A Multiprotein Trafficking Complex Composed of SAP97, CASK, Veli, and Mint1 Is Associated with Inward Rectifier Kir2 Potassium Channels

Abstract

Strong inward rectifier potassium (Kir2) channels are important in the control of cell excitability, and their functions are modulated by interactions with intracellular proteins. Here we identified a complex of scaffolding/trafficking proteins in brain that associate with Kir2.1, Kir2.2, and Kir2.3 channels. By using a combination of affinity interaction pulldown assays and co-immunoprecipitations from brain and transfected cells, we demonstrated that a complex composed of SAP97, CASK, Veli, and Mint1 associates with Kir2 channels via the C-terminal PDZ-binding motif. We further demonstrated by using in vitro protein interaction assays that SAP97, Veli-1, or Veli-3 binds directly to the Kir2.2 C terminus and recruits CASK. Co-immunoprecipitations indicated that specific Veli isoforms participate in forming distinct protein complexes in brain, where Veli-1 stably associates with CASK and SAP97, Veli-2 associates with CASK and Mint1, and Veli-3 associates with CASK, SAP97, and Mint1. Additionally, immunocytochemistry of rat cerebellum revealed overlapping expression of Kir2.2, SAP97, CASK, Mint1, with Veli-1 in the granule cell layer and Veli-3 in the molecular layer. We propose a model whereby Kir2.2 associates with distinct SAP97-CASK-Veli-Mint1 complexes. In one complex, SAP97 interacts directly with the Kir2 channels and recruits CASK, Veli, and Mint1. Alternatively, Veli-1 or Veli-3 interacts directly with the Kir2 channels and recruits CASK and SAP97; association of Mint1 with the complex requires Veli-3. Expression of Kir2.2 in polarized epithelial cells resulted in targeting of the channels to the basolateral membrane and co-localization with SAP97 and CASK, whereas a dominant interfering form of CASK caused the channels to mislocalize. Therefore, CASK appears to be a central protein of a macromolecular complex that participates in trafficking and plasma membrane localization of Kir2 channels. Strong inward rectifier potassium (Kir2) channels are important in the control of cell excitability, and their functions are modulated by interactions with intracellular proteins. Here we identified a complex of scaffolding/trafficking proteins in brain that associate with Kir2.1, Kir2.2, and Kir2.3 channels. By using a combination of affinity interaction pulldown assays and co-immunoprecipitations from brain and transfected cells, we demonstrated that a complex composed of SAP97, CASK, Veli, and Mint1 associates with Kir2 channels via the C-terminal PDZ-binding motif. We further demonstrated by using in vitro protein interaction assays that SAP97, Veli-1, or Veli-3 binds directly to the Kir2.2 C terminus and recruits CASK. Co-immunoprecipitations indicated that specific Veli isoforms participate in forming distinct protein complexes in brain, where Veli-1 stably associates with CASK and SAP97, Veli-2 associates with CASK and Mint1, and Veli-3 associates with CASK, SAP97, and Mint1. Additionally, immunocytochemistry of rat cerebellum revealed overlapping expression of Kir2.2, SAP97, CASK, Mint1, with Veli-1 in the granule cell layer and Veli-3 in the molecular layer. We propose a model whereby Kir2.2 associates with distinct SAP97-CASK-Veli-Mint1 complexes. In one complex, SAP97 interacts directly with the Kir2 channels and recruits CASK, Veli, and Mint1. Alternatively, Veli-1 or Veli-3 interacts directly with the Kir2 channels and recruits CASK and SAP97; association of Mint1 with the complex requires Veli-3. Expression of Kir2.2 in polarized epithelial cells resulted in targeting of the channels to the basolateral membrane and co-localization with SAP97 and CASK, whereas a dominant interfering form of CASK caused the channels to mislocalize. Therefore, CASK appears to be a central protein of a macromolecular complex that participates in trafficking and plasma membrane localization of Kir2 channels. Strong inward rectifier potassium (Kir2) 1The abbreviations used are: Kir2, inward rectifier potassium channel 2; MAGUK, membrane-associated guanylate