Andersen-Tawil syndrome

Andersen-Tawil syndrome (ATS) is a rare condition consisting of ventricular arrhythmias, periodic paralysis, and dysmorphic features. In 2001, mutations in KCNJ2, which encodes the a subunit of the potassium channel Kir2.1, were identified in patients with ATS. To date, KCNJ2 is the only gene implicated in ATS, accounting for approximately 60% of cases. ATS is a unique channelopathy, and represents the first link between cardiac and skeletal muscle excitability. The arrhythmias observed in ATS are distinctive; patients may be asymptomatic, or minimally symptomatic despite a high arrhythmia burden with frequent ventricular ectopy and bidirectional ventricular tachycardia. However, patients remain at risk for life-threatening arrhythmias, including torsades de pointes and ventricular fibrillation, albeit less commonly than observed in other genetic arrhythmia syndromes. The characteristic heterogeneity at both the genotypic and phenotypic levels contribute to the continued difficulties with appropriate diagnosis, risk stratification, and effective therapy. The initial recognition of a syndromic association of clinically diverse symptoms, and the subsequent identification of the underlying molecular genetic basis of ATS has enhanced both clinical care, and our understanding of the critical function of Kir2.1 on skeletal muscle excitability and cardiac action potential.     

Kir2.6 regulates the surface expression of Kir2.x inward rectifier potassium channels

Precise trafficking, localization, and activity of inward rectifier potassium Kir2 channels are important for shaping the electrical response of skeletal muscle. However, how coordinated trafficking occurs to target sites remains unclear. Kir2 channels are tetrameric assemblies of Kir2.x subunits. By immunocytochemistry we show that endogenous Kir2.1 and Kir2.2 are localized at the plasma membrane and T-tubules in rodent skeletal muscle. Recently, a new subunit, Kir2.6, present in human skeletal muscle, was identified as a gene in which mutations confer susceptibility to thyrotoxic hypokalemic periodic paralysis. Here we characterize the trafficking and interaction of wild type Kir2.6 with other Kir2.x in COS-1 cells and skeletal muscle in vivo. Immunocytochemical and electrophysiological data demonstrate that Kir2.6 is largely retained in the endoplasmic reticulum, despite high sequence identity with Kir2.2 and conserved endoplasmic reticulum and Golgi trafficking motifs shared with Kir2.1 and Kir2.2. We identify amino acids responsible for the trafficking differences of Kir2.6. Significantly, we show that Kir2.6 subunits can coassemble with Kir2.1 and Kir2.2 in vitro and in vivo. Notably, this interaction limits the surface expression of both Kir2.1 and Kir2.2. We provide evidence that Kir2.6 functions as a dominant negative, in which incorporation of Kir2.6 as a subunit in a Kir2 channel heterotetramer reduces the abundance of Kir2 channels on the plasma membrane.     

Regulation of Kir2.1 channels by the Rho-GTPase, Rac1

Mutations in Kir2.1 inwardly rectifying potassium channels are associated with Andersen syndrome, a disease characterized by potentially fatal cardiac arrhythmias. While several Andersen-associated mutations affect membrane expression, the cytoplasmic signals that regulate Kir2.1 trafficking are poorly understood. Here, we investigated whether the Rho-family of small GTPases regulates trafficking of Kir2.1 channels expressed in HEK-293 cells. Treatment with Clostridium difficile toxin B, an inhibitor of Rho-family GTPases, or co-expression of the dominant-negative mutant of Rac1 (Rac1(DN)) increased Kir2.1 channels approximately 2-fold. However, the dominant-negative forms of other Rho-family GTPases, RhoA or Cdc42, did not alter Kir2.1 currents, suggesting a selective effect of Rac1 on Kir2.1 channels. Single-channel properties (gamma, tau(o), tau(c)) and total protein levels of Kir2.1 were unchanged with co-expression of Rac1(DN); however, studies using TIRF microscopy and CFP-tagged Kir2.1 revealed increased channel surface expression. Immunohistochemical detection of extracellularly tagged HA-Kir2.1 channels showed that Rac1(DN) reduced channel internalization when co-expressed. Finally, the dominant-negative mutant of dynamin, which interferes with endocytosis, occluded the Rac1(DN)-induced potentiation of Kir2.1 currents. These data suggest that inhibition of Rac1 increases Kir2.1 surface expression by interfering with endocytosis, likely via a dynamin-dependent pathway. Surprisingly, Rac1(DN) did not alter Kir2.2 current density or internalization, suggesting subunit specific modulation of Kir2.1 channels. Consistent with this, construction of Kir2.1/2.2 chimeras implicated the C-terminal domain of Kir2.1 in mediating the potentiating effect of Rac1(DN). This novel pathway for regulating surface expression of cardiac Kir2.1 channels could have implications for normal and diseased cardiac states.     

