Rho Small GTPases Activate the Epithelial Na+ Channel

Abstract

Small G proteins in the Rho family are known to regulate diverse cellular processes, including cytoskeletal organization and cell cycling, and more recently, ion channel activity and activity of phosphatidylinositol 4-phosphate 5-kinase (PI(4)P 5-K). The present study investigates regulation of the epithelial Na+ channel (ENaC) by Rho GTPases. We demonstrate here that RhoA and Rac1 markedly increase ENaC activity. Activation by RhoA was suppressed by the C3 exoenzyme. Inhibition of the downstream RhoA effector Rho kinase, which is necessary for RhoA activation of PI(4)P 5-K, abolished ENaC activation. Similar to RhoA, overexpression of PI(4)P 5-K increased ENaC activity suggesting that production of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in response to RhoA-Rho kinase signaling stimulates ENaC. Supporting this idea, inhibition of phosphatidylinositol 4-kinase, but not the RhoA effector phosphatidylinositol 3-kinase and MAPK cascades, markedly attenuated RhoA-dependent activation of ENaC. RhoA increased ENaC activity by increasing the plasma membrane levels of this channel. We conclude that RhoA activates ENaC via Rho kinase and subsequently activates PI(4)P 5-K with concomitant increases in PI(4,5)P2 levels promoting channel insertion into the plasma membrane. Small G proteins in the Rho family are known to regulate diverse cellular processes, including cytoskeletal organization and cell cycling, and more recently, ion channel activity and activity of phosphatidylinositol 4-phosphate 5-kinase (PI(4)P 5-K). The present study investigates regulation of the epithelial Na+ channel (ENaC) by Rho GTPases. We demonstrate here that RhoA and Rac1 markedly increase ENaC activity. Activation by RhoA was suppressed by the C3 exoenzyme. Inhibition of the downstream RhoA effector Rho kinase, which is necessary for RhoA activation of PI(4)P 5-K, abolished ENaC activation. Similar to RhoA, overexpression of PI(4)P 5-K increased ENaC activity suggesting that production of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in response to RhoA-Rho kinase signaling stimulates ENaC. Supporting this idea, inhibition of phosphatidylinositol 4-kinase, but not the RhoA effector phosphatidylinositol 3-kinase and MAPK cascades, markedly attenuated RhoA-dependent activation of ENaC. RhoA increased ENaC activity by increasing the plasma membrane levels of this channel. We conclude that RhoA activates ENaC via Rho kinase and subsequently activates PI(4)P 5-K with concomitant increases in PI(4,5)P2 levels promoting channel insertion into the plasma membrane. The amiloride-sensitive epithelial Na+ channel (ENaC) 1The abbreviations used are: ENaC, epithelial Na+ channel; hENaC, human ENaC; PI, phosphatidylinositol; MAPK, mitogen-activated protein kinase; CHO, Chinese hamster ovary; pF, picofarad; GDPβS, guanyl-5′-yl thiophosphate; PI3K, phosphatidylinositol 3-kinase; PI4K, phosphatidylinositol 4-kinase; NHS-LC, succinimidyl-6-(biotinamido) hexanoate; PI(4)P 5-K, phosphatidylinositol 4-phosphate 5-kinase; TRP, transient receptor potential.1The abbreviations used are: ENaC, epithelial Na+ channel; hENaC, human ENaC; PI, phosphatidylinositol; MAPK, mitogen-activated protein kinase; CHO, Chinese hamster ovary; pF, picofarad; GDPβS, guanyl-5′-yl thiophosphate; PI3K, phosphatidylinositol 3-kinase; PI4K, phosphatidylinositol 4-kinase; NHS-LC, succinimidyl-6-(biotinamido) hexanoate; PI(4)P 5-K, phosphatidylinositol 4-phosphate 5-kinase; TRP, transient receptor potential. is localized to the luminal plasma membrane of epithelial cells where its activity is rate-limiting for Na+ transport (1Alvarez de la Rosa D. Canessa C.M. Fyfe G.K. Zhang P. Annu. ENaC in Na+ and concomitant epithelial effector for and that and epithelial is that ENaC activity is by regulation of channel to the luminal plasma membrane and channel