Participation of the Chaperone Hsc70 in the Trafficking and Functional Expression of ASIC2 in Glioma Cells

High-grade glioma cells express subunits of the ENaC/Deg superfamily, including members of ASIC subfamily. Our previous work has shown that glioma cells exhibit a basally active cation current, which is not present in low-grade tumor cells or normal astrocytes, and that can be blocked by amiloride. When ASIC2 is present within the channel complex in the plasma membrane, the channel is rendered non-functional because of inherent negative effectors that require ASIC2. We have previously shown that high-grade glioma cells functionally express this current because of the lack of ASIC2 in the plasma membrane. We now hypothesize that ASIC2 trafficking in glioma cells is regulated by a specific chaperone protein, namely Hsc70. Our results demonstrated that Hsc70 co-immunoprecipitates with ASIC2 and that it is overexpressed in glioma cells as compared with normal astrocytes. In contrast, there was no difference in the expression of calnexin, which also co-immunoprecipitates with ASIC2. In addition, glycerol and sodium 4-phenylbutyrate reduced the amount of Hsc70 expressed in glioma cells to in normal astrocytes. of Hsc70 the current, and ASIC2 expression in glioma results Hsc70 and ASIC2 that the of ASIC2 in the of glioma also that Hsc70 expression of a high-grade glioma to a normal High-grade glioma cells express subunits of the ENaC/Deg superfamily, including members of ASIC subfamily. Our previous work has shown that glioma cells exhibit a basally active cation current, which is not present in low-grade tumor cells or normal astrocytes, and that can be blocked by amiloride. When ASIC2 is present within the channel complex in the plasma membrane, the channel is rendered non-functional because of inherent negative effectors that require ASIC2. We have previously shown that high-grade glioma cells functionally express this current because of the lack of ASIC2 in the plasma membrane. We now hypothesize that ASIC2 trafficking in glioma cells is regulated by a specific chaperone protein, namely Hsc70. Our results demonstrated that Hsc70 co-immunoprecipitates with ASIC2 and that it is overexpressed in glioma cells as compared with normal astrocytes. In contrast, there was no difference in the expression of calnexin, which also co-immunoprecipitates with ASIC2. In addition, glycerol and sodium 4-phenylbutyrate reduced the amount of Hsc70 expressed in glioma cells to in normal astrocytes. of Hsc70 the current, and ASIC2 expression in glioma results Hsc70 and ASIC2 that the of ASIC2 in the of glioma also that Hsc70 expression of a high-grade glioma to a normal sodium sodium the of of the or cells have normal and In addition, to normal Our has that members of the channel and in the of as have that cells high-grade express a basally current that is to amiloride. ENaC/Deg members the ASIC subfamily. We that the glioma cation current is by ASIC and including ASIC2 as and can be glioma cells cation channel is a specific of high-grade In normal astrocytes, no current can be have shown that ASIC and subunits the difference glioma cells and normal astrocytes, have previously shown that the current in is to the of plasma expression of ASIC2 in plasma ASIC2 expression is as in because can ASIC2 in of the glioma that have or as a in trafficking of this to in the plasma membrane, as ASIC2 can be in is not the plasma the of this work was to and the that ASIC2 and it is in that of the or the sodium 4-phenylbutyrate results in expression of a in and and of the current in the glioma previous that a of ASIC2 to to the the active cation current in glioma cells and to the and of the glioma of trafficking that glycerol and and it to the that the by was to of the that trafficking of by it to with in the and and is as the Hsc70 to it can the to Hsc70 a with the and and Hsc70 to channel as and and trafficking including and the present the that ASIC2 trafficking is regulated by with Hsc70 in a of and demonstrated that expression of Hsc70 is in glioma and that this chaperone ASIC2. Our results demonstrated that of Hsc70 is with of ASIC2 to the glioma and of the active cation of normal astrocytes, cells and cells the of cells and cells of and cells in with cells in a and the or the and the and and cells with the Hsc70 with the or a a of with the ASIC2 cation channel with the or a a of and the or the and of normal astrocytes, and with and cells with in with was and with a of the was with of the a by the of of with and with and of the was demonstrated by of the and with blocked with and with the in with and was by the of the cells in and with Hsc70 or Hsc70 and by plasma was to the to the plasma the We have previously shown that this results in a in plasma plasma was in in and plasma was the or cells with Hsc70 or Hsc70 with ASIC2 or ASIC2 in a with and and the of the cells to the of the to and with and the plasma of of normal astrocytes, a was