Characterization of the Selectivity Filter of the Epithelial Sodium Channel

Abstract

The epithelial sodium channel (ENaC) is composed of three homologous subunits termed α, β, and γ. Previous studies suggest that selected residues within a hydrophobic region immediately preceding the second membrane-spanning domain of each subunit contribute to the conducting pore of ENaC. We probed the pore of mouse ENaC by systematically mutating all 24 amino acids within this putative pore region of the α-subunit to cysteine and co-expressing these mutants with wild type β- and γ-subunits of mouse ENaC inXenopus laevis oocytes. Functional of these mutants by and channel the with domain cysteine cysteine within the domain and The to and of a to a suggest that the of the pore region within the α-subunit to the of ENaC. The epithelial sodium channel (ENaC) is composed of three homologous subunits termed α, β, and γ. Previous studies suggest that selected residues within a hydrophobic region immediately preceding the second membrane-spanning domain of each subunit contribute to the conducting pore of ENaC. We probed the pore of mouse ENaC by systematically mutating all 24 amino acids within this putative pore region of the α-subunit to cysteine and co-expressing these mutants with wild type β- and γ-subunits of mouse ENaC inXenopus laevis oocytes. Functional of these mutants by and channel the with domain cysteine cysteine within the domain and The to and of a to a suggest that the of the pore region within the α-subunit to the of ENaC. epithelial sodium channel mouse ENaC sodium sodium of epithelial that the the and and the composed of three homologous termed and and a subunit of subunit The three subunits a with amino and and and that by a hydrophobic and immediately and preceding The region preceding the to the region of the and of selected residues within the region and the and of the γ-subunits of ENaC channel to a putative pore of ENaC that all three subunits pore and that the region preceding the domain a of the a the of of the pore region of the of ENaC. and that the pore of ENaC is that of a by this to the hydrophobic region preceding the domain the pore region of ENaC. We systematically all 24 residues within the pore region to cysteine to residues within the mouse α-subunit that the ENaC The of with wild type β- and γ-subunits of to that of the channel that to and to the residues within the of the pore region of studies that the domain within the putative pore region of the α-subunit to the of of cysteine systematically the pore region of the α-subunit of to to the of residues within this region within this pore region of these the of this to residues this a with the and with the with all of the mutants within the of this domain to with the of studies of of ENaC suggest that this is a pore and with the of the pore to within the channel within the with the channel is that selected within the pore region of ENaC the with this We that within the of the pore region cysteine of residues with these with the with a within the pore region of the β- of mouse and and ENaC and residues within β- and within the pore and that is that of the pore region of the and within a region that is the three ENaC subunits The within to and is and studies that of a cysteine this a We this this the of the and within the of within the is that these the of the pore and with the the of within the pore region of the by the of the pore cysteine residues and and a cysteine with and the and mutants these and the and cysteine within the pore region of the α-subunit channel with the of The with within this region channel with the that these with with the of and the of to to and with wild type β- and these with wild type and mutants that that with wild type suggest that these residues a a within the pore that with and a amino residues to with the that and residues within a domain of the pore region that and the residues the pore and with residues within the channel to the pore to to The of these pore and of and ENaC is with studies with amino residues within pore that to a We a cysteine of that is all three subunits of ENaC and a of with with selected the residues within the β- and γ-subunits and and with the within these is this within cysteine to of ENaC is to a that is within the of the of the that and with by the with selected by and that this that this the that the is with the of a with and a to mouse and a the the suggest that the within the three ENaC subunits is of the within the pore region of that a and studies of with the residues the of that the channel with a of and is the that of the mutants that within the pore region of the suggest that the ENaC pore of with the pore of the of amino residues with and that wild type We that of residues to The mouse a within the of the pore region of a channel with to and that the of a this to the channel these suggest that residues within the of the α-subunit of ENaC a is that to ENaC ENaC within the with within the of the pore domain suggest that residues within this is to the of the We a ENaC is within the pore region suggest that a of within the pore region of that and to the is by the within is and to the of the and to the We that is this the of is to this the of the pore to with and to the with the and We that a second that of a within the this second to residues to and is that the the and of residues the pore and the with residues within the channel to channel a of the ENaC with by the and The is to the a to the is to of the of amino acids within the pore and of ENaC. that the of the pore and that the of the pore the The pore domain and the of the to the channel is that the of the and within the and of the pore region and these the pore and the to and within the region The membrane-spanning pore region is residues these residues within the that the domain to the and with of within the The of the of the pore region the that within the and of the putative pore region of the channel suggest of the ENaC pore is by and the pore region the second membrane-spanning to by this the pore the and to a region the that is of to residues We that the pore to second membrane-spanning domain and that the pore this to of a putative suggest that that this We that residues and that residues membrane-spanning