is known the cellular this PI(4,5)P2 the activity of ion including ENaC, by channel and membrane levels We that G proteins the family ENaC activity by signaling P. with the of this kinase and increasing channel of ENaC by G proteins not and Rac1 are G proteins the Rho family of Similar to G and the Rho with effector Rho kinase to downstream RhoA and Rac1 Rho kinase increase PI(4,5)P2 levels by PI(4)P 5-K P. this and Rho of plasma membrane proteins D. Rho signaling to activity of of ion including and and Rho signaling increases the membrane levels of the transient receptor channel PI(4)P the that RhoA ENaC activity not of ion are to for RhoA and this study Rho ENaC activity and signaling was of this We that RhoA and Rac1 increase ENaC activity with Rho kinase necessary for this activation. the downstream RhoA-Rho kinase effector PI(4)P 5-K ENaC and RhoA is to ENaC activity of PI(4,5)P2 is The of RhoA ENaC was to increase the plasma membrane levels of this channel. We conclude that RhoA-Rho kinase signaling stimulates PI(4)P 5-K to increase PI(4,5)P2 levels with increases in the plasma membrane levels of of and The and human ENaC P. and RhoA, and Rac1 the The PI(4)P 5-K was used in and The was was and cells with and the P. cell of ENaC in cells P. P. ENaC was the amiloride-sensitive Na+ with and and and with GDPβS, the in the was with in which proteins to the C3 was in the cells with and with via to the of family of by of to to and to used to and to ENaC activity cell was and was for cells with ENaC in the and of and cells with and subsequently with for in the with and in and for protein with of with and subsequently in and for in the of to and with in with and are and with ENaC the of Rho ENaC, the family RhoA and human ENaC in cells in the and of and RhoA and was by and channel of Na+ by of in cell ENaC and the of and the of this channel cells ENaC and with of the RhoA GTPases. by to in the and of in the of ENaC in amiloride-sensitive Na+ that not present in cells and P. of and but not RhoA markedly increased ENaC activity. The amiloride-sensitive in in was of RhoA and with increased to and are with RhoA in the ENaC activity. of which activation of RhoA and its by with Rho and effector proteins in in activity with in this was to that RhoA the of RhoA the of RhoA ENaC of Rho by ENaC of Rac1 with ENaC to RhoA in increased ENaC activity of RhoA and its and Rac1 cell RhoA and Rac1 of ENaC activity that this by of the Rho the of RhoA ENaC the C3 which and Rho G proteins the Rho are not for the C3 the C3 is protein that not the cell plasma this protein to the the of C3 in cells and to the C3 ENaC activity in the of of activation of ENaC by of the amiloride-sensitive for and with of RhoA in the and of C3 in the are the in ENaC ENaC ENaC in RhoA ENaC of the in the of in cell ENaC and the ENaC activity that is necessary for RhoA to channel activity. of the of in RhoA-dependent activation of activates ENaC in of cell RhoA the of the and and the with was the of the by of to and to of the amiloride-sensitive for RhoA in the and of in Activation of ENaC by Rho RhoA ENaC signaling via of its downstream effector Rho kinase and the MAPK and was that RhoA with ENaC signaling and ENaC via the which is known to by Rho and to Rho GTPases. of cell by and are cells RhoA in the and of with which is of Rho kinase in cells and RhoA with RhoA-dependent ENaC activity to the MAPK the and RhoA ENaC. We conclude that Rho kinase not and is necessary for RhoA-dependent activation of ENaC suggesting that this RhoA effector is the G protein and the increases ENaC activity via Rho of by and cells with RhoA in the and of with the Rho kinase of the amiloride-sensitive for RhoA and with with of MAPK and and Rho kinase ENaC for RhoA-dependent Activation of which with PI(4)P 5-K, stimulates the activity of this kinase via Rho kinase D. activation of PI(4)P 5-K in response to RhoA-Rho