Sodium Channel β1 Subunit-mediated Modulation of Nav1.2 Currents and Cell Surface Density Is Dependent on Interactions with Contactin and Ankyrin

Abstract

Voltage-gated sodium channels are composed of a pore-forming α subunit and at least one auxiliary β subunit. Both β1 and β2 are cell adhesion molecules that interact homophilically, resulting in ankyrin recruitment. In contrast, β1, but not β2, interacts heterophilically with contactin, resulting in increased levels of cell surface sodium channels. We took advantage of these results to investigate the molecular basis of β1-mediated enhancement of sodium channel cell surface density, including elucidating structure-function relationships for β1 association with contactin, ankyrin, and Nav1.2. β1/β2 subunit chimeras were used to assign putative sites of contactin interaction to two regions of the β1 Ig loop. Recent studies have shown that glutathione S-transferase fusion proteins containing portions of Nav1.2 intracellular domains interact directly with ankyrinG. We show that Nav1.2 with in in the of β and that interaction in the of β1 but not a that not interact with ankyrinG. not Nav1.2 channel association with Nav1.2 and Nav1.2 cell surface but not β1/β2 chimeras regions of the β1 Ig were in Nav1.2 cell surface results that β1 for channel and enhancement of sodium channel cell surface Voltage-gated sodium channels are composed of a pore-forming α subunit and at least one auxiliary β subunit. Both β1 and β2 are cell adhesion molecules that interact homophilically, resulting in ankyrin recruitment. In contrast, β1, but not β2, interacts heterophilically with contactin, resulting in increased levels of cell surface sodium channels. We took advantage of these results to investigate the molecular basis of β1-mediated enhancement of sodium channel cell surface density, including elucidating structure-function relationships for β1 association with contactin, ankyrin, and Nav1.2. β1/β2 subunit chimeras were used to assign putative sites of contactin interaction to two regions of the β1 Ig loop. Recent studies have shown that glutathione S-transferase fusion proteins containing portions of Nav1.2 intracellular domains interact directly with ankyrinG. We show that Nav1.2 with in in the of β and that interaction in the of β1 but not a that not interact with ankyrinG. not Nav1.2 channel association with Nav1.2 and Nav1.2 cell surface but not β1/β2 chimeras regions of the β1 Ig were in Nav1.2 cell surface results that β1 for channel and enhancement of sodium channel cell surface Voltage-gated sodium channels of a pore-forming α subunit and at least one β subunit β1, and with β2 and are to α β have shown to channel and cell surface of α channel β are channel auxiliary in that cell adhesion molecules β1 and β2 interact with and resulting in and in cell adhesion resulting in ankyrin to the at of β1 of intracellular of of the to a β1-mediated ankyrin and results in β1 to domains for for β in with Ig cell adhesion β1 interacts with contactin in resulting in increased Nav1.2 cell surface channel Both β1 and interact with and the intracellular of β1 shown to interact with the β of the to the of β1-mediated cell in β1-mediated channel and of sodium channel cell surface We that and are for the of β1 sodium channel and We in β1, β1/β2 and results that β1 are proteins that and intracellular sites for α subunit Ig that for and and for ankyrin and channel channel a of the intracellular domains and of the α and the Nav1.2 were of β1, to the of β2, and the of were to contactin were and and used glutathione a were and of in domains were the β1 and β2 were of the two to the and were with of the that the resulting in and the subunit the and of the β1 Ig with the of the β2 Ig loop. β1 Ig for the β2 Ig for and the and sites of the β2 Ig with the β1 Ig loop. β1 Ig and the β2 and the and sites of the β1 and intracellular domains with the domains of β2 the the β1 and the of a the and of the and sites of and and the were with and the were with and for were in to fusion and of cell the and have β1, β2 were the and sites in and of of were at the were in the of with and were and the for β1, and the for β2 and and the for were to and were in of and containing and were with a at for of α of β with the with at for were with and containing a with were in a containing and for and proteins were to and with the proteins of β1 and β2 were with to and the of Nav1.2 and β1 with were with of the were and with for α with a cell of cell a at a of with the of to were to in Nav1.2 channels were at cell were and were the were the were the were at and at and to with and to with to the a of for levels to the of channel the in the of to in a to a of to the and the were in the of to in a to with a of and were with a the of the and a to the of