within for the in we within and the of TOXCAT of to identify a of that been to a in and of which in to in the a an that of in the and that and that of that to of the within the and We that important for the of to within the for that and the of to and in and we that to the of the of the the TOXCAT We the of the of ENaC in either or with and subunits we Na+ for the and channels We that Na+ that the of and and the in Na+ for of of a in the of channels the or a in the of we the for two a subunit and a assay and assay with of to that of the and of ENaC that in that We that an has been which a which may the TOXCAT the be in ENaC, the may be or either or the or we that or with a in and the we and we have residues to to the in and in has a large be that with in interactions with residues within the the large in be that with that with that with or that with for that have a or subunits of ENaC with and subunits and Na+ with a in the and of and the the to the for ENaC the of a to a in Na+ to ENaC residues a in the Na+ that and or Na+ with ENaC, and of the of the we to a a the and and and the for for residues to a of for an and the a with an residues the of residues Na+ to the of Each to and with which to Na+ for and the and to the Na+ for ENaC the Each the for residues to a of the an the Na+ and the of the be the of Each the of for Na+ or to a for residues and of the of the of for and we with with or in the and of and the the channels and Na+ for to a for with ENaC, the of the to for residues with Na+ and of and a for the for residues for Na+ for residues and either Na+ or of ENaC the in Na+ for the to in the of channels the we the of a subunit with an to with and residues and to ENaC the three and with three for that to for Na+ the and for in that that two of of or subunits with and subunits and ENaC subunits used to Each the and of ENaC that the Na+ and with ENaC to ENaC the of We that the of be to in the of the we of the in which the and the and the the with a of for ENaC we the to be which the of may for ENaC that a for the of the of the the and and the an and of the for Each the of three of the a of to be of to that within the segment of an to the of to ENaC we subunit residues an and to to the to in the of with the of of the in the and to two of the putative We the a the of the to that to have that the transmembrane ENaC subunit and the with residues the to for the of in the of the ENaC an the Na+ a that with ENaC that the of the residues in to a an a that with ENaC identified the of that to to or to channels and have that and may with within the subunit a for the of the and residues and which with the of for an the residues to to that or residues with the Na+ of that that two and of transmembrane may be structural or may have a the that the may be a in the to the in the of the segment of channels in the that to that in either or for of in ENaC for and and and two with the the and the subunit for subunit residues and within a the may be We that the may be or a and may with the may in a to for has been proposed for of the the of of the that either residues the or residues the to for ENaC with for may be to a ENaC structural in the of or a of We to the intracellular of ENaC of the large of residues that to to the of of with an ENaC in which with a of ENaC the TOXCAT assay the of a to residues identified to be important in and and the that the TOXCAT assay the of two of and and or the of and and of be to the of of may be to the that in the TOXCAT the of to we that the residues in interactions with residues membrane-spanning segments within for the for a in which with a of the of we that the of with that the that Na+ or higher the in which with a to the that the of the structural and of that with to have or in in and Na+ and the structural a in of the a in and a large in in may be to the of the in the of and Na+ channels the the of the in a the and have residues and the the of of the be to three the we the of the and the of the which we that which to and with ENaC structural the TOXCAT the of the ENaC a an the of the and to the segments of subunit the and the segment of subunit may the segment the subunit a of to interactions have the in the of to interactions be a ENaC the two an a be with a of two and two subunits ENaC to a ENaC that the that which for for in an the or segments either the or to either the or we that of that and we that in the of We that of or with the in the TOXCAT assay that the of has an important in the of with residues within and the be in a to a for in and to the in the of we We the of the amino to and the the within the of the in to or in with with the of the amino to with a in which of with that in that and the within the and or we have in to to be to the of and that two with the an of the and to the may be or may have a a that or to in the TOXCAT identified to be the that within an structural within Na+ used ENaC, Na+ membrane-spanning membrane-spanning of used ENaC, Na+ membrane-spanning membrane-spanning of in the of that the and and and the of the in ENaC a in Na+ and and ENaC and subunits arranged in either a α2βγ or higher ordered Each subunit has two membrane-spanning and M2), intracellular amino and carboxyl termini, and large extracellular of the of channels that have in and of the ENaC has been the of has been to be of the segments with the residues extracellular to segments three subunits the α2βγ the two subunits to be of the that the of three subunits the to have that three subunits and to the that residues with segments that have the of within of the that of may be proposed for the of the ENaC the a with the the and a that the of the the of has been to be and to the to the of in channels we have used the TOXCAT assay to identify residues within the ENaC transmembrane that may participate in important structural interactions within the assay the of to of a to the of a transmembrane we identified a candidate within We and the ENaC the an