kinase signaling in ENaC activity. this overexpression of PI(4)P 5-K ENaC in to and cells and with PI(4)P are the with by in of PI(4)P 5-K with ENaC channel activity to demonstrate that to RhoA, overexpression of its downstream effector PI(4)P 5-K ENaC activity suggesting that this effector is the G protein and the channel and that the of this kinase the to 5-K increases ENaC of by and cells with and with of the amiloride-sensitive for and with with PI(4)P PI(4)P 5-K and its PI(4,5)P2 in RhoA-dependent activation of ENaC, the of this G protein the channel in the of PI4K, which PI(4,5)P2 ENaC activity in the and of RhoA in cells not and with for to this and and in in ENaC, and was used to the of and of PI(4,5)P2 RhoA-dependent activation of ENaC. activity ENaC was to with with RhoA, channel activity to with that PI(4,5)P2 is necessary for RhoA-dependent activation of the channel with this for RhoA downstream of the in its to the channel. We the is more the in and the of and Rho activation of PI(4)P 5-K and increases in PI(4,5)P2 P. of PI(4,5)P2 RhoA activation of ENaC. of the amiloride-sensitive for and with of RhoA in the and of for with ENaC by the of in the PI(4,5)P2 is known to and of membrane proteins in the plasma membrane D. PI(4,5)P2 downstream of RhoA signaling to ENaC, RhoA and of this ENaC levels in the plasma membrane. with cell cells not and ENaC and with of and The membrane of and cellular of of ENaC are for The membrane was by of cell cells that plasma membrane proteins with plasma membrane proteins by protein but P. and and which is to that P. P. in the of ENaC in the plasma membrane in cells the channel was with and RhoA increased membrane levels to and RhoA the membrane levels of ENaC demonstrate that RhoA increases the membrane levels of ENaC by which is to the increase in channel activity ENaC is with increases ENaC levels in the plasma with the plasma membrane and cell cells protein and and in the and of and membrane proteins with and with ENaC in the plasma membrane in cells ENaC and with and ENaC in the membrane was with of with the of the of the membrane of ENaC was by the cellular of demonstrate for the that ENaC is effector of Rho GTPases. G protein ENaC in and C3 The of RhoA to increase the membrane levels of ENaC. Activation of ENaC in response to Rho was to inhibition of Rho kinase and of PI(4,5)P2 and was by PI(4)P 5-K, suggesting that Rho kinase and its known effector PI(4)P 5-K are the G protein and the channel with production of PI(4,5)P2 in response to activation of PI(4)P 5-K increasing membrane levels of that ENaC in P. activation of ENaC by the for protein that activates ENaC is is for RhoA that the of ENaC activity in the is not to RhoA activation of the channel. is that RhoA increases ENaC levels in the plasma membrane is not to the channel ENaC activity by this of We that the of the of are via the activity of which is to channel We that the of this ENaC is to of channel activity by Rho via increasing channel the channel to channel of Rho ENaC are to for in the that increase channel activity P. the of activation of increase channel Rho increase channel membrane is that to signaling in is for Rho via signaling cytoskeletal organization to insertion and of membrane Rho and Rho kinase are of PI(4)P 5-K, which is rate-limiting for PI(4,5)P2 P. Rho signaling via PI(4)P 5-K and production of PI(4,5)P2 and organization of the receptor and is to ENaC activity D. C.M. C.M. and RhoA via signaling into this ENaC activity. Rho via Rho kinase of to the plasma membrane of which ENaC D. is to that Rho the cellular in and of insertion of insertion of ENaC. Rho to ENaC, insertion of into the plasma membrane is Rho kinase signaling and PI(4)P 5-K PI(4,5)P2 in of channel necessary and for channel D. regulation of ENaC that PI(4,5)P2 channel of PI(4,5)P2 in channel suggesting that this is necessary for channel The are not to ENaC