and to Nav1.2 and β1, and were of in to a to a to a of were with to the cell the of the β1 Ig for that β1 interacts heterophilically with contactin, β2 not in the of β1 and β2 to chimeras these the and the Ig of β1 and β2 to investigate the molecular basis of interaction of β1 with contactin regions of the Ig were in We the to the of the β1 intracellular in with β subunit in of the were in and with with that and β1 were to with contactin and β2 were for contactin, of and β2 with β subunit results show that a of the β1 Ig to for contactin with Both Nav1.2 α and β1 ankyrin to the in to and β1 with and in have shown that fusion proteins containing intracellular domains of Nav1.2 to a fusion containing the of and Nav1.2 at and of not shown Nav1.2 interact directly with of contactin, and β1 were with cell containing β1 and contactin were the cell that of α subunit were and with α subunit with that Nav1.2 with in the of β1, in with the fusion studies of Nav1.2 with β1 the interaction of Nav1.2 with In contrast, of Nav1.2 with a that to interact with ankyrin not the for that with Nav1.2 and and β1 interact with ankyrinG. Nav1.2 were with were and with a and with for at cell were used for with Nav1.2 but not to ankyrin in to adhesion We subunit and β1 of sodium that sodium channel Nav1.2 and that these two in not Nav1.2 and the of a to Nav1.2 the for and for were with Nav1.2 the of with β1 We have shown that in adhesion in but to ankyrin adhesion the to at of β1 cell with contactin for the not for β1-mediated cell adhesion for subunit but for ankyrin association and for of Nav1.2 with Nav1.2 and contactin but not channel cell were with were with for β1 at at are the cell and were with were with for contactin at for and for the of are to the for and of the in the in the of β1 the for of and for and for Nav1.2 and are for and the not sodium Nav1.2 channels with β1, with contactin and β1, with contactin and were cell of to in a of were the the the and the were to in a to a of were with a the of the and a of the the β1, the the β1 the the the the the in a β1 with and to of of Nav1.2 with β1 and contactin results in in sodium channel cell surface and In the the of a of β1/β2 and Nav1.2 cell surface to interact with contactin not cell surface with Nav1.2 contactin with the β1 Ig were not to β1-mediated enhancement of cell surface channels. studies have that the of the β1 Ig for α subunit interaction the β1 in to the of the Ig contactin interaction not Nav1.2 cell surface density, that the intracellular domains of β1 for β1-mediated in channel of a β1 with and domains that the intracellular in a in cell surface α that for β1, a that for sodium channels at the cell that and increased cell surface with Nav1.2 that β1 with ankyrin are of the for sodium channels at the cell not and were to in Nav1.2 cell surface in to interact with and Nav1.2 and in these results that β1 and intracellular domains that to in sodium channel cell surface in with ankyrin and contactin are to Nav1.2 at the cell surface in to Nav1.2 Nav1.2 with a of contactin and β for Nav1.2 and cell at least two used to to Nav1.2 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Nav1.2 and in a in the of β of for Nav1.2 the of the of sodium channel cell surface in the Nav1.2 cell that a of sodium channels are at the and to interact with ankyrinG. of β1 with Nav1.2 the of that with Nav1.2 In contrast, and results of with Nav1.2 not the association of sodium channels with the of cell surface sodium channel that Nav1.2 and interact in the of β but that interaction the of β1 In fusion proteins containing the β1 intracellular not We that are to sodium channel α ankyrin are the β1 Ig and with Nav1.2 but Nav1.2 ankyrin the of not for but for of Nav1.2 channel not the of Nav1.2 channel are to in studies that show that to the of of Nav1.2 Nav1.2 with to with Nav1.2. and interact with contactin, resulting in increased Nav1.2 cell surface density, to a with β1 the for a intracellular that molecules and that in of proteins in of in cell adhesion to proteins and sites for molecules that are sites of adhesion molecules that are at the in the interact with ankyrin and interact with a that in of Nav1.2 and ankyrin a for interaction with the that sodium channel β1 are in channel of channel cell surface and cell in with have to a for β1 β1 Ig for and and the Ig and intracellular domains are for β1-mediated enhancement of channel cell surface for of channel and in intracellular of β1 for channel and for ankyrin in to cell of of to results in the of β1-mediated ankyrin recruitment. results in of β1 and β1 to and in for for to have not to regions of β1 that channel results the domains of β1 in cell adhesion and sodium channel and to of β1 structure-function relationships in the structure-function relationships of