Identification of Novel Cholesterol-binding Regions in Kir2 Channels

Inwardly rectifying potassium (Kir) channels play an important role in setting the resting membrane potential and modulating membrane excitability. We have recently shown that cholesterol regulates representative members of the Kir family and that in the majority of the cases, cholesterol suppresses channel function. Furthermore, recent data indicate that cholesterol regulates Kir channels by specific sterol-protein interactions, yet the location of the cholesterol binding site in Kir channels is unknown. Using a combined computational-experimental approach, we show that cholesterol may bind to two nonanular hydrophobic regions in the transmembrane domain of Kir2.1 located between adjacent subunits of the channel. The location of the binding regions suggests that cholesterol modulates channel function by affecting the hinging motion at the center of the pore-lining transmembrane helix that underlies channel gating either directly or through the interface between the N and C termini of the channel.     

Initiation of human myoblast differentiation via dephosphorylation of Kir2.1 K+ channels at tyrosine 242

Myoblast differentiation is essential to skeletal muscle formation and repair. The earliest detectable event leading to human myoblast differentiation is an upregulation of Kir2.1 channel activity, which causes a negative shift (hyperpolarization) of the resting potential of myoblasts. After exploring various mechanisms, we found that this upregulation of Kir2.1 was due to dephosphorylation of the channel itself. Application of genistein, a tyrosine kinase inhibitor, increased Kir2.1 activity and triggered the differentiation process, whereas application of bpV(Phen), a tyrosine phosphatase inhibitor, had the opposite effects. We could show that increased Kir2.1 activity requires dephosphorylation of tyrosine 242; replacing this tyrosine in Kir2.1 by a phenylalanine abolished inhibition by bpV(Phen). Finally, we found that the level of tyrosine phosphorylation in endogenous Kir2.1 channels is considerably reduced during differentiation when compared with proliferation. We propose that Kir2.1 channels are already present at the membrane of proliferating, undifferentiated human myoblasts but in a silent state, and that Kir2.1 tyrosine 242 dephosphorylation triggers differentiation.     

Identification of gamma-aminobutyric acid receptor-interacting factor 1 (TRAK2) as a trafficking factor for the K+ channel Kir2.1

To identify proteins that regulate potassium channel activity and expression, we performed functional screening of mammalian cDNA libraries in yeast that express the mammalian K(+) channel Kir2.1. Growth of Kir2.1-expressing yeast in media with low K(+) concentration is a function of K(+) uptake via Kir2.1 channels. Therefore, the host strain was transformed with a human cDNA library, and cDNA clones that rescued growth at low K(+) concentration were selected. One of these clones was identical to the protein of unknown function isolated previously as gamma-aminobutyric acid receptor-interacting factor 1 (GRIF-1) (Beck, M., Brickley, K., Wilkinson, H., Sharma, S., Smith, M., Chazot, P., Pollard, S., and Stephenson, F. (2002) J. Biol. Chem. 277, 30079-30090). GRIF-1 specifically enhanced Kir2.1-dependent growth in yeast and Kir2.1-mediated (86)Rb(+) efflux in HEK293 cells. Quantitative microscopy and flow cytometry analysis of immunolabeled surface Kir2.1 channel showed that GRIF-1 significantly increased the number of Kir2.1 channels in the plasma membrane of COS and HEK293 cells. Physical interaction of Kir2.1 channel and GRIF-1 was demonstrated by co-immunoprecipitation from HEK293 lysates and yeast two-hybrid assay. In vivo association of Kir2.1 and GRIF-1 was demonstrated by co-immunoprecipitation from brain lysate. Yeast two-hybrid assays showed that an N-terminal region of GRIF-1 interacts with a C-terminal region of Kir2.1. These results indicate that GRIF-1 binds to Kir2.1 and facilitates trafficking of this channel to the cell surface.     