by that not is PI(4,5)P2 for channel but that this signaling the of promoting channel the membrane the of PI(4,5)P2 channel to that the of RhoA via PI(4,5)P2 membrane levels of ENaC is to organization and of that ENaC is to in that Rho and PI(4,5)P2 signaling and membrane The amiloride-sensitive epithelial Na+ channel (ENaC) 1The abbreviations used are: ENaC, epithelial Na+ channel; hENaC, human ENaC; PI, phosphatidylinositol; MAPK, mitogen-activated protein kinase; CHO, Chinese hamster ovary; pF, picofarad; GDPβS, guanyl-5′-yl thiophosphate; PI3K, phosphatidylinositol 3-kinase; PI4K, phosphatidylinositol 4-kinase; NHS-LC, succinimidyl-6-(biotinamido) hexanoate; PI(4)P 5-K, phosphatidylinositol 4-phosphate 5-kinase; TRP, transient receptor potential.1The abbreviations used are: ENaC, epithelial Na+ channel; hENaC, human ENaC; PI, phosphatidylinositol; MAPK, mitogen-activated protein kinase; CHO, Chinese hamster ovary; pF, picofarad; GDPβS, guanyl-5′-yl thiophosphate; PI3K, phosphatidylinositol 3-kinase; PI4K, phosphatidylinositol 4-kinase; NHS-LC, succinimidyl-6-(biotinamido) hexanoate; PI(4)P 5-K, phosphatidylinositol 4-phosphate 5-kinase; TRP, transient receptor potential. is localized to the luminal plasma membrane of epithelial cells where its activity is rate-limiting for Na+ transport (1Alvarez de la Rosa D. Canessa C.M. Fyfe G.K. Zhang P. Annu. ENaC in Na+ and concomitant epithelial effector for and that and epithelial is that ENaC activity is by regulation of channel to the luminal plasma membrane and channel is known the cellular this PI(4,5)P2 the activity of ion including ENaC, by channel and membrane levels We that G proteins the family ENaC activity by signaling P. with the of this kinase and increasing channel of ENaC by G proteins not RhoA and Rac1 are G proteins the Rho family of Similar to G and the Rho with effector Rho kinase to downstream RhoA and Rac1 Rho kinase increase PI(4,5)P2 levels by PI(4)P 5-K P. this and Rho of plasma membrane proteins D. Rho signaling to activity of of ion including and and Rho signaling increases the membrane levels of the transient receptor channel PI(4)P the that RhoA ENaC activity not of ion are to for RhoA and this The study Rho ENaC activity and signaling was of this We that RhoA and Rac1 increase ENaC activity with Rho kinase necessary for this activation. the downstream RhoA-Rho kinase effector PI(4)P 5-K ENaC and RhoA is to ENaC activity of PI(4,5)P2 is The of RhoA ENaC was to increase the plasma membrane levels of this channel. We conclude that RhoA-Rho kinase signaling stimulates PI(4)P 5-K to increase PI(4,5)P2 levels with increases in the plasma membrane levels of ENaC. of and The and human ENaC P. and RhoA, and Rac1 the The PI(4)P 5-K was used in and The was was and cells with and the P. cell of ENaC in cells P. P. ENaC was the amiloride-sensitive Na+ with and and and with GDPβS, the in the was with in which proteins to the C3 was in the cells with and with via to the of family of by of to to and to used to and to ENaC activity cell was and was for cells with ENaC in the and of and cells with and subsequently with for in the with and in and for protein with of with and subsequently in and for in the of to and with in with and are and with of and The and human ENaC P. and RhoA, and Rac1 the The PI(4)P 5-K was used in and The was was and cells with and the P. cell of ENaC in cells P. P. ENaC was the amiloride-sensitive Na+ with and and and with GDPβS, the in the was with in which proteins to the C3 was in the cells with and with via to the of family of by of to to and to used to and to ENaC activity cell was and was for cells with ENaC in the and of and cells with and subsequently with for in the with and in and for protein with of with and subsequently in and for in the of to and with in with and are and with ENaC the of Rho ENaC, the family RhoA and human ENaC in cells in the and of and