kinase protein family; GST, glutathione S-transferase; PBS, phosphate-buffered saline; SH3, Src homology 3; GK, guanylate kinase-like; GFP, green fluorescent protein; FITC, fluorescein isothiocyanate; MDCK, Madin-Darby canine kidney cells; NMDA, N-methyl-d-aspartic acid. 1The abbreviations used are: Kir2, inward rectifier potassium channel 2; MAGUK, membrane-associated guanylate kinase protein family; GST, glutathione S-transferase; PBS, phosphate-buffered saline; SH3, Src homology 3; GK, guanylate kinase-like; GFP, green fluorescent protein; FITC, fluorescein isothiocyanate; MDCK, Madin-Darby canine kidney cells; NMDA, N-methyl-d-aspartic acid. channels are a widely expressed family of ion channel proteins distinguished by their ability to pass K+ current in the inward direction more readily than outward. Kir2 channels are key components in control of neuronal excitability in brain, electrical activity in heart, vascular tone, and glial buffering of potassium (1Isomoto S. Kondo C. Kurachi Y. Jpn. J. Physiol. 1997; 47: 11-39Google Scholar, 2Nichols C.G. Lopatin A.N. Annu. Rev. Physiol. 1997; 59: 171-191Google Scholar). Kir2 channels are important in the modulation of cell excitability, repolarization of the action potential, and determination of the cellular resting potential. Furthermore, Kir2 mutations are implicated in at least one genetic disease causing periodic paralysis and heart arrhythmias (Andersen's syndrome (3Plaster N.M. Tawil R. Tristani-Firouzi M. Canun S. Bendahhou S. Tsunoda A. Donaldson M.R. Iannaccone S.T. Brunt R. J. A. A. C. and trafficking of ion channels are by interactions of their intracellular with of the protein proteins a to a Src homology and a guanylate with proteins proteins to participate in the of macromolecular or complexes M. C. Annu. Rev. Scholar, Scholar). Kir2.1, Kir2.2, and Kir2.3 to the of of the family via their PDZ-binding at the C terminus Scholar, J. Scholar). Kir2 channels demonstrated to with the proteins Scholar, M. A. J. and SAP97 in a J. Scholar). to Kir2 channel in the plasma membrane M. A. J. the of the interaction of Kir2 channels with SAP97 in are SAP97 in the of macromolecular complexes M. we that one of SAP97 be to protein complexes to to Kir2 channels. SAP97 to with a of proteins S. S. S. Scholar, J. Scholar). CASK a terminus that interacts with Veli and and Mint1 and forming a complex implicated in basolateral localization and trafficking Scholar, M. J. Scholar, S. M. Scholar, M. Scholar). J. J. Physiol. demonstrated that the Veli-2 associates with Kir2.3 in epithelial cells, that Veli-2 directly interacts with Kir2 channels. Additionally, by using that and CASK proteins are expressed at the basolateral membrane of epithelial cells J. J. Physiol. complex that Kir2 channels form a complex with SAP97, CASK, Veli, and Mint1. association of CASK with SAP97 to that the complex to Kir2 channels by SAP97 and participates in channel trafficking or of a Additionally, we identified SAP97, CASK, Veli, and Mint1 in association with Kir2 channels in a S. J. Scholar). Here we by that a complex composed of CASK, Veli, Mint1, and SAP97 associates with Kir2 channels in brain by a combination of affinity interaction and co-localization expressed in polarized epithelial cells, Kir2.2 channels are to the basolateral a of the of complex, we expressed a form of CASK. of Kir2.2 that a of the complex in Kir2.2 trafficking and localization in the plasma potassium channel C-terminal to the of glutathione in the expression J. Scholar). SAP97 proteins SAP97 and rat Kir2.2 from Y. and the C-terminal rat CASK a from C. of CASK, the kinase and with a at the terminus S. S. S. and the expression rat SAP97 from C. C. the expression Kir2.1, and the expression that to the terminus of the Kir2 channels. Veli-1 Veli-2 and Veli-3 from S. with a at the terminus by and and J. Scholar, J. Scholar). used the and and family and and and to used where and from and in and at in of to a of and at at and the more at to and used affinity and proteins expressed in and J. Scholar). brain, or with to protein to to the protein and with by and with in and the proteins by or