Andersen's syndrome mutation effects on the structure and assembly of the cytoplasmic domains of Kir2.1

Kir2.1 channels play a key role in maintaining the correct resting potential in eukaryotic cells. Recently, specific amino acid mutations in the Kir2.1 inwardly rectifying potassium channel have been found to cause Andersen's Syndrome in humans. Here, we have characterized individual Andersen's Syndrome mutants R218Q, G300V, E303K, and delta314-315 and have found multiple effects on the ability of the cytoplasmic domains in Kir2.1 channels to form proper tetrameric assemblies. For the R218Q mutation, we identified a second site mutation (T309K) that restored tetrameric assembly but not function. We successfully crystallized and solved the structure (at 2.0 A) of the N- and C-terminal cytoplasmic domains of Kir2.1-R218Q/T309K(S). This new structure revealed multiple conformations of the G-loop and CD loop, providing an explanation for channels that assemble but do not conduct ions. Interestingly, Glu303 forms both intra- and intersubunit salt bridges, depending on the conformation of the G-loop, suggesting that the E303K mutant stabilizes both closed and open G-loop conformations. In the Kir2.1-R218Q/T309K(S) structure, we discovered that the DE loop forms a hydrophobic pocket that binds 2-methyl-2,4-pentanediol, which is located near the putative G(betagamma)-activation site of Kir3 channels. Finally, we observed a potassium ion bound to the cytoplasmic domain for this class of K+ channels.     

Cholesterol sensitivity and lipid raft targeting of Kir2.1 channels

This study investigates how changes in the level of cellular cholesterol affect inwardly rectifying K+ channels belonging to a family of strong rectifiers (Kir2). In an earlier study we showed that an increase in cellular cholesterol suppresses endogenous K+ current in vascular endothelial cells, presumably due to effects on underlying Kir2.1 channels. Here we show that, indeed, cholesterol increase strongly suppressed whole-cell Kir2.1 current when the channels were expressed in a null cell line. However, cholesterol level had no effect on the unitary conductance and only little effect on the open probability of the channels. Moreover, no cholesterol effect was observed either on the total level of Kir2.1 protein or on its surface expression. We suggest, therefore, that cholesterol modulates not the total number of Kir2.1 channels in the plasma membrane but rather the transition of the channels between active and silent states. Comparing the effects of cholesterol on members of the Kir2.x family shows that Kir2.1 and Kir2.2 have similar high sensitivity to cholesterol, Kir2.3 is much less sensitive, and Kir2.4 has an intermediate sensitivity. Finally, we show that Kir2.x channels partition virtually exclusively into Triton-insoluble membrane fractions indicating that the channels are targeted into cholesterol-rich lipid rafts.     

Direct interaction between the actin-binding protein filamin-A and the inwardly rectifying potassium channel,Kir2.1

The role of filamins in actin cross-linking and membrane stabilization is well established, but recently their ability to interact with a variety of transmembrane receptors and signaling proteins has led to speculation of additional roles in scaffolding and signal transduction. Here we report a direct interaction between filamin-A and Kir2.1, an isoform of inwardly rectifying potassium channel expressed in vascular smooth muscle and an important regulator of vascular tone. Yeast two-hybrid screening of a porcine coronary artery cDNA library using the carboxyl terminus of Kir2.1 as bait yielded cDNA encoding a fragment of filamin-A (residues 2481-2647). Interaction between filamin-A and Kir2.1 was confirmed by in vitro overlay assay of membrane-bound Kir2.1 with glutathione S-transferase fusion protein of the isolated filamin clone. Additionally, antibodies directed against Kir2.1 coimmunoprecipitated filamin-A from arterial smooth muscle cell lysates, and immunocytochemical analysis of individual arterial smooth muscle cells showed that Kir2.1 and filamin co-localize in "hotspots" at the cell membrane. Interaction with filamin-A was found to have no effect on Kir2.1 channel behavior but, rather, increased the number of functional channels resident within the membrane. We conclude that filamin-A is potentially an important regulator of Kir2.1 surface expression and location within vascular smooth muscle.     