RhoA and was by and channel of Na+ by of in cell ENaC and the of and the of this channel cells ENaC and with of the RhoA GTPases. by to in the and of in the of ENaC in amiloride-sensitive Na+ that not present in cells and P. of and but not RhoA markedly increased ENaC activity. The amiloride-sensitive in in was of RhoA and with increased to and are with RhoA in the ENaC activity. of which activation of RhoA and its by with Rho and effector proteins in in activity with in this was to that RhoA the of RhoA the of RhoA ENaC of Rho by ENaC of Rac1 with ENaC to RhoA in increased ENaC activity of RhoA and its and Rac1 cell RhoA and Rac1 of ENaC activity that this by of the Rho the of RhoA ENaC the C3 which and Rho G proteins the Rho are not for the C3 the C3 is protein that not the cell plasma this protein to the the of C3 in cells and to the C3 ENaC activity in the of of ENaC in RhoA ENaC of the in the of in cell ENaC and the ENaC activity that is necessary for RhoA to channel activity. of the of in RhoA-dependent activation of activates ENaC in of cell RhoA the of the and and the with was the of the by of to and to of the amiloride-sensitive for RhoA in the and of in Activation of ENaC by Rho RhoA ENaC signaling via of its downstream effector Rho kinase and the MAPK and was that RhoA with ENaC signaling and ENaC via the which is known to by Rho and to Rho GTPases. of cell by and are cells RhoA in the and of with which is of Rho kinase in cells and RhoA with RhoA-dependent ENaC activity to the MAPK the and RhoA ENaC. We conclude that Rho kinase not and is necessary for RhoA-dependent activation of ENaC suggesting that this RhoA effector is the G protein and the increases ENaC activity via Rho of by and cells with RhoA in the and of with the Rho kinase of the amiloride-sensitive for RhoA and with with of MAPK and and Rho kinase ENaC for RhoA-dependent Activation of which with PI(4)P 5-K, stimulates the activity of this kinase via Rho kinase D. activation of PI(4)P 5-K in response to RhoA-Rho kinase signaling in ENaC activity. this overexpression of PI(4)P 5-K ENaC in to and cells and with PI(4)P are the with by in of PI(4)P 5-K with ENaC channel activity to demonstrate that to RhoA, overexpression of its downstream effector PI(4)P 5-K ENaC activity suggesting that this effector is the G protein and the channel and that the of this kinase the to 5-K increases ENaC of by and cells with and with of the amiloride-sensitive for and with with PI(4)P PI(4)P 5-K and its PI(4,5)P2 in RhoA-dependent activation of ENaC, the of this G protein the channel in the of PI4K, which PI(4,5)P2 ENaC activity in the and of RhoA in cells not and with for to this and and in in ENaC, and was used to the of and of PI(4,5)P2 RhoA-dependent activation of ENaC. activity ENaC was to with with RhoA, channel activity to with that PI(4,5)P2 is necessary for RhoA-dependent activation of the channel with this for RhoA downstream of the in its to the channel. We the is more the in and the of and Rho activation of PI(4)P 5-K and increases in PI(4,5)P2 P. of PI(4,5)P2 RhoA activation of ENaC. of the amiloride-sensitive for and with of RhoA in the and of for with ENaC by the of in the PI(4,5)P2 is known to and of membrane proteins in the plasma membrane D. PI(4,5)P2 downstream of RhoA signaling to ENaC, RhoA and of this ENaC levels in the plasma membrane. with cell cells not and ENaC and with of and The membrane of and cellular of of ENaC are for The membrane was by of cell cells that plasma membrane proteins with plasma membrane proteins by protein but P. and and which is to that P. P. in the of ENaC in the plasma membrane in cells the channel was with and RhoA increased membrane levels to and RhoA the membrane levels of ENaC demonstrate that RhoA increases the membrane levels of ENaC by which is to the increase in channel activity ENaC is with increases ENaC levels in the plasma with the