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) 1 channels are a widely expressed family of ion channel proteins distinguished by their ability to pass K ϩ 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 (1, 2). 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 (3)).The function, localization, and trafficking behavior of many ion channels are regulated by interactions of their intracellular domains with members of the MAGUK protein family. MAGUK proteins have a characteristic domain structure containing up to three PDZ domains, a Src homology 3 (SH3) domain, and a catalytically inactive guanylate kinase-like domain (GK). These domains interact with numerous other proteins allowing MAGUK proteins to particip...

Protein Trafficking and Anchoring Complexes Revealed by Proteomic Analysis of Inward Rectifier Potassium Channel (Kir2.x)-associated Proteins

“…4). Mint1 is known to form a complex with CASK and Veli in brain (40,41), and our recent studies demonstrate that this complex is associated with Kir2 channels in brain and heart (18). No DAPC proteins or Mint1 were detected in the control Kir2.2⌬3 brain eluates, demonstrating that the intact PDZ binding motif is essential for their association with Kir2.2 (Fig.…”

“…The C-terminal segments of the fusion constructs coded for Kir2.1 (mouse Kir2.1 amino acids 372-428), Kir2.2 (rat Kir2.2 amino acids 362-427), Kir2.2⌬3 (rat Kir2.2 amino acids 362-424), Kir2.3 (human Kir2.3 amino acids 390 -445), and rat Kir4.1 (amino acids 329 -379). cDNAs encoding full-length Kir2.2 and Kir2.2⌬3 were subcloned into pcDNA1 as described previously (18). cDNAs encoding ␣1-, ␤1-, and ␤2-syntrophin were subcloned into pGW1.…”

“…CASK, Dlg2, Dlg3, and Pals2 are similar to one another in domain structure, with a single PDZ domain followed by SH3 and GK domains, but with significantly different N termini. The PDZ domains of CASK, Dlg2, Dlg3, and Pals2 are class II PDZ domains, and, unlike the PDZ domains of SAP97-like MAGUK proteins, they are predicted to be incapable of direct interaction with Kir2.2 Indeed, we recently have shown that CASK does not bind directly to the channel, but instead interacts with the channel through its tight association with SAP97 or Veli (18,36,37).…”

“…COS-1 Cell Protein Expression and Co-immunoprecipitation-COS-1 cells were co-transfected with ␣1-, ␤1-, or ␤2-syntrophin with Kir2.2 or Kir2.2⌬3 utilizing FuGENE 6 (Roche Applied Science) and used for co-immunoprecipitation as described (18).…”

“…We identified eight members of the MAGUK family of proteins (SAP97, PSD-95, Chapsyn-110, SAP102, CASK, Dlg2, Dlg3, and Pals2), two isoforms of Veli (Veli-1 and Veli-3), Mint1, and the actinbinding LIM protein (abLIM) as Kir2.2-associated proteins. Several of these proteins were investigated in detail in a parallel study in which we demonstrated that SAP97, CASK, Veli-3, and Mint1 form a multiprotein complex with Kir2 channels in brain and heart and showed that a CASK-containing complex is involved in channel trafficking (18). Here, we also show for the first time that components of the dystrophinassociated protein complex (DAPC), including ␣1-, ␤1-, and ␤2-syntrophin, dystrophin, and dystrobrevin, associate with the C terminus of Kir2 channels in muscle and brain.…”