Zacopride selectively activates the Kir2.1 channel via a PKA signaling pathway in rat cardiomyocytes

We recently reported that zacopride is a selective inward rectifier potassium current (IK1 ) channel agonist, suppressing ventricular arrhythmias without affecting atrial arrhythmias. The present study aimed to investigate the unique pharmacological properties of zacopride. The whole-cell patch-clamp technique was used to study IK1 currents in rat atrial myocytes and Kir2.x currents in human embryonic kidney (HEK)-293 cells transfected with inward rectifier potassium channel (Kir)2.1, Kir2.2, Kir2.3, or mutated Kir2.1 (at phosphorylation site S425L). Western immunoblots were performed to estimate the relative protein expression levels of Kir2.x in rat atria and ventricles. Results showed that zacopride did not affect the IK1 and transmembrane potential of atrial myocytes. In HEK293 cells, zacopride increased Kir2.1 homomeric channels by 40.7%±9.7% at 50 mV, but did not affect Kir2.2 and Kir2.3 homomeric channels, and Kir2.1-Kir2.2, Kir2.1-Kir2.3 and Kir2.2-Kir2.3 heteromeric channels. Western immunoblots showed that similar levels of Kir2.3 protein were expressed in rat atria and ventricles, but atrial Kir2.1 protein level was only 25% of that measured in the ventricle. In addition, 5-hydroxytryptamine (5-HT) 3 receptor was undetectable, whereas 5-HT 4 receptor was weakly expressed in HEK293 cells. The Kir2.1-activating effect of zacopride in these cells was abolished by inhibition of protein kinase A (PKA), but not PKC or PKG. Furthermore, zacopride did not activate the mutant Kir2.1 channel in HEK293 cells but selectively activated the Kir2.1 homomeric channel via a PKA-dependent pathway, independent to that of the 5-HT receptor.     

Stoichiometry of Kir channels with phosphatidylinositol bisphosphate

Phosphatidylinositol bisphosphate (PIP2) is the most abundant phosphoinositide in the plasma membranes of cells and its interaction with many ion channel proteins has proven to be a critical factor enabling ion channel gating. All members of the inwardly rectifying potassium (Kir) channel family depend on PIP2 for their activity, displaying distinct affinities and stereospecificities of interaction with the phosphoinositide. Here, we explored the stoichiometry of Kir channels with PIP2. We first showed that PIP2 regulated the activity of Kir3.4 channels mainly by altering their bursting behavior. Detailed burst analysis indicates that the channels assumed up to four open states and a connectivity of four between open and closed states depending on the available PIP2 levels. Moreover, by controlling the number of PIP2-sensitive subunits in the stoichiometry of a tetrameric Kir2.1 channel, we showed that characteristic channel activity was obtained when at least two wild-type subunits were present. Our studies support a kinetic model for gating of Kir channels by PIP2, where each of the four open states corresponds to the channel activated by one to four PIP2 molecules.     

Heteromeric assembly of inward rectifier channel subunit Kir2.1 with Kir3.1 and with Kir3.4

Heteromultimerization of different pore-forming subunits is known to contribute to the diversity of inward rectifier K⁺ channels. We examined if the subunits belonging to different subfamilies Kir2 and Kir3 can co-assemble to form heteromultimers in heterologous expression systems. We observed co-immunoprecipitation of Kir2.1 and Kir3.1 as well as Kir2.1 and Kir3.4 in HEK293T cells. Furthermore, analyses of subcellular localization using confocal microscopy revealed that co-expression of Kir2.1 promoted the cell surface localization of Kir3.1 and Kir3.4 in HEK293T cells. In electrophysiological experiments, co-expression of Kir2.1 with Kir3.1 and/or Kir3.4 in Xenopus oocytes and HEK293T cells did not yield currents with distinguishable features. However, co-expression of a dominant-negative Kir2.1 with the wild-type Kir3.1/3.4 decreased the Kir3.1/3.4 current amplitude in Xenopus oocytes. The results indicate that Kir2.1 is capable of forming heteromultimeric channels with Kir3.1 and with Kir3.4.     