proteins J. Scholar). proteins from with glutathione PBS, and to of protein of to the protein to a and with C C with C with with and with and at brain by with of by a of protein to of rat brain protein and in at with a and with by with to and proteins with of with by proteins with with at and cells with CASK and Kir2.2 cells with PBS, in at with and used protein by the of protein of or to the protein and of protein and with proteins with and proteins by In CASK binds directly or with Kir2 in vitro assays and SAP97 from by with by with CASK the expression and expressed in in and at at and the CASK used the interaction of GST, or protein to with or of SAP97 at by of CASK and at with with and with with proteins in and the of Veli in CASK to the interaction assays that Veli-1, or Veli-3 Veli-1 Veli-2 and Veli-3 the the to a expressed in and with and in at and to by in in and at with a and to in in at with in at with with in at by with with using a and cells and in with cells stably transfected with or and with and transfected the with or SAP97 using to and with with with in and using and C-terminal of Kir2 with the of CASK, Veli, and we identified SAP97 a protein in the brain and heart J. Scholar). SAP97 a protein that a to we to of a Kir2 complex M. Scholar, Scholar). that the protein CASK interacts with the terminus and of SAP97 S. S. S. Scholar, J. Scholar). Additionally, CASK to with the proteins Mint1 and Veli in the the that a SAP97 be to CASK and proteins Mint1 and Veli to Kir2 channels. CASK and Kir2.2 are expressed in the a with and We expression of CASK and Kir2.2 in brain, and In the cell cell from kidney MDCK, and expressed CASK Kir2.2, whereas the cell expressed Kir2.2 CASK protein expression indicated expression of SAP97 in J. Scholar). Veli proteins are expressed in brain, heart, and S. M. Scholar, R. M. J. Scholar). Mint1 expressed in neuronal M. J. 1997; and heart S. J. Scholar). Therefore, a complex composed of Kir2.2, SAP97, CASK, Veli, and Mint1 in brain and heart, whereas complexes Mint1 form in CASK associate with the C-terminal of Kir2 we protein affinity with C of Kir2.1, Kir2.2, and to C-terminal of Kir2.1, Kir2.2, and Kir2.3 proteins with cerebellum or heart and proteins by to and with C of of Kir2 channels with CASK from cerebellum and heart and with the be with interaction with CASK specific the C-terminal of Kir2.1, Kir2.2, and Kir2.3 a PDZ-binding with CASK and the of a C-terminal PDZ-binding CASK we a C-terminal protein of a potassium channel a PDZ-binding CASK cerebellum or heart with the the of PDZ-binding in ion Kir2 and interactions with the proteins Scholar, M. A. M. Scholar, and SAP97 J. Scholar). CASK directly to the channel via or via protein a we that of the C-terminal of Kir2.2 the ability of Kir2.2 to with CASK. of the C-terminal of the C-terminal resulted in a of CASK the C-terminal CASK association with the channel demonstrated that CASK directly associate with SAP97 S. S. S. Scholar, J. a SAP97 with to the terminus of SAP97 used a control CASK SAP97 protein with CASK from cerebellum and heart the with the protein of proteins used and CASK, Veli, and Mint1 are to Kir2 affinity interactions by using and proteins to SAP97, CASK, Mint1, and Veli a the Veli-1 protein the molecular of from in from the control that the association of proteins specific and via the at the C terminus of interacts with SAP97 J. with the Kir2.2 that a of a macromolecular complex with Additionally, we from and we the or of the proteins S. J. Scholar). that CASK, Veli, and Mint1 associate with the Kir2.2 C terminus via interaction with the channel motif. the Veli with the Kir2 pulldown and with pulldown with rat cerebellum with Kir2.2 and Kir2.3 proteins from the affinity readily indicated that SAP97, CASK, Veli-1, and Veli-3 associate with the Kir2.2 and Kir2.3 C and with the or the the C-terminal and Veli-2 in pulldown assays in the to expression in with where Veli-1 and Veli-3 with the channel C terminus Veli-2 S. J. Scholar). Mint1 in pulldown assays with the Kir2 channel complex to interactions or expression in the affinity interaction that SAP97, CASK, and at least isoforms