to the of in the of ENaC, we with the of we to We that within the of and with a with a in to in in the of the we a in the putative that the of the of and large a in which the of and structural that to the ENaC that the of may be either or a and may with structural with the of and the and subunits of the of the for the of the a of the membrane-spanning and for the transmembrane in which the transmembrane segment an to the for the membrane-spanning segments of the and subunits the and and to the and of of in the to and of two in the of to and ENaC to to of of the and of the for in the of and with a of with and and and to in for and of the to that an for of membrane-spanning of the to within the of the a within the the membrane-spanning of that for and ENaC ENaC used for ENaC subunits a the of for and ENaC subunits with and of with of for ENaC and for in and a and a and a with a and a of with and the in a with of and with a of either or and the in to the in for to ENaC the of ENaC with of or or subunits with and for in with and for with with for in and with with for and to in of and for in a of of the in with to a with and to for and with of in an an and a to a a in the and the with a for and of channels of with and TOXCAT and the and subunits of the of the for the of the a of the membrane-spanning and for the transmembrane in which the transmembrane segment an to the for the membrane-spanning segments of the and subunits the and and to the and of of in the to and of two in the of to TOXCAT and ENaC to to of of the and of the for in the of and with a of with and and and to in for and of the to that an for of membrane-spanning of the to within the of the a within the the membrane-spanning of that for and ENaC ENaC used for ENaC subunits a the of for and ENaC subunits with and of with of for ENaC and for in and a and a and a with a and a of with and the in a with of and with a of either or and the in to the in for to ENaC the of ENaC with of or or subunits with and for in with and for with with for in and with with for and to in of and for in a of of the in with to a with and to for and with of in an an and a to a a in the and the with a for and of channels of with and of ENaC the of of the ENaC membrane-spanning segments to within the of a we a TOXCAT assay the transmembrane and of transmembrane and the putative membrane-spanning to the the to with the of a reporter be the of the to a for the TOXCAT assay for of the transmembrane segments (M1 and for ENaC subunits and the to that ENaC has the to of the a which residues within for the in we within and the of TOXCAT of to identify a of that been to a in and of which in to in the a an that that to of the within the and We that important for the of to within the for that and the of to and in and we that to the of the of the the TOXCAT We the of the of ENaC in either or with and subunits we Na+ for the and channels We that Na+ that the of and and the in Na+ for of of a in the of channels the or a in the of we the for two a subunit and a assay and assay with of to that of the and of ENaC that in that We that an has been which a which may the TOXCAT the be in ENaC, the may be or either or the or we that or with a in and the we and we have residues to to the in and in has a large be that with in interactions with residues within the the large in be that with that with that with or that with for that have a or subunits of ENaC with and subunits and Na+ with a in the and of and the the to the for ENaC the of a to a in Na+ to ENaC residues a in the Na+ that and or Na+ with ENaC, and of the of the we to a a the and and and the for for residues to a of for an and the a with an residues the of residues Na+ to the of Each to and with which to Na+ for and the and to the Na+ for ENaC the Each the for residues to a of the an the Na+ and the of the be the of Each the of for Na+ or to a for residues and of the of the of for and we with with or in the and of and the the channels and Na+ for to a for with ENaC, the of the to for residues with Na+ and of and a for the for residues for Na+ for residues and either Na+ or of ENaC the in Na+ for the to in the of channels the we the of a subunit with an to with and residues and to ENaC the three and with three for that to for Na+ the and for in that that two of of or subunits with and subunits and ENaC subunits used to Each the and of ENaC that the Na+ and with ENaC to ENaC the of We that the of be to in the of the we of the in which the and the and the the with a of for ENaC we the to be which the of may for ENaC that a for the of the of the the and and the an and of the for Each the of three of the a of to be of to that within the segment of an to the of to ENaC we subunit residues an and to to the to in the of with the of of the in the and to two of the putative We the a the of the to that to TOXCAT of ENaC the of of the ENaC membrane-spanning segments to within the of a we a TOXCAT assay the transmembrane and of transmembrane and the putative membrane-spanning to the the to with the of a reporter be the of the to a We for the TOXCAT assay for of the transmembrane segments (M1 and for ENaC subunits and the to that ENaC has the to of the a which residues within for the in we within and the of TOXCAT of to identify a of that been to a in and of which in to in the a an that We that to of the within the and We that important for the of to within the for that and the of to and in and we that to the of the of the the TOXCAT We the of the of ENaC in either or with and subunits we Na+ for the and channels We that Na+ that the of and and the in Na+ for of of a in the of channels the or a in the of we the for two a subunit and a assay and assay with of to that of the and of ENaC that in that We that an has been which a which may the TOXCAT the be in ENaC, the may be or either or the or we that or with a in and the we and we have residues to to the in and in has a large be that with in interactions with