β1 are to the Ig are shown to that have to for α subunit association are to the in in in sites of are shown to for subunit cell adhesion the intracellular shown in in ankyrin α subunit and interaction with β Voltage-gated sodium channels of a pore-forming α subunit and at least one β subunit β1, and with β2 and are to α β have shown to channel and cell surface of α channel β are channel auxiliary in that cell adhesion molecules β1 and β2 interact with and resulting in and in cell adhesion resulting in ankyrin to the at of β1 of intracellular of of the to a β1-mediated ankyrin and results in β1 to domains for for β in with Ig cell adhesion β1 interacts with contactin in resulting in increased Nav1.2 cell surface channel Both β1 and interact with and the intracellular of β1 shown to interact with the β of the to the of β1-mediated cell in β1-mediated channel and of sodium channel cell surface We that and are for the of β1 sodium channel and We in β1, β1/β2 and results that β1 are proteins that and intracellular sites for α subunit Ig that for and and for ankyrin and channel channel a of the intracellular domains and of the α and the Nav1.2 were of β1, to the of β2, and the of were to contactin were and and used glutathione a were and of in domains were the β1 and β2 were of the two to the and were with of the that the resulting in and the subunit the and of the β1 Ig with the of the β2 Ig loop. β1 Ig for the β2 Ig for and the and sites of the β2 Ig with the β1 Ig loop. β1 Ig and the β2 and the and sites of the β1 and intracellular domains with the domains of β2 the the β1 and the of a the and of the and sites of and and the were with and the were with and for were in to fusion and of cell the and have β1, β2 were the and sites in and of of were at the were in the of with and were and the for β1, and the for β2 and and the for were to and were in of and containing and were with a at for of α of β with the with at for were with and containing a with were in a containing and for and proteins were to and with the proteins of β1 and β2 were with to and the of Nav1.2 and β1 with were with of the were and with for α with a cell of cell a at a of with the of to were to in Nav1.2 channels were at cell were and were the were the were the were at and at and to with and to with to the a of for levels to the of channel the in the of to in a to a of to the and the were in the of to in a to with a of and were with a the of the and a to the of and to Nav1.2 and β1, and were of in to a to a to a of were with to the cell the of the channel a of the intracellular domains and of the α and the Nav1.2 were of β1, to the of β2, and the of were to contactin were and and used glutathione a were and of in domains were the β1 and β2 were of the two to the and were with of the that the resulting in and the subunit the and of the β1 Ig with the of the β2 Ig loop. β1 Ig for the β2 Ig for and the and sites of the β2 Ig with the β1 Ig loop. β1 Ig and the β2 and the and sites of the β1 and intracellular domains with the domains of β2 the the β1 and the of a the and of the and sites of and and the were with and the were with and for were in to fusion and of cell the and have β1, β2 were the and sites in and of of were at the were in the of with and were and the for β1, and the for β2 and and the for were to and were in of and containing and were with a at for of α of β with the with at for were with and containing a with were in a containing and for and proteins were to and with the proteins of β1 and β2 were with to and the of Nav1.2 and β1 with were with of the were and with for α with a cell of cell a at a of with the of to were to in Nav1.2 channels were at cell were and were the were the were the were at and at and to with and to with to the a of for levels to the of channel the in the of to in a to a of to the and the were in the of to in a to with a of and were with a the of the and a to the of and to Nav1.2 and β1, and were of in to a to a to a of were with to the cell the of the β1 Ig for that β1 interacts heterophilically with contactin, β2 not in the of β1 and β2 to chimeras these the and the Ig of β1 and β2 to investigate the molecular basis of interaction of β1 with contactin regions of the Ig were in We the to the of the β1 intracellular in with β subunit in of the were in and with with that and β1 were to with contactin and β2 were for contactin, of and β2 with β subunit results show that a of the β1 Ig to for contactin with Both Nav1.2 α and β1 ankyrin to the in to and β1 with and in have shown that fusion proteins containing intracellular domains of Nav1.2 to a fusion containing the of and Nav1.2 at and of not shown Nav1.2 interact directly with of contactin, and β1 were with cell containing β1 and contactin were the cell that of α subunit were and with α