of Veli and and Mint1 associate with the C-terminal PDZ-binding of Kir2 channels. CASK with Kir2.2 in a that Kir2.2 interacts with CASK in a cellular we expressed proteins and in combination in cells SAP97 and CASK. By using the terminus of we Kir2.2 from cell CASK transfected CASK with Kir2.2 and CASK with a Kir2.2 with the C-terminal CASK be Additionally, CASK Kir2.2 transfected or used the and Expression of Kir2.2, and CASK indicated by a of the that Kir2.2 associate with CASK in cells, and the C terminus of Kir2.2 the PDZ-binding the association with CASK. and in to Kir2.2, with CASK in a cellular we co-immunoprecipitations with green fluorescent protein Kir2.1, Kir2.2, and Kir2.3 with CASK in with and CASK that channels with CASK that Kir2.1, Kir2.2, and Kir2.3 channels the C-terminal PDZ-binding associate with CASK in Kir2.2 with CASK in the and Kir2 channels and CASK associate in brain Kir2.2 and CASK are expressed in the cerebellum C. A. J. Scholar, C. A. 1997; Scholar, S. M. J. Scholar, M. J. Scholar, Y. S. J. we co-immunoprecipitations with the from rat cerebellum expressed in the cerebellum C. A. J. Scholar, C. A. 1997; of the protein be of CASK with the with control and the that SAP97 in J. Scholar). that Kir2.2 with CASK in the cerebellum and that CASK and SAP97 be with Kir2 channels. We with the from rat heart of CASK with the with control that the Kir2 channel association with CASK to brain be important in Furthermore, the that a Kir2 channel complex CASK and SAP97 in heart C of Kir2.2 with CASK with are CASK associates with directly with the channel or binds interactions with proteins. We demonstrated that the of SAP97 binds to the Kir2.2 C terminus J. and S. S. S. Scholar, J. the interaction of SAP97 with CASK in epithelial to the that SAP97 binds to CASK and Kir2.2 a SAP97 and CASK associate in brain, with by CASK. Here we that CASK with SAP97 in brain using with control CASK. that Kir2.2, SAP97, and CASK form a complex in their the the and the a A. J. Scholar, C. J. A. 1997; Scholar). CASK a interaction with Kir2 channels. a or at the C-terminal of C. J. A. 1997; whereas the Kir2 channel C a at to the that CASK directly with Kir2 channels via association with SAP97, directly interacts with the PDZ-binding of Kir2 channels. we the ability of expressed proteins to with the C terminus of Kir2.2 in GST, or proteins to with or expressed and SAP97 by with expressed CASK protein and proteins by and with CASK in the pulldown with the Kir2.2 C terminus with the of SAP97 Kir2.2 associate with CASK SAP97 the with CASK in the or of SAP97 and that association of CASK with the Kir2.2 C terminus requires the PDZ-binding motif. a a the to with CASK more than SAP97 J. CASK that CASK directly the Kir2 channel C terminus with the potassium channel via interaction with SAP97 the of Kir2.2 with the demonstrated that the terminus of SAP97 interaction with CASK, with the to the association in epithelial cells S. S. S. Scholar, J. Scholar). that interactions in brain, we affinity using proteins of SAP97 CASK in the using the SAP97 terminus and a that the to with a that CASK interacts with SAP97 via terminus in epithelial cells S. S. S. Scholar). association with or the terminus and interaction to CASK Veli-1 and Veli-3 with the terminus of SAP97 Mint1 to associate with SAP97 and the SAP97 and to that the of SAP97 participate in the association with Mint1. further the that Mint1 associates with Kir2 channels via interactions by we to Kir2.2 from brain with SAP97 that the J. further the that Kir2.2, SAP97, CASK, Veli, and Mint1 form a macromolecular that the terminus of SAP97 association with CASK and Veli, Mint1 interactions with SAP97 to form a further the that SAP97 associates with the complex in brain, we co-immunoprecipitations with from brain proteins with Mint1, CASK, and a that the SAP97, and Mint1, CASK, and Veli-1 molecular Veli-1 Veli-2 and Veli-3 with SAP97 using the of SAP97 and the molecular proteins and are by the Veli-1, and Veli-3 with the C of Kir2.2 and isoforms of Veli in cells, we Kir2.2 with Veli-1, and Veli-3. cell with by with the Kir2.2 Veli-1, and Veli-3 with Kir2.2 