residues within the the large in be that with that with that with or that with for that have a or subunits of ENaC with and subunits and Na+ with a in the and of and the the to the for ENaC the of a to a in Na+ to ENaC residues a in the Na+ that and or Na+ with ENaC, and of the of the we to a a the and and and the for for residues to a of for an and the a with an residues the of residues Na+ to the We the of the of for and we with with or in the and of and the the channels and Na+ for to a for with ENaC, the of the to for residues with Na+ and of and a for the for residues for Na+ for residues and either Na+ or of ENaC the in Na+ for the to in the of channels the we the of a subunit with an to with and residues and to ENaC the three and with three for that to for Na+ the and for in that that two of ENaC that the Na+ and with ENaC to ENaC the of We that the of be to in the of the we of the in which the and the and the the with a of for ENaC we the to be which the of may for ENaC that a for the of the of to that within the segment of an to the of to ENaC we subunit residues an and to to the to in the of with the of of the in the and to two of the putative We the a the of the to that to have that the transmembrane ENaC subunit and the with residues the to for the of in the of the ENaC an the Na+ a that with ENaC that the of the residues in to a an a that with ENaC identified the of that to to or to channels and have that and may with within the subunit a for the of the and residues and which with the of for an the residues to to that or residues with the Na+ of that that two and of transmembrane may be structural or may have a the that the may be a in the to the in the of the segment of channels in the that to that in either or the of of the that either residues the or residues the to for ENaC with for may be to a ENaC structural in the of or a of We to the intracellular of ENaC of the large of residues that to to the of of with an ENaC in which with a of ENaC the TOXCAT assay the of a to residues identified to be important in and and the that the TOXCAT assay the of two of and and or the of and and of be to the of of may be to the that in the TOXCAT the of to we that the residues in interactions with residues membrane-spanning segments within for the for a in which with a of the of we that the of with that the that Na+ or higher the in which with a to the that the of the structural and of that with to have or in in and Na+ and the structural a in of the a in and a large in in may be to the of the in the of and Na+ channels the the of the in a the and have residues and the the of of the be to three the we the of the and the of the which we that which to and with ENaC structural the TOXCAT the of the ENaC a an the of the and to the segments of subunit the and the segment of subunit may the segment the subunit a of to interactions have the in the of to interactions be a ENaC the two an a be with a of two and two subunits ENaC to a ENaC that the that which for for in an the or segments either the or to either the or we that of that and we that in the of We that of or with the in the TOXCAT assay that the of has an important in the of with residues within and the be in a to a for in and to the in the of we We the of the amino to and the the within the of the in to or in with with the of the amino to with a in which of with that in that and the within the and or we have in to to be to the of and that two with the an of the and to the may be or may have a a that or to in the TOXCAT identified to be the that within an structural within have that the transmembrane ENaC subunit and the with residues the to for the of in the of the ENaC an the Na+ a that with ENaC that the of the residues in to a an a that with ENaC identified the of that to to or to channels and have that and may with within the subunit a for the of the and residues and which with the of for an the residues to to that or residues with the Na+ of that that two and of transmembrane may be structural or may have a the that the may be a in the to the in the of the segment of channels in the that to that in either or the of of the that either residues the or residues the to for ENaC with for may be to a ENaC structural in the of or a of We to the intracellular of ENaC of the large of residues that to to the of of with an ENaC in which with a of ENaC the TOXCAT assay the of a to residues identified to be important in and and the that the TOXCAT assay the of two of and and or the of and and of be to the of of may be to the that in the TOXCAT the of to we that the residues in interactions with residues membrane-spanning segments within for the for a in which with a of the of we that the of with that the that Na+ or higher the in which with a to the that the of the structural and of that with to have or in in and Na+ and the structural a in of the a in and a large in in may be to the of the in the of and Na+ channels the the of the in a the and have residues and the the of of the be to three the we the of the and the of the which we that which to and with ENaC structural the TOXCAT the of the ENaC a an the of the and to the segments of subunit the and the segment of subunit may the segment the subunit a of to interactions have the in the of to interactions be a ENaC the two an a be with a of two and two subunits ENaC to a ENaC We that the that which for for in an the or segments either the or to either the or we that of that and we that in the of We that of or with the in the TOXCAT assay that the of has an important in the of with residues within and the be in a to a for in and to the in the of we We the of the amino to and the the within the of the in to or in with with the of the amino to with a in which of with that in that and the within the and or we have in to to be to the of and that two with the an of the and to the may be or may have a a that or to in the TOXCAT identified to be the that within an structural within