Comparative analysis of cholesterol sensitivity of Kir channels: Role of the CD loop

Kir channels are important in setting the resting membrane potential and modulating membrane excitability. A common feature of Kir2 channels and several other ion channels that has emerged in recent years is that they are regulated by cholesterol, a major lipid component of the plasma membrane whose excess is associated with multiple pathological conditions. Yet, the mechanism by which cholesterol affects channel function is not clear. We have recently shown that the sensitivity of Kir2 channels to cholesterol depends on residues in the CD loop of the cytosolic domain of the channels with one of the mutations, L222I, abrogating cholesterol sensitivity of the channels completely. Here we show that in addition to Kir2 channels, members of other Kir subfamilies are also regulated by cholesterol. Interestingly, while similarly to Kir2 channels, several Kir channels, Kir1.1, Kir4.1 and Kir6.2Delta36 were suppressed by an increase in membrane cholesterol, the function of Kir3.4* and Kir7.1 was enhanced following cholesterol enrichment. Furthermore, we show that independent of the impact of cholesterol on channel function, mutating residues in the corresponding positions of the CD loop in Kir2.1 and Kir3.4*, inhibits cholesterol sensitivity of Kir channels, thus extending the critical role of the CD loop beyond Kir2 channels.     

Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel

Tyrosine side chains participate in several distinct signaling pathways, including phosphorylation and membrane trafficking. A nonsense suppression procedure was used to incorporate a caged tyrosine residue in place of the natural tyrosine at position 242 of the inward rectifier channel Kir2.1 expressed in Xenopus oocytes. When tyrosine kinases were active, flash decaging led both to decreased K(+) currents and also to substantial (15-26%) decreases in capacitance, implying net membrane endocytosis. A dominant negative dynamin mutant completely blocked the decaging-induced endocytosis and partially blocked the decaging-induced K(+) channel inhibition. Thus, decaging of a single tyrosine residue in a single species of membrane protein leads to massive clathrin-mediated endocytosis; in fact, membrane area equivalent to many clathrin-coated vesicles is withdrawn from the oocyte surface for each Kir2.1 channel inhibited. Oocyte membrane proteins were also labeled with the thiol-reactive fluorophore tetramethylrhodamine-5-maleimide, and manipulations that decreased capacitance also decreased surface membrane fluorescence, confirming the net endocytosis. In single-channel studies, tyrosine kinase activation decreased the membrane density of active Kir2.1 channels per patch but did not change channel conductance or open probability, in agreement with the hypothesis that tyrosine phosphorylation results in endocytosis of Kir2.1 channels. Despite the Kir2.1 inhibition and endocytosis stimulated by tyrosine kinase activation, neither Western blotting nor (32)P labeling produced evidence for direct tyrosine phosphorylation of Kir2.1. Therefore, it is likely that tyrosine phosphorylation affects Kir2.1 function indirectly, via interactions between clathrin adaptor proteins and a tyrosine-based sorting motif on Kir2.1 that is revealed by decaging the tyrosine side chain. These interactions inhibit a fraction of the Kir2.1 channels, possibly via direct occlusion of the conduction pathway, and also lead to endocytosis, which further decreases Kir2.1 currents. These data establish that side chain decaging can provide valuable time-resolved data about intracellular signaling systems.     

Characterization of a novel, dominant negative KCNJ2 mutation associated with Andersen-Tawil syndrome

Andersen-Tawil syndrome is characterized by periodic paralysis, ventricular ectopy, and dysmorphic features. Approximately 60% of patients exhibit loss-of-function mutations in KCNJ2, which encodes the inwardly rectifying K(+) channel pore forming subunit Kir2.1. Here, we report the identification of a novel KCNJ2 mutation (G211T), resulting in the amino acid substitution D71Y, in a patient presenting with signs and symptoms of Andersen-Tawil syndrome. The functional properties of the mutant subunit were characterized using voltage-clamp experiments on transiently transfected HEK-293 cells and neonatal mouse ventricular myocytes. Whole-cell current recordings of transfected HEK-293 cells demonstrated that the mutant protein Kir2.1-D71Y fails to form functional ion channels when expressed alone, but co-assembles with wild-type Kir2.1 subunits and suppresses wild-type subunit function. Further analysis revealed that current suppression requires at least two mutant subunits per channel. The D71Y mutation does not measurably affect the membrane trafficking of either the mutant or the wild-type subunit or alter the kinetic properties of the currents. Additional experiments revealed that expression of the mutant subunit suppresses native I(K1) in neonatal mouse ventricular myocytes. Simulations predict that the D71Y mutation in human ventricular myocytes will result in a mild prolongation of the action potential and potentially increase cell excitability. These experiments indicate that the Kir2.1-D71Y mutant protein functions as a dominant negative subunit resulting in reduced inwardly rectifying K(+) current amplitudes and altered cellular excitability in patients with Andersen-Tawil syndrome.     