to the the and the cells by to the the to the a was to the within the the a to the the the of was and by the to and negative cells a of and to of to of to the of in the of as and as a the of the and in the a was cells and normal in and with of and Hsc70 or ASIC2 or ASIC2 to was by expression the cells was in with in the with of the to the and the the of to cells of the membrane, cells the can be cells and in with cells to the cells was to the normal cells that the to the of the in a of was and have previously shown that glioma cells exhibit a basally active cation current because trafficking of not to the is and ASIC2 that exhibit of to chaperone with ASIC2 not that exhibit and ASIC2. and with ASIC subunits and to with ASIC2 this chaperone not to to not with or ASIC2 in ASIC2 with Hsc70 expression in glioma cells compared with normal astrocytes. of glioma cells in and normal and of Hsc70 to that Hsc70 is overexpressed in glioma and cells as compared with normal In contrast, of calnexin, in is overexpressed in glioma expression of Hsc70 in glioma cells as compared with normal astrocytes. of with and that expression of in glioma cells is not that in normal astrocytes. the of expression of chaperone to as by to expression in normal astrocytes. and of Hsc70 in has previously demonstrated that and glycerol expression of Hsc70 in cells this also in glioma be to this in of Hsc70. of normal astrocytes, of and cells with and glycerol of Hsc70 to that Hsc70 expression was by and In contrast, of not by of We have previously shown that and glycerol ASIC2 trafficking to the and the current of glioma cells to the that the Hsc70 and ASIC2. with and with of normal astrocytes, and of cells with and glycerol with and glycerol reduced the amount of Hsc70 that was with In cells and in the the of in the of and glycerol was to in normal and glycerol expression of Hsc70 in and cells to the as that in normal expression of with or and with of the normal with with the normal with with of cells in of in and the of expression of or Hsc70 to as by of normal astrocytes, or with and with normal with with with with and is to of the Hsc70 expression as by of of Hsc70 in with and glycerol Hsc70 expression We to the that the specific of Hsc70 expression plasma expression of ASIC2 and the current as as glioma and cells with Hsc70 and a Hsc70 as a negative and with and Hsc70 reduced expression of Hsc70 protein, the Hsc70 no in glioma of of and with and cells with Hsc70 cells with Hsc70 cells in of the of ASIC2 to the cells with Hsc70 or Hsc70 as previously by and with of Hsc70 was with the of ASIC2 in the plasma of cells of Hsc70 in glioma cells expression of ASIC2. plasma as and by with to amount of in of the with ASIC2 expression the plasma a as by of Hsc70 with of in have shown that the current of the glioma cells results the of ASIC2 expression the plasma membrane. of Hsc70 is with of ASIC2 the plasma membrane, that Hsc70 also the cation and of cells with Hsc70 or a by and was with Hsc70 the current in and current in normal and of and with Hsc70 and with Hsc70 or a Hsc70 by and and in of in the with difference current and current in the of the of cells and of and with Hsc70 and with Hsc70 or a Hsc70 by and and in of in the with difference current and current in the of the of cells and of normal with ASIC2 with ASIC2 or a ASIC2 and and in of in the with difference current and current in the of the of cells of ASIC2 the of in also to that ASIC2 is with the of the current in normal astrocytes. normal with ASIC2 or a ASIC2 as a negative and with ASIC2 or because the amount of present in a it to the channel by ASIC2 reduced expression of ASIC2 the ASIC2 no ASIC2 was with ASIC2 the of current in normal in normal astrocytes. and of of normal with or with ASIC2 or ASIC2 by cells in that ASIC2 ASIC2 expression ASIC2 of cells by of and normal exhibit of and ASIC2 is of the glioma and ASIC2 trafficking is by a in Hsc70 that in glioma cells of Hsc70 be with of a of ASIC2 be with of in normal astrocytes. cells with Hsc70 or Hsc70 and as a with ASIC2 or ASIC2 and cells and the with the of cells that have the can be to by of Hsc70 the of in In contrast, of or the Hsc70 not have of ASIC2 in normal of of or the ASIC2 not the of normal of Hsc70 of cells and ASIC2 of normal astrocytes. with as a and Hsc70 or ASIC Hsc70 or ASIC2 to that the and a that to of a that the channel be in that it is of a of ASIC and including and regulated trafficking as a of plasma of the in is and in the subunits there is a of that is In to is trafficking of to and the plasma membrane. is to the of trafficking of ASIC2 in this channel and of to is that the cation current, present in glioma cells not in normal astrocytes, a to the glioma by to cells by of and the and the of and in as is to and be to the of and is in this that hypothesize that the glioma channel a and it be that of the current glioma and previous that in of glioma and have ASIC2 is in the and is the plasma membrane. In