plasma membrane and cell cells protein and and in the and of and membrane proteins with and with ENaC in the plasma membrane in cells ENaC and with and ENaC in the membrane was with of with the of the of the membrane of ENaC was by the cellular of Rho ENaC the of Rho ENaC, the family RhoA and human ENaC in cells in the and of and RhoA and was by and channel of Na+ by of in cell ENaC and the of and the of this channel cells ENaC and with of the RhoA GTPases. by to in the and of in the of ENaC in amiloride-sensitive Na+ that not present in cells and P. of and but not RhoA markedly increased ENaC activity. The amiloride-sensitive in in was of RhoA and with increased to and are with RhoA in the ENaC activity. of which activation of RhoA and its by with Rho and effector proteins in in activity with in this was to that RhoA the of RhoA We the of RhoA ENaC of Rho by ENaC of Rac1 with ENaC to RhoA in increased ENaC activity of RhoA and its and Rac1 cell RhoA and Rac1 of ENaC activity that this by of the Rho We the of RhoA ENaC the C3 which and Rho G proteins the Rho are not for the C3 the C3 is protein that not the cell plasma this protein to the the of C3 in cells and to the C3 ENaC activity in the of of RhoA ENaC in RhoA ENaC of the in the of in cell ENaC and the ENaC activity that is necessary for RhoA to channel activity. of the of in RhoA-dependent activation of ENaC. RhoA Activation of ENaC by Rho RhoA ENaC signaling via of its downstream effector Rho kinase and the MAPK and was that RhoA with ENaC signaling and ENaC via the which is known to by Rho and to Rho GTPases. of cell by and are cells RhoA in the and of with which is of Rho kinase in cells and RhoA with RhoA-dependent ENaC activity to the MAPK the and RhoA ENaC. We conclude that Rho kinase not and is necessary for RhoA-dependent activation of ENaC suggesting that this RhoA effector is the G protein and the channel. PI(4,5)P2 for RhoA-dependent Activation of which with PI(4)P 5-K, stimulates the activity of this kinase via Rho kinase D. activation of PI(4)P 5-K in response to RhoA-Rho kinase signaling in ENaC activity. this overexpression of PI(4)P 5-K ENaC in to and cells and with PI(4)P are the with by in of PI(4)P 5-K with ENaC channel activity to demonstrate that to RhoA, overexpression of its downstream effector PI(4)P 5-K ENaC activity suggesting that this effector is the G protein and the channel and that the of this kinase the to ENaC. PI(4)P 5-K and its PI(4,5)P2 in RhoA-dependent activation of ENaC, the of this G protein the channel in the of PI4K, which PI(4,5)P2 ENaC activity in the and of RhoA in cells not and with for to this and and in in ENaC, and was used to the of and of PI(4,5)P2 RhoA-dependent activation of ENaC. activity ENaC was to with with RhoA, channel activity to with that PI(4,5)P2 is necessary for RhoA-dependent activation of the channel with this for RhoA downstream of the in its to the channel. We the is more the in and the of and Rho activation of PI(4)P 5-K and increases in PI(4,5)P2 P. RhoA ENaC by the of in the PI(4,5)P2 is known to and of membrane proteins in the plasma membrane D. PI(4,5)P2 downstream of RhoA signaling to ENaC, RhoA and of this ENaC levels in the plasma membrane. with cell cells not and ENaC and with of and The membrane of and cellular of of ENaC are for The membrane was by of cell cells that plasma membrane proteins with plasma membrane proteins by protein but P. and and which is to that P. P. in the of ENaC in the plasma membrane in cells the channel was with and RhoA increased membrane levels to and RhoA the membrane levels of ENaC demonstrate that RhoA increases the membrane levels of ENaC by which is to the increase in channel activity ENaC is with demonstrate for the that ENaC is effector of Rho GTPases. G protein ENaC in and C3 The of RhoA to increase the membrane levels of ENaC. Activation of ENaC in response to Rho was to inhibition