Expression of Kir2.1 channels in astrocytes under pathophysiological conditions

Astrocyte ion channels participate in ionic homeostasis in the brain. Inward rectifying potassium channels (Kir channels) in astrocytes have been particularly implicated in K(+) homeostasis because of their high open probability at resting potential and their increased conductance at high concentrations of extracellular K(+). We examined the expression of the Kir2.1 subunit, one of the Kir channel subunits, in the mouse brain by immunohistochemistry. Kir2.1 channels were widely distributed throughout the brain, with high expression in the olfactory bulb and the cerebellum. Interestingly, they were abundantly expressed in astrocytes of the olfactory bulb, while astrocytes in other brain regions including the hippocampus did not show any detectable expression. However, Kir2.1 channel-expressing cells were dramatically increased in the hippocampus by kainic acid-induced seizure and the cells were glial fibrillary acidic protein (GFAP)-positive, which confirms that astrocytes in the hippocampus express Kir2.1 channels under pathological conditions. Our results imply that Kir2.1 channels in astrocyte may be involved in buffering K(+) against accumulated extracellular K(+) caused by neuronal hyperexcitability under phathophysiological conditions.     

Dual-mode phospholipid regulation of human inward rectifying potassium channels

The lipid bilayer is a critical determinant of ion channel activity; however, efforts to define the lipid dependence of channel function have generally been limited to cellular expression systems in which the membrane composition cannot be fully controlled. We reconstituted purified human Kir2.1 and Kir2.2 channels into liposomes of defined composition to study their phospholipid dependence of activity using ⁸⁶Rb⁺ flux and patch-clamp assays. Our results demonstrate that Kir2.1 and Kir2.2 have two distinct lipid requirements for activity: a specific requirement for phosphatidylinositol 4,5-bisphosphate (PIP₂) and a nonspecific requirement for anionic phospholipids. Whereas we previously showed that PIP₂ increases the channel open probability, in this work we find that activation by POPG increases both the open probability and unitary conductance. Oleoyl CoA potently inhibits Kir2.1 by antagonizing the specific requirement for PIP₂, and EPC appears to antagonize activation by the nonspecific anionic requirement. Phosphatidylinositol phosphates can act on both lipid requirements, yielding variable and even opposite effects on Kir2.1 activity depending on the lipid background. Mutagenesis experiments point to the role of intracellular residues in activation by both PIP₂ and anionic phospholipids. In conclusion, we utilized purified proteins in defined lipid membranes to quantitatively determine the phospholipid requirements for human Kir channel activity.     

Protein trafficking and anchoring complexes revealed by proteomic analysis of inward rectifier potassium channel (Kir2.x)-associated proteins

Inward rectifier potassium (Kir) channels play important roles in the maintenance and control of cell excitability. Both intracellular trafficking and modulation of Kir channel activity are regulated by protein-protein interactions. We adopted a proteomics approach to identify proteins associated with Kir2 channels via the channel C-terminal PDZ binding motif. Detergent-solubilized rat brain and heart extracts were subjected to affinity chromatography using a Kir2.2 C-terminal matrix to purify channel-interacting proteins. Proteins were identified with multidimensional high pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry, N-terminal microsequencing, and immunoblotting with specific antibodies. 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 actin-binding LIM protein (abLIM) as Kir2.2-associated brain proteins. From heart extract purifications, SAP97, CASK, Veli-3, and Mint1 also were found to associate with Kir2 channels. Furthermore, we demonstrate for the first time that components of the dystrophin-associated protein complex, including alpha1-, beta1-, and beta2-syntrophin, dystrophin, and dystrobrevin, interact with Kir2 channels, as demonstrated by immunoaffinity purification and affinity chromatography from skeletal and cardiac muscle and brain. Affinity pull-down experiments revealed that Kir2.1, Kir2.2, Kir2.3, and Kir4.1 all bind to scaffolding proteins but with different affinities for the dystrophin-associated protein complex and SAP97, CASK, and Veli. Immunofluorescent localization studies demonstrated that Kir2.2 co-localizes with syntrophin, dystrophin, and dystrobrevin at skeletal muscle neuromuscular junctions. These results suggest that Kir2 channels associate with protein complexes that may be important to target and traffic channels to specific subcellular locations, as well as anchor and stabilize channels in the plasma membrane.