is to the pore by and that the is by the is to with a of the channel within the membrane-spanning domain and with residues within the β- and γ-subunits to all residues within the and domain within the region of the channel the the putative membrane-spanning region a of residues membrane-spanning the residues a and the second membrane-spanning domain of the channel is residues and is with to the by pore region with to the pore of the is by a pore that the by region the of the channel region the We that this region within is residues and is to the of the residues region is by the second membrane-spanning is with the that with and these residues the of the pore that that of the with to a channel is with the that the ENaC pore to the pore of the channel and residues homologous within the and γ-subunits that the pore of the channel the that residues of ENaC. residues the by to the of the a the with the the channel pore a to the within the pore and with residues within the of pore and the of the and the and with of a of the within to within mouse and the the of the pore region of the α-subunit to the domain We that this by the of the the that is within the within the putative domain of the channel the and residues preceding the pore region the of α-subunit suggest that a the of suggest that the within the α-subunit of ENaC is by region of the pore residues suggest that within this domain that and with the that that the ENaC pore that a sodium sodium of epithelial that the the and and the composed of three homologous termed and and a subunit of subunit The three subunits a with amino and and and that by a hydrophobic and immediately and preceding The region preceding the to the region of the and of selected residues within the region and the and of the γ-subunits of ENaC channel to a putative pore of ENaC that all three subunits pore and that the region preceding the domain a of the a the of of the pore region of the of ENaC. and that the pore of ENaC is that of a by this to the hydrophobic region preceding the domain the pore region of ENaC. We systematically all 24 residues within the pore region to cysteine to residues within the mouse α-subunit that the ENaC The of with wild type β- and γ-subunits of to that of the channel that to and to the residues within the of the pore region of studies that the domain within the putative pore region of the α-subunit to the of ENaC. of cysteine systematically the pore region of the α-subunit of to to the of residues within this region within this pore region of these the of this to residues this a with the and with the with all of the mutants within the of this domain to with the of studies of of ENaC suggest that this is a pore and with the of the pore to within the channel within the with the channel is that selected within the pore region of ENaC the with this We that within the of the pore region cysteine of residues with these with the with a within the pore region of the β- of mouse and and ENaC and residues within β- and within the pore and that is that of the pore region of the and within a region that is the three ENaC subunits The within to and is and studies that of a cysteine this a We this this the of the and within the of within the is that these the of the pore and with the the of within the pore region of the by the of the pore cysteine residues and and a cysteine with and the and mutants these and the and cysteine within the pore region of the α-subunit channel with the of The with within this region channel with the that these with with the of and the of to to and with wild type β- and these with wild type and mutants that that with wild type suggest that these residues a a within the pore that with and a amino residues to with the that and residues within a domain of the pore region that and the residues the pore and with residues within the channel to the pore to to The of these pore and of and ENaC is with studies with amino residues within pore that to a We a cysteine of that is all three subunits of ENaC and a of with with selected the residues within the β- and γ-subunits and and with the within these is this within cysteine to of ENaC is to a that is within the of the of the that and with by the with selected by and that this that this the that the is with the of a with and a to mouse and a the the suggest that the within the three ENaC subunits is of the within the pore region of that a and studies of with the residues the of that the channel with a of and is the that of the mutants that within the pore region of the suggest that the ENaC pore of with the pore of the of amino residues with and that wild type We that of residues to The mouse a within the of the pore region of a channel with to and that the of a this to the channel these suggest that residues within the of the α-subunit of ENaC a is that to ENaC ENaC within the with within the of the pore domain suggest that residues within this is to the of the We a ENaC is within the pore region suggest that a of within the pore region of that and to the is by the within is and to the of the and to the We that is this the of is to this the of the pore to with and to the with the and We that a second that of a within the this second to residues to and is that the the and of residues the pore and the with residues within the channel to channel and of the putative pore region of the channel suggest of the ENaC pore is by and the pore region the second membrane-spanning to by this the pore the and to a region the that is of to residues We that the pore to second membrane-spanning domain and that the pore this to of a putative suggest that that this We that residues and that residues membrane-spanning is to the pore by and that the is by the is to with a of the channel within the membrane-spanning domain and with residues within the β- and γ-subunits to all residues within the and domain within the region of the channel the the putative membrane-spanning region a of residues membrane-spanning the residues a and the second membrane-spanning domain of the channel is residues and is with to the by pore region with to the pore of the is by a pore that the by region the of the channel region the We that this region within is residues and is to the of the residues region is by the second