the ASIC2 is not because of within the ASIC2 We hypothesize that of the tumor and is to the in expression of ASIC2. is of that with of and the of is to by the a of and to be and is the as with Hsc70 is to be a in a of by the Hsc70 in the the of of of a of ASIC2 has not that that ASIC2 is also a of Hsc70. chaperone is in there has and is the of ASIC Our results the of in glioma cells and in normal astrocytes. is to ASIC as it is with of the tumor results in this that not with the and with the that of this ASIC by Hsc70 ASIC2 expression the plasma membrane. chaperone is in glioma cells as compared with normal astrocytes. We that in the of glioma that express the is to in the plasma membrane. in Hsc70 expression also with ASIC2 and it ASIC2 of the channel the of plasma is a active cation that the of Hsc70 the active current in a with because of Hsc70 of ASIC2 to the membrane, of the basally active current in glioma and a in glioma In contrast, of ASIC2 in normal the current and the of normal astrocytes. results with that trafficking of ASIC2 to the plasma of glioma cells the cation current and that this current is of the glioma sodium sodium the of of the or cells have normal and In addition, to normal Our has that members of the channel and in the of as have that cells high-grade express a basally current that is to amiloride. ENaC/Deg members the ASIC subfamily. We that the glioma cation current is by ASIC and including ASIC2 as and can be glioma cells cation channel is a specific of high-grade In normal astrocytes, no current can be have shown that ASIC and subunits In the difference glioma cells and normal astrocytes, have previously shown that the current in is to the of plasma expression of ASIC2 in plasma ASIC2 expression is as in because can ASIC2 in of the glioma that have or as a in trafficking of this to in the plasma membrane, as ASIC2 can be in is not the plasma the of this work was to and the that ASIC2 and it is in Our that of the or the sodium 4-phenylbutyrate results in expression of a in and and of the current in the glioma previous that a of ASIC2 to to the the active cation current in glioma cells and to the and of the glioma of trafficking that glycerol and and it to the that the by was to of the that trafficking of by it to with Hsc70. Hsc70 in the and and is as the Hsc70 to it can the to Hsc70 a with the and and Hsc70 to channel as and and trafficking including and In the present the that ASIC2 trafficking is regulated by with Hsc70 in a of and demonstrated that expression of Hsc70 is in glioma and that this chaperone ASIC2. Our results demonstrated that of Hsc70 is with of ASIC2 to the glioma and of the active cation of normal astrocytes, cells and cells the of cells and cells of and cells in with cells in a and the or the and the and and cells with the Hsc70 with the or a a of with the ASIC2 cation channel with the or a a of and the or the and of normal astrocytes, and with and cells with in with was and with a of the was with of the a by the of of with and with and of the was demonstrated by of the and with blocked with and with the in with and was by the of the cells in and with Hsc70 or Hsc70 and by plasma was to the to the plasma the We have previously shown that this results in a in plasma plasma was in in and plasma was the or cells with Hsc70 or Hsc70 with ASIC2 or ASIC2 in a with and and the of the cells to the of the to and with and the plasma of of normal astrocytes, a was to the the and the cells by to the the to the a was to the within the the a to the the the of was and by the to and negative cells a of and to of to of to the of in the of as and as a the of the and in the a was cells and normal in and with of and Hsc70 or ASIC2 or ASIC2 to was by expression the cells was in with in the with of the to the and the the of to cells of the membrane, cells the can be cells and in with cells to the cells was to the normal cells that the to the of the in a of was of normal astrocytes, cells and cells the of cells and cells of and cells in with cells in a and the or the and the and and cells with the Hsc70 with the or a a of with the ASIC2 cation channel with the or a a of and the or the and of normal astrocytes, and with and cells with in with was and with a of the was with of the a by the of of with and with and of the was demonstrated by of the and with blocked with and with the in with and was by the of the cells in and with Hsc70 or Hsc70 and by plasma was to the to the plasma the We have previously shown that this results in a in plasma plasma was in in and plasma was the or cells with Hsc70 or Hsc70 with ASIC2 or ASIC2 in a with and and the of the cells to the of the to and with and the plasma of of normal astrocytes, a was to the the and the cells by to the the to the a was to the within the the a to the the the of was and by the to and negative cells a of and to of to of to the of in the of as and as a the of the and in the a was cells and normal in and with of and