and the control with of the Veli with be and of Kir2.2 and Veli proteins expressed in the cells used in that isoforms of Veli associate with Kir2.2 in a cellular and interaction requires the C-terminal of the channel Veli proteins a that to with in brain R. M. J. and Kir2.3 in epithelial cells J. J. Physiol. we that at least the Veli-1 and Veli-3 to associate with Kir2.2 C terminus from brain C and with the C terminus of Kir2.2 a interaction with the channel C we expressed isoforms of Veli proteins in and the proteins with affinity We proteins with GST, Kir2.2, or SAP97 proteins. Kir2.2 directly with Veli-1, and Veli-3 the Veli proteins with or SAP97 and that Veli-1, and Veli-3 are of directly with the Kir2.2 C In proteins directly with SAP97, that Veli association with SAP97, in via CASK. Veli-1 and Veli-3 Veli-2 CASK to the C of demonstrated that SAP97 CASK to the Kir2.2 C terminus the Veli proteins directly to the C-terminal Kir2.2 and to with the of CASK S. S. S. we that Veli isoforms CASK to Kir2.2 proteins to with CASK with and Veli-1, Veli-2 or Veli-3. proteins by and with control protein or CASK interaction with or Veli proteins and CASK with Kir2.2 directly be Veli-1 or Veli-3 the of the Veli-2 of CASK and Veli-1, or that Veli protein with the Kir2.2 in assays the ability of Veli-1 and SAP97, to directly to the Kir2.2 C terminus and CASK to the used a control interaction with CASK, CASK in the and of Veli and the Veli isoforms to in the of CASK that Veli-1 and Veli-3 directly to the Kir2.2 C terminus and participate in CASK to the of Veli in isoforms of Veli are to be in complex with CASK, SAP97, Mint1, and Kir2.2 in brain, using rat cerebellum or brain with from co-immunoprecipitations from and brain that CASK associates with Veli proteins and and SAP97 and and with more SAP97 in with Veli-3. SAP97 be in co-immunoprecipitations with from cerebellum or brain Veli-2 more and Mint1 with and and and Mint1 be in with and that Mint1 associates with Veli-2 or Veli-3 in from co-immunoprecipitations that the complexes in brain are composed of Kir2.2, SAP97, CASK, with Mint1 and a complex with Kir2.2, SAP97, CASK, and Veli-1 Mint1. Additionally, a complex composed of CASK, and Mint1 SAP97 and Kir2 channels We directly the of Kir2.2 in the Kir2.2 in the the In to a the of Kir2.2 at by the of the Kir2.2 Expression with SAP97, CASK, Veli-1, and Mint1 in the SAP97, CASK, Veli-1, and Mint1 to be expressed in the cerebellum J. Scholar, C. A. 1997; Scholar, S. M. J. Scholar, Y. S. J. Scholar, M. J. Scholar, Y. J. J. Scholar, C. J. Scholar). protein affinity pulldown and from the association of proteins in a macromolecular complex of proteins identified in the we that their expression we rat in control and with in A. J. S. Scholar). We the of the cerebellum where Kir2.2 expression J. Scholar, C. A. 1997; Scholar). Kir2.2 a from the cell layer to the molecular layer with in the granule cell layer J. Scholar, C. A. 1997; Scholar). we demonstrated that the Kir2.2 expression with the glial cell in in the molecular layer and in the granule cell the of Kir2.2 in glial cells of the cerebellum J. Scholar). CASK to be in J. Scholar, C. J. SAP97 in the molecular layer expression in the granule cell layer of CASK with SAP97 overlapping the cerebellum and with the association of proteins by the S. S. S. Scholar, J. Scholar). Mint1 to be expressed in the molecular layer and granule cell layer that Kir2.2 J. Scholar, C. A. J. Scholar). Veli-1 expression in the cell with in the granule cell layer with the with in the molecular layer and more in the granule cell layer Veli-3 to be expressed in the molecular layer in to Veli are in with Y. J. J. Scholar). of the cerebellum the or of the with the that Kir2.2, CASK, and SAP97 associate with Veli-1 in the granule cell layer and with Veli-3 in the molecular layer. Mint1 in association with SAP97, CASK, and Veli-3 and that a complex with Kir2.2, SAP97, CASK, and Mint1 be in the molecular layer. CASK of Kir2.2 in the of CASK in Kir2 channel we the polarized kidney epithelial cell in membrane proteins PDZ-binding and to the basolateral membrane