of Rho kinase and of PI(4,5)P2 and was by PI(4)P 5-K, suggesting that Rho kinase and its known effector PI(4)P 5-K are the G protein and the channel with production of PI(4,5)P2 in response to activation of PI(4)P 5-K increasing membrane levels of that ENaC in P. activation of ENaC by the for protein that activates ENaC is is for RhoA that the of ENaC activity in the is not to RhoA activation of the channel. is that RhoA increases ENaC levels in the plasma membrane is not to the channel ENaC activity by this of We that the of the of are via the activity of which is to channel We that the of this ENaC is to of channel activity by Rho via increasing channel the channel to channel of Rho ENaC are to for in the that increase channel activity P. the of activation of increase channel Rho increase channel membrane is that to signaling in is for Rho via signaling cytoskeletal organization to insertion and of membrane Rho and Rho kinase are of PI(4)P 5-K, which is rate-limiting for PI(4,5)P2 P. Rho signaling via PI(4)P 5-K and production of PI(4,5)P2 and organization of the receptor and is to ENaC activity D. C.M. C.M. and RhoA via signaling into this ENaC activity. Rho via Rho kinase of to the plasma membrane of which ENaC D. is to that Rho the cellular in and of insertion of insertion of ENaC. Rho to ENaC, insertion of into the plasma membrane is Rho kinase signaling and PI(4)P 5-K PI(4,5)P2 in of channel necessary and for channel D. regulation of ENaC that PI(4,5)P2 channel of PI(4,5)P2 in channel suggesting that this is necessary for channel The are not to ENaC by that not is PI(4,5)P2 for channel but that this signaling the of promoting channel the membrane the of PI(4,5)P2 channel to that the of RhoA via PI(4,5)P2 membrane levels of ENaC is to organization and of that ENaC is to in that Rho and PI(4,5)P2 signaling and membrane The demonstrate for the that ENaC is effector of Rho GTPases. G protein ENaC in and C3 The of RhoA to increase the membrane levels of ENaC. Activation of ENaC in response to Rho was to inhibition of Rho kinase and of PI(4,5)P2 and was by PI(4)P 5-K, suggesting that Rho kinase and its known effector PI(4)P 5-K are the G protein and the channel with production of PI(4,5)P2 in response to activation of PI(4)P 5-K increasing membrane levels of ENaC. that ENaC in P. activation of ENaC by the for protein that activates ENaC is is for RhoA that the of ENaC activity in the is not to RhoA activation of the channel. is that RhoA increases ENaC levels in the plasma membrane is not to the channel ENaC activity by this of We that the of the of are via the activity of which is to channel We that the of this ENaC is to of channel activity by Rho via increasing channel the channel to channel The of Rho ENaC are to for in the that increase channel activity P. the of activation of increase channel Rho increase channel membrane is that to signaling in is for Rho via signaling cytoskeletal organization to insertion and of membrane Rho and Rho kinase are of PI(4)P 5-K, which is rate-limiting for PI(4,5)P2 P. Rho signaling via PI(4)P 5-K and production of PI(4,5)P2 and organization of the receptor and is to ENaC activity D. C.M. C.M. and RhoA via signaling into this ENaC activity. Rho via Rho kinase of to the plasma membrane of which ENaC D. is to that Rho the cellular in and of insertion of insertion of ENaC. Rho to ENaC, insertion of into the plasma membrane is Rho kinase signaling and PI(4)P 5-K PI(4,5)P2 in of channel necessary and for channel D. regulation of ENaC that PI(4,5)P2 channel of PI(4,5)P2 in channel suggesting that this is necessary for channel The are not to ENaC by that not is PI(4,5)P2 for channel but that this signaling the of promoting channel the membrane the of PI(4,5)P2 channel to that the of RhoA via PI(4,5)P2 membrane levels of ENaC is to organization and of that ENaC is to in that Rho and PI(4,5)P2 signaling and membrane