Hsc70 or ASIC2 or ASIC2 to was by expression the cells was in with in the with of the to the and the the of to cells of the membrane, cells the can be cells and in with cells to the cells was to the normal cells that the to the of the in a of was and have previously shown that glioma cells exhibit a basally active cation current because trafficking of not to the is and ASIC2 that exhibit of to chaperone with ASIC2 not that exhibit and ASIC2. and with ASIC subunits and to with ASIC2 this chaperone not to to not with or ASIC2 in ASIC2 with Hsc70 expression in glioma cells compared with normal astrocytes. of glioma cells in and normal and of Hsc70 to that Hsc70 is overexpressed in glioma and cells as compared with normal In contrast, of calnexin, in and of Hsc70 in has previously demonstrated that and glycerol expression of Hsc70 in cells this also in glioma be to this in of Hsc70. of normal astrocytes, of and cells with and glycerol of Hsc70 to that Hsc70 expression was by and In contrast, of not by of We have previously shown that and glycerol ASIC2 trafficking to the and the current of glioma cells to the that the Hsc70 and ASIC2. with and with of normal astrocytes, and of cells with and glycerol with and glycerol reduced the amount of Hsc70 that was with In cells and in the the of in the of and glycerol was to in normal and glycerol expression of Hsc70 in and cells to the as that in normal expression of with or and with of the normal with with the normal with with of cells in of in and the of expression of or Hsc70 to as by of normal astrocytes, or with and with normal with with with with and is to of the Hsc70 expression as by of of Hsc70 in with and glycerol Hsc70 expression We to the that the specific of Hsc70 expression plasma expression of ASIC2 and the current as as glioma and cells with Hsc70 and a Hsc70 as a negative and with and Hsc70 reduced expression of Hsc70 protein, the Hsc70 no in glioma of of and with and cells with Hsc70 cells with Hsc70 cells in of the of ASIC2 to the cells with Hsc70 or Hsc70 as previously by and with of Hsc70 was with the of ASIC2 in the plasma of cells of Hsc70 in glioma cells expression of ASIC2. plasma as and by with to amount of in of the with ASIC2 expression the plasma a as by of Hsc70 with of in have shown that the current of the glioma cells results the of ASIC2 expression the plasma membrane. of Hsc70 is with of ASIC2 the plasma membrane, that Hsc70 also the cation and of cells with Hsc70 or a by and was with Hsc70 the current in and current in normal and of and with Hsc70 and with Hsc70 or a Hsc70 by and and in of in the with difference current and current in the of the of cells and of and with Hsc70 and with Hsc70 or a Hsc70 by and and in of in the with difference current and current in the of the of cells and of normal with ASIC2 with ASIC2 or a ASIC2 and and in of in the with difference current and current in the of the of cells of ASIC2 the of in also to that ASIC2 is with the of the current in normal astrocytes. normal with ASIC2 or a ASIC2 as a negative and with ASIC2 or because the amount of present in a it to the channel by ASIC2 reduced expression of ASIC2 the ASIC2 no ASIC2 was with ASIC2 the of current in normal in normal astrocytes. and of of normal with or with ASIC2 or ASIC2 by cells in that ASIC2 ASIC2 expression ASIC2 of cells by of and normal exhibit of and ASIC2 is of the glioma and ASIC2 trafficking is by a in Hsc70 that in glioma cells of Hsc70 be with of a of ASIC2 be with of in normal astrocytes. cells with Hsc70 or Hsc70 and as a with ASIC2 or ASIC2 and cells and the with the of cells that have the can be to by of Hsc70 the of in In contrast, of or the Hsc70 not have of ASIC2 in normal of of or the ASIC2 not the of normal of Hsc70 of cells and ASIC2 of normal astrocytes. with as a and Hsc70 or ASIC Hsc70 or ASIC2 to that the and a that to of a and have previously shown that glioma cells exhibit a basally active cation current because trafficking of not to the is and ASIC2 that exhibit of to chaperone with ASIC2 not that exhibit and ASIC2. and with ASIC subunits and to with ASIC2 this chaperone not to to not with or ASIC2 Hsc70 in ASIC2 with Hsc70 expression in glioma cells compared with normal astrocytes. of glioma cells in and normal and of Hsc70 to that Hsc70 is overexpressed in glioma and cells as compared with normal In contrast, of calnexin, in and of Hsc70 in has previously demonstrated that and glycerol expression of Hsc70 in cells this also in glioma be to this in of Hsc70. of normal astrocytes, of and cells with and glycerol of Hsc70 to that Hsc70 expression was by and In contrast, of not by of We have previously shown that and glycerol ASIC2 trafficking to the and the current of glioma cells to the that the Hsc70 and ASIC2. with and with of normal astrocytes, and of cells with and glycerol with and glycerol reduced the amount of Hsc70 that was with In cells and in the the of in the of and glycerol was to in normal astrocytes. of