J. J. Physiol. Scholar, C. C. A. R. A. J. Scholar, Scholar). the membrane localization of Kir2.2, we polarized cells in the and by of the in cells resulted in basolateral expression of the channel and in with Kir2.3 localization in cells J. J. Physiol. Scholar). the PDZ-binding basolateral with of the channel expressed and to the and that the PDZ-binding of Kir2.2 important targeting of the channels to the basolateral plasma targeting and of to the basolateral membrane that proteins a in of the proteins SAP97 and CASK are expressed at the basolateral membrane in cells and S. S. S. Scholar, J. J. Physiol. proteins in channel targeting and membrane S. S. S. demonstrated that a form of CASK that the in a dominant and the of SAP97 in SAP97 directly interacts with Kir2.2 we that CASK a central in the localization and of the protein Therefore, of a dominant CASK with Kir2.2 in cells in a Kir2.2 in the channels are from the basolateral of the stably transfected cells resulted in of intracellular Kir2.2 protein membrane localization and whereas cells transfected with the dominant CASK basolateral a of SAP97 and the localization and that the of the dominant CASK of protein that a the association of Kir2.2 with the complex in polarized epithelial cells in trafficking localization of channels at the plasma In we the that the inward rectifier potassium channels Kir2.1, Kir2.2, and Kir2.3 associate in brain with a complex of proteins that SAP97, CASK, Veli-1, and Mint1. that the complex of proteins to Kir2 channels via of SAP97 or Furthermore, that the isoforms of Veli participate in the of distinct protein complexes in Veli-1 associates with Kir2 SAP97, and CASK, whereas Veli-3 associates with Kir2 SAP97, CASK, and Mint1. Veli-2 to form of complex with Kir2 channels or dominant interfering CASK resulted in the of Kir2.2 from the basolateral membrane of cells and of intracellular Kir2.2, CASK and proteins in Kir2 channel Kir2 with SAP97, CASK, Veli, and pulldown assays and co-immunoprecipitations demonstrated that the C-terminal of the Kir2.2 channel that a interaction are the association of of the identified complex proteins CASK, Veli-1, and with the channel and using cerebellum that the identified Kir2 protein SAP97 interacts with CASK, and of proteins associate with Kir2.2 in C and pulldown assays co-immunoprecipitations with demonstrated that the complex associates with SAP97 Kir2.2 In vitro protein interaction assays that CASK to the C terminus of Kir2 channels directly associates with the channel interaction with SAP97 We the association of the Veli protein isoforms with Kir2.2, and we demonstrated that the proteins in cells directly with the Kir2.2 C terminus using in vitro interaction assays interaction via and proteins. SAP97, we demonstrated that Veli-1 and Veli-3 CASK to the Kir2 channel C terminus we that complexes of Kir2 with SAP97-CASK-Veli-Mint1 are in brain, with proteins Kir2 to the proteins In one complex, SAP97 the a model in the of SAP97 interacts directly with the Kir2 channel C terminus and recruits CASK J. Scholar). interaction to of the of SAP97 to the of CASK S. S. S. Scholar, M. J. Scholar, S. M. Scholar, M. J. Scholar). and and affinity interaction assays and that Veli and Mint1 associate with complex, by Veli with the of CASK S. S. S. and Mint1 with the kinase of CASK M. J. Scholar, S. M. Scholar, M. J. In the complex, isoforms of Veli directly to the a model in the of Veli-1 or Veli-3 directly with the Kir2 channel C terminus and CASK of the Veli to the CASK C-terminal S. S. S. Scholar, S. M. Scholar). In complex, SAP97 and Mint1 are to the Kir2 channel via interactions with the CASK terminus S. S. S. Scholar). Kir2 channels with Veli-1 and Veli-3 in the brain, and Veli isoforms distinct protein complexes. Co-immunoprecipitations with brain using that Veli-1 and Veli-3 associate with SAP97 Veli-3 stably with Mint1. that Kir2 channel complexes that Veli-1 Mint1, whereas Kir2 complexes that Veli-3 Mint1 We Veli-2 association with the C terminus of Kir2.2 or Kir2.3 or complex with SAP97 and in pulldown assays using brain that a complex Kir2 channels and SAP97 to Veli-2 in the expression