membrane-spanning is with the that with and these residues the of the pore that that of the with to a channel is with the that the ENaC pore to the pore of the channel and residues homologous within the and γ-subunits that the pore of the channel the that residues of ENaC. residues the by to the of the a the with the the channel pore a to the within the pore and with residues within the of pore and the of the and the and with of a of the within to within mouse and the the of the pore region of the α-subunit to the domain We that this by the of the the that is within the within the putative domain of the channel the and residues preceding the pore region the of α-subunit suggest that a the of suggest that the within the α-subunit of ENaC is by region of the pore residues suggest that within this domain that and with the that that the ENaC pore that a The of cysteine systematically the pore region of the α-subunit of to to the of residues within this region within this pore region of these the of this to residues this a with the and with the with all of the mutants within the of this domain to with the of Previous studies of of ENaC suggest that this is a pore and with the of the pore to within the channel within the with the channel is that selected within the pore region of ENaC the with this We that within the of the pore region cysteine of residues with these with the with a within the pore region of the β- of mouse and and ENaC and residues within β- and within the pore and that is that of the pore region of the and within a region that is the three ENaC subunits The within to and is and studies that of a cysteine this a We this this the of the and within the of within the is that these the of the pore and with the We the of within the pore region of the by the of the pore cysteine residues and and a cysteine with and the and mutants these and the and cysteine within the pore region of the α-subunit channel with the of The with within this region channel with the that these with with the of and the of to to and with wild type β- and these with wild type ENaC. The and mutants that that with wild type suggest that these residues a a within the pore that with and a amino residues to with the that and residues within a domain of the pore region that and the residues the pore and with residues within the channel to the pore to to The of these pore and of and ENaC is with studies with amino residues within pore that to a We a cysteine of that is all three subunits of ENaC and a of with with selected the residues within the β- and γ-subunits and and with the within these is this within The cysteine to of ENaC is to a that is within the of the of the that and with by the with selected by and that this that this the that the is with the of a with and a to mouse and a the the suggest that the within the three ENaC subunits is of the within the pore region of that a and studies of with the residues the of that the channel with a of and is the that of the mutants that within the pore region of the suggest that the ENaC pore of with the pore of the of amino residues with and that wild type We that of residues to The mouse a within the of the pore region of a channel with to and that the of a this to the channel these suggest that residues within the of the α-subunit of ENaC a is that to oocytes. ENaC ENaC within the with within the of the pore domain suggest that residues within this is to the of the We a ENaC is within the pore region suggest that a of within the pore region of that and to the is by the within is and to the of the and to the We that is this the of is to this the of the pore to with and to the with the and We that a second that of a within the this second to residues to and is that the the and of residues the pore and the with residues within the channel to channel and of the putative pore region of the channel suggest of the ENaC pore is by and the pore region the second membrane-spanning to by this the pore the and to a region the that is of to residues We that the pore to second membrane-spanning domain and that the pore this to of a putative suggest that that this We that residues and that residues membrane-spanning is to the pore by and that the is by the is to with a of the channel within the membrane-spanning domain and with residues within the β- and γ-subunits to all residues within the and domain within the region of the channel the the putative membrane-spanning region a of residues membrane-spanning the residues a and the second membrane-spanning domain of the channel is residues and is with to the by pore region with to the pore of the is by a pore that the by region the of the channel region the We that this region within is residues and is to the of the residues region is by the second membrane-spanning is with the that with and these residues the of the pore that that of the with to a channel is with the that the ENaC pore to the pore of the channel and residues homologous within the and γ-subunits that the pore of the channel the that residues of ENaC. residues the by to the of the a the with the the channel pore a to the within the pore and with residues within the of pore and the of the and the and with of a of the within to within mouse and the the of the pore region of the α-subunit to the domain We that this by the of the the that is within the within the putative domain of the channel the and residues preceding the pore region the of α-subunit suggest that a the of suggest that the within the α-subunit of ENaC is by region of the pore residues suggest that within this domain that and with the that that the ENaC pore that a ENaC ENaC within the with within the of the pore domain suggest that residues within this is to the of the We a ENaC is within the pore region suggest that a of within the pore region of that and to the is by the within is and to the of the and to the We that is this the of is to this the of the pore to with and to the with the and We that a second that of a within the this second to residues to and is that the the and of residues the pore and the with residues within the channel to channel and of the putative pore region of the channel suggest of the ENaC pore is by and the pore region the second membrane-spanning to by this the pore the and to