subunit with that Nav1.2 with in the of β1, in with the fusion studies of Nav1.2 with β1 the interaction of Nav1.2 with In contrast, of Nav1.2 with a that to interact with ankyrin not the for that with Nav1.2 and with Nav1.2 but not to ankyrin in to adhesion We subunit and β1 of sodium that sodium channel Nav1.2 and that these two in not Nav1.2 and the of a to Nav1.2 the for and for were with Nav1.2 the of with β1 We have shown that in adhesion in but to ankyrin adhesion the to at of β1 cell with contactin for the not for β1-mediated cell adhesion for subunit but for ankyrin association and for of Nav1.2 with Nav1.2 and contactin but not channel cell were with were with for β1 at at are the cell and were with were with for contactin at for and for the of are to the for and of the in the in the of β1 the for of and for and for Nav1.2 and are for and the not sodium Nav1.2 channels with β1, with contactin and β1, with contactin and were cell of to in a of were the the the and the were to in a to a of were with a the of the and a of the the β1, the the β1 the the the the the in a β1 with and to of of Nav1.2 with β1 and contactin results in in sodium channel cell surface and In the the of a of β1/β2 and Nav1.2 cell surface to interact with contactin not cell surface with Nav1.2 contactin with the β1 Ig were not to β1-mediated enhancement of cell surface channels. studies have that the of the β1 Ig for α subunit interaction the β1 in to the of the Ig contactin interaction not Nav1.2 cell surface density, that the intracellular domains of β1 for β1-mediated in channel of a β1 with and domains that the intracellular in a in cell surface α that for β1, a that for sodium channels at the cell that and increased cell surface with Nav1.2 that β1 with ankyrin are of the for sodium channels at the cell not and were to in Nav1.2 cell surface in to interact with and Nav1.2 and in these results that β1 and intracellular domains that to in sodium channel cell surface in with ankyrin and contactin are to Nav1.2 at the cell surface in to Nav1.2 Nav1.2 with a of contactin and β for Nav1.2 and cell at least two used to to Nav1.2 Nav1.2 and Nav1.2 and β1 Ig for that β1 interacts heterophilically with contactin, β2 not in the of β1 and β2 to chimeras these the and the Ig of β1 and β2 to investigate the molecular basis of interaction of β1 with contactin regions of the Ig were in We the to the of the β1 intracellular in with β subunit in of the were in and with with that and β1 were to with contactin and β2 were for contactin, of and β2 with β subunit results show that a of the β1 Ig to for contactin with Both Nav1.2 α and β1 ankyrin to the in to and β1 with and in have shown that fusion proteins containing intracellular domains of Nav1.2 to a fusion containing the of and Nav1.2 at and of not shown Nav1.2 interact directly with of contactin, and β1 were with cell containing β1 and contactin were the cell that of α subunit were and with α subunit with that Nav1.2 with in the of β1, in with the fusion studies of Nav1.2 with β1 the interaction of Nav1.2 with In contrast, of Nav1.2 with a that to interact with ankyrin not the for that with Nav1.2 and with Nav1.2 but not to ankyrin in to adhesion We subunit and β1 of sodium that sodium channel Nav1.2 and that these two in not Nav1.2 and the of a to Nav1.2 the for and for were with Nav1.2 the of with β1 We have shown that in adhesion in but to ankyrin adhesion the to at of β1 cell with contactin for the not for β1-mediated cell adhesion for subunit but for ankyrin association and for of Nav1.2 β1 with and to of of Nav1.2 with β1 and contactin results in in sodium channel cell surface and In the the of a of β1/β2 and Nav1.2 cell surface to interact with contactin not cell surface with Nav1.2 contactin with the β1 Ig were not to β1-mediated enhancement of cell surface channels. studies have that the of the β1 Ig for α subunit interaction the β1 in to the of the Ig contactin interaction not Nav1.2 cell surface density, that the intracellular domains of β1 for β1-mediated in channel of a β1 with and domains that the intracellular in a in cell surface α that for β1, a that for sodium channels at the cell that and increased cell surface with Nav1.2 that β1 with ankyrin are of the for sodium channels at the cell not and were to in Nav1.2 cell surface in to interact with and Nav1.2 and in these results that β1 and intracellular domains that to in sodium channel cell surface in and of β1 for α β1 and domains are for channel and of the of sodium channels in In the β1 including the intracellular for of and sodium channel β1 for with that a Ig to results in the of β1-mediated of Nav1.2 and in the intracellular of that results in the of β1 of α in We that not Nav1.2 and results show that to Nav1.2 the interaction of these two these