in rat cerebellum of Kir2.2, SAP97, CASK, Mint1, and Veli-1, in the granule cell and of Kir2.2, SAP97, CASK, Mint1 and in the molecular using that distinct complexes in specific of the of SAP97-CASK-Veli-Mint1 proteins SAP97 and CASK to functions in trafficking and targeting ion channels and in of proteins in epithelial cells where are and to the basolateral membrane at of of SAP97 to be targeting S. J. Scholar). the of SAP97 that interacts with the of CASK S. S. S. Scholar). S. S. S. used a CASK the to SAP97 dominant CASK caused SAP97 to from basolateral in epithelial cells S. S. S. Scholar). genetic of CASK and SAP97 in Scholar, C. M. and Scholar, C. Scholar, C. A. proteins in targeting and localization in polarized that SAP97 and CASK in the trafficking and targeting of ion channels and We expressed a in cells Kir2.3 J. J. Physiol. the channels to and to the basolateral membrane and Expression of the PDZ-binding a of specific membrane localization with of the channel expressed and that the PDZ-binding of Kir2.2 a in targeting or of the channels in the basolateral the of CASK, the central protein in Kir2 complex, we a dominant CASK in cells stably resulted in the and of Kir2.2 from the basolateral membrane to intracellular and appears that the of Kir2.2 with complexes trafficking Kir2 channels. We that the complex functions in with SAP97 a in trafficking of Kir2 channels. CASK be in targeting the complex, Mint1 the complex to and Veli the complex at the plasma from and channels of the In C. the are proteins in the basolateral targeting and localization the kinase and in epithelial cells C. A. a key in Scholar). of Mint1 in trafficking demonstrated in by M. identified the protein a Mint1, association of Mint1, CASK, Veli, and and demonstrated that be by Additionally, Veli to the of the at the basolateral of epithelial cells C. C. A. R. A. J. and demonstrated plasma membrane by Veli-2 of Kir2.3 expressed in J. J. Physiol. Scholar). that Kir2 channels expressed in brain, in a to channels are to the plasma we Veli-2 with Kir2 channels or SAP97 in brain, a of the channels Veli-1 and Veli-3 in interaction of Kir2 channels with the SAP97-CASK-Veli-Mint1 complex to channels to SAP97 and CASK the protein or their and with the of with protein S. A. Scholar, J. Scholar, J. Scholar). in plasma membrane components to the C. S. 1997; Scholar). In a CASK demonstrated to to a neuronal form of protein and the C of membrane proteins J. Scholar). In and proteins to associate with SAP97 J. J. and S. J. C. M. J. Scholar). the that Kir2 channels be via SAP97, CASK, and proteins to the or and the complex of proteins in intracellular channel or channel to at the plasma of the SAP97-CASK-Veli-Mint1 complex be to form a molecular that recruits to Kir2 the channel demonstrated to associate with a of protein kinase M. Src family J. 1997; Scholar, S. S. A. and proteins M. J. Scholar, Scholar). Kir2 complex be that the complex by that protein kinase of the the PDZ-binding at the C terminus of Kir2 channels the ability of the channels to with proteins Scholar, J. Scholar). that Kir2 channels are to the plasma membrane by the SAP97-CASK-Veli-Mint1 complex, by protein kinase to Kir2 channels from the complex, by interaction with a membrane-associated and the channels. Additionally, that complex be by interactions and Y. S. J. Scholar, M. J. Scholar, C. S. S. S. J. Scholar, M. J. Scholar, J. Scholar, C. Scholar, Scholar). In we identified macromolecular complexes SAP97, CASK, Veli-1, and Mint1 that are at the C of Kir2 channels in complexes are of to Kir2 channels one to the C terminus of Kir2 channels SAP97, and the with the complex via by In distinct protein complexes are by Veli Veli-1 with CASK, SAP97, and Kir2 whereas Veli-3 with Kir2 CASK, SAP97, and Mint1. proteins to be in ion channel and localization M. Scholar, J. J. Physiol. Scholar, R. M. J. Scholar, C. J. J. Scholar). CASK a key of complex in with complex proteins to and channels at the plasma important in the of Kir2 and a of from neuronal excitability to of using the of SAP97, CASK, Veli, and Mint1 in trafficking and of Kir2 channels. We and and of the