a region the that is of to residues We that the pore to second membrane-spanning domain and that the pore this to of a putative suggest that that this We that residues and that residues membrane-spanning is to the pore by and that the is by the is to with a of the channel within the membrane-spanning domain and with residues within the β- and γ-subunits to all residues within the and domain within the region of the channel the the putative membrane-spanning region a of residues membrane-spanning the residues a and the second membrane-spanning domain of the channel is residues and is with to the by pore region with to the pore of the is by a pore that the by region the of the channel region the We that this region within is residues and is to the of the residues region is by the second membrane-spanning is with the that with and these residues the of the pore that that of the with to a channel is with the that the ENaC pore to the pore of the channel and residues homologous within the and γ-subunits that the pore of the channel the that residues of ENaC. residues the by to the of the a the with the the channel pore a to the within the pore and with residues within the of pore and the of the and the and with of a of the within to within mouse and the the of the pore region of the α-subunit to the domain We that this by the of the the that is within the within the putative domain of the channel the and residues preceding the pore region the of α-subunit suggest that a the of suggest that the within the α-subunit of ENaC is by region of the pore residues suggest that within this domain that and with the that that the ENaC pore that a ENaC ENaC within the with within the of the pore domain suggest that residues within this is to the of the We a ENaC is within the pore region suggest that a of within the pore region of that and to the is by the within is and to the of the and to the We that is this the of is to this the of the pore to with and to the with the and We that a second that of a within the this second to residues to and is that the the and of residues the pore and the with residues within the channel to channel and of the putative pore region of the channel suggest of the ENaC pore is by and the pore region the second membrane-spanning to by this the pore the and to a region the that is of to residues We that the pore to second membrane-spanning domain and that the pore this to of a putative suggest that that this We that residues and that residues membrane-spanning is to the pore by and that the is by the is to with a of the channel within the membrane-spanning domain and with residues within the β- and γ-subunits to all residues within the and domain within the region of the channel the the putative membrane-spanning region a of residues membrane-spanning the residues a and the second membrane-spanning domain of the channel is residues and is with to the by pore region with to the pore of the is by a pore that the by region the of the channel region the We that this region within is residues and is to the of the residues region is by the second membrane-spanning is with the that with and these residues the of the pore that that of the with to a channel is with the that the ENaC pore to the pore of the channel and residues homologous within the and γ-subunits that the pore of the channel the that residues of ENaC. residues the by to the of the a the with the the channel pore a to the within the pore and with residues within the of pore and the of the and the and with of a of the within to within mouse and the the of the pore region of the α-subunit to the domain We that this by the of the the that is within the within the putative domain of the channel the and residues preceding the pore region the of α-subunit suggest that a the of suggest that the within the α-subunit of ENaC is by region of the pore residues suggest that within this domain that and with the that that the ENaC pore that a The with within the of the pore domain suggest that residues within this is to the of the We a ENaC is within the pore region suggest that a of within the pore region of that and to the is by the within is and to the of the and to the We that is this the of is to this the of the pore to with and to the with the and We that a second that of a within the this second to residues to and is that the the and of residues the pore and the with residues within the channel to channel and of the putative pore region of the channel suggest of the ENaC pore is by and the pore region the second membrane-spanning to by this the pore the and to a region the that is of to residues We that the pore to second membrane-spanning domain and that the pore this to of a putative suggest that that this We that residues and that residues membrane-spanning is to the pore by and that the is by the is to with a of the channel within the membrane-spanning domain and with residues within the β- and γ-subunits to all residues within the and domain within the region of the channel the the putative membrane-spanning region a of residues membrane-spanning the residues a and the second membrane-spanning domain of the channel is residues and is with to the by pore region with to the pore of the is by a pore that the by region the of the channel region the We that this region within is residues and is to the of the residues region is by the second membrane-spanning is with the that with and these residues the of the pore that that of the with to a channel is with the that the ENaC pore to the pore of the channel We and residues homologous within the and γ-subunits that the pore of the channel the that residues of ENaC. residues the by to the of the a the with the the channel pore a to the within the pore and with residues within the of pore and the of the and the and with of a of the within to within mouse and the the of the pore region of the α-subunit to the domain We that this by the of the the that is within the within the putative domain of the channel the and residues preceding the pore region the of α-subunit suggest that a the of ENaC. suggest that the within the α-subunit of ENaC is by region of the pore residues suggest that within this domain that and with the that that the ENaC pore that a We and the of these studies and the of