studies not that of sodium channels β1 in with but that and sodium channel α and β1 have intracellular and sites of to of sodium channel β1 channel at the of and β1 and contactin in in channel cell surface in that β1 the of β1 for and for interaction with contactin were not in We show that the β1 Ig for and that the Ig and intracellular domains of β1 are for β1-mediated enhancement of cell surface sodium channel and β1 with fusion proteins containing intracellular regions of Nav1.2 interact directly with a fusion containing the of We show that Nav1.2 interacts with the fusion studies and to show the association of Nav1.2 and in a in the of β of for Nav1.2 the of the of sodium channel cell surface in the Nav1.2 cell that a of sodium channels are at the and to interact with ankyrinG. of β1 with Nav1.2 the of that with Nav1.2 In contrast, and results of with Nav1.2 not the association of sodium channels with the of cell surface sodium channel that Nav1.2 and interact in the of β but that interaction the of β1 In fusion proteins containing the β1 intracellular not We that are to sodium channel α ankyrin are the β1 Ig and with Nav1.2 but Nav1.2 ankyrin the of not for but for of Nav1.2 channel not the of Nav1.2 channel are to in studies that show that to the of of Nav1.2 Nav1.2 with to with Nav1.2. and interact with contactin, resulting in increased Nav1.2 cell surface density, to a with β1 the for a intracellular that molecules and that in of proteins in of in cell adhesion to proteins and sites for molecules that are sites of adhesion molecules that are at the in the interact with ankyrin and interact with a that in of Nav1.2 and ankyrin a for interaction with the that sodium channel β1 are in channel of channel cell surface and cell in with have to a for β1 β1 Ig for and and the Ig and intracellular domains are for β1-mediated enhancement of channel cell surface for of channel and in intracellular of β1 for channel and for ankyrin in to cell of of to results in the of β1-mediated ankyrin recruitment. results in of β1 and β1 to and in for for to have not to regions of β1 that channel results the domains of β1 in cell adhesion and sodium channel and to of β1 structure-function relationships in the and of β1 for α β1 and domains are for channel and of the of sodium channels in In the β1 including the intracellular for of and sodium channel β1 for with that a Ig to results in the of β1-mediated of Nav1.2 and in the intracellular of that results in the of β1 of α in We that not Nav1.2 and results show that to Nav1.2 the interaction of these two these studies not that of sodium channels β1 in with but that and sodium channel α and β1 have intracellular and sites of In to of sodium channel β1 channel at the of and β1 and contactin in in channel cell surface in that β1 the of β1 for and for interaction with contactin were not in We show that the β1 Ig for and that the Ig and intracellular domains of β1 are for β1-mediated enhancement of cell surface sodium channel Nav1.2 and β1 with fusion proteins containing intracellular regions of Nav1.2 interact directly with a fusion containing the of We show that Nav1.2 interacts with the fusion studies and to show the association of Nav1.2 and in a in the of β of for Nav1.2 the of the of sodium channel cell surface in the Nav1.2 cell that a of sodium channels are at the and to interact with ankyrinG. of β1 with Nav1.2 the of that with Nav1.2 In contrast, and results of with Nav1.2 not the association of sodium channels with the of cell surface sodium channel that Nav1.2 and interact in the of β but that interaction the of β1 In fusion proteins containing the β1 intracellular not We that are to sodium channel α ankyrin are the β1 Ig and with Nav1.2 but Nav1.2 ankyrin the of not for but for of Nav1.2 channel not the of Nav1.2 channel are to in studies that show that to the of of Nav1.2 Nav1.2 with to with Nav1.2. and interact with contactin, resulting in increased Nav1.2 cell surface density, to a with β1 the for a intracellular that molecules and that in of proteins in of in cell adhesion to proteins and sites for molecules that are sites of adhesion molecules that are at the in the interact with ankyrin and interact with a that in of Nav1.2 and ankyrin a for interaction with the that sodium channel β1 are in channel of channel cell surface and cell in with have to a for β1 β1 Ig for and and the Ig and intracellular domains are for β1-mediated enhancement of channel cell surface for of channel and in intracellular of β1 for channel and for ankyrin in to cell of of to results in the of β1-mediated ankyrin recruitment. results in of β1 and β1 to and in for for to have not to regions of β1 that channel results the domains of β1 in cell adhesion and sodium channel and to of β1 structure-function relationships in the