Hsc70 in with and glycerol Hsc70 expression We to the that the specific of Hsc70 expression plasma expression of ASIC2 and the current as as glioma and cells with Hsc70 and a Hsc70 as a negative and with and Hsc70 reduced expression of Hsc70 protein, the Hsc70 no Hsc70 the of ASIC2 to the cells with Hsc70 or Hsc70 as previously by and with of Hsc70 was with the of ASIC2 in the plasma of cells of Hsc70 with of in have shown that the current of the glioma cells results the of ASIC2 expression the plasma membrane. of Hsc70 is with of ASIC2 the plasma membrane, that Hsc70 also the cation and of cells with Hsc70 or a by and was with Hsc70 the current in and current in normal of ASIC2 the of in also to that ASIC2 is with the of the current in normal astrocytes. normal with ASIC2 or a ASIC2 as a negative and with ASIC2 or because the amount of present in a it to the channel by ASIC2 reduced expression of ASIC2 the ASIC2 no ASIC2 was with ASIC2 the of current in normal Hsc70 ASIC2 of cells by of and normal exhibit of and ASIC2 is of the glioma and ASIC2 trafficking is by a in Hsc70 that in glioma cells of Hsc70 be with of a of ASIC2 be with of in normal astrocytes. cells with Hsc70 or Hsc70 and as a with ASIC2 or ASIC2 and cells and the with the of cells that have the can be to by of Hsc70 the of in In contrast, of or the Hsc70 not have of ASIC2 in normal of of or the ASIC2 not the of normal astrocytes. that the channel be in that it is of a of ASIC and including and regulated trafficking as a of plasma of the in is and in the subunits there is a of that is In to is trafficking of to and the plasma membrane. is to the of trafficking of ASIC2 in this channel and of to is that the cation current, present in glioma cells not in normal astrocytes, a to the glioma by to cells by of and the and the of and in as is to and be to the of and is in this that hypothesize that the glioma channel a and it be that of the current glioma and previous that in of glioma and have ASIC2 is in the and is the plasma membrane. In the ASIC2 is not because of within the ASIC2 We hypothesize that of the tumor and is to the in expression of ASIC2. is of that with of and the of is to by the a of and to be and is the as with Hsc70 is to be a in a of by the Hsc70 in the the of of of a of ASIC2 has not that that ASIC2 is also a of Hsc70. chaperone is in there has and is the of ASIC Our results the of in glioma cells and in normal astrocytes. is to ASIC as it is with of the tumor results in this that not with the and with the that of this ASIC by Hsc70 ASIC2 expression the plasma membrane. chaperone is in glioma cells as compared with normal astrocytes. We that in the of glioma that express the is to in the plasma membrane. in Hsc70 expression also with ASIC2 and it ASIC2 of the channel the of plasma is a active cation that the of Hsc70 the active current in a with because of Hsc70 of ASIC2 to the membrane, of the basally active current in glioma and a in glioma In contrast, of ASIC2 in normal the current and the of normal astrocytes. results with that trafficking of ASIC2 to the plasma of glioma cells the cation current and that this current is of the glioma Our that the channel be in that it is of a of ASIC and including and regulated trafficking as a of plasma of the in is and in the subunits there is a of that is In to is trafficking of to and the plasma membrane. is to the of trafficking of ASIC2 in this channel and of to is that the cation current, present in glioma cells not in normal astrocytes, a to the glioma by to cells by of and the and the of and in as is to and be to the of and is in this that hypothesize that the glioma channel a and it be that of the current glioma and Our previous that in of glioma and have ASIC2 is in the and is the plasma membrane. In the ASIC2 is not because of within the ASIC2 We hypothesize that of the tumor and is to the in expression of ASIC2. is of that with of and the of is to by the a of and to be and is the as with Hsc70 is to be a in a of by the Hsc70 in the the of of of a of ASIC2 has not that that ASIC2 is also a of Hsc70. chaperone is in there has and is the of ASIC Our results the of in glioma cells and in normal astrocytes. is to ASIC as it is with of the tumor results in this that not with the and with the that of this ASIC by Hsc70 ASIC2 expression the plasma membrane. chaperone is in glioma cells as compared with normal astrocytes. We that in the of glioma that express the is to in the plasma membrane. in Hsc70 expression also with ASIC2 and it ASIC2 of the channel the of plasma is a active cation In that the of Hsc70 the active current in a with because of Hsc70 of ASIC2 to the membrane, of the basally active current in glioma and a in glioma In contrast, of ASIC2 in normal the current and the of normal astrocytes. results with that trafficking of ASIC2 to the plasma of glioma cells the cation current and that this current is of the glioma We of and and of of of normal and of of chaperone and of