Mapping of the physical interaction between the intracellular domains of an inwardly rectifying potassium channel, Kir6.2

The amino-terminal and carboxyl-terminal domains of inwardly rectifying potassium (Kir) channel subunits are both intracellular. There is increasing evidence that both of these domains are required for the regulation of Kir channels by agents such as G-proteins and nucleotides. Kir6.2 is the pore-forming subunit of the ATP-sensitive K(+) (K(ATP)) channel. Using an in vitro protein-protein interaction assay, we demonstrate that the two intracellular domains of Kir6.2 physically interact with each other, and we map a region within the N terminus that is responsible for this interaction. "Cross-talk" through this interaction may explain how mutations in either the N or C terminus can influence the intrinsic ATP-sensitivity of Kir6.2. Interestingly, the "interaction domain" is highly conserved throughout the superfamily of Kir channels. The N-terminal interaction domain of Kir6.2 can also interact with the C terminus of both Kir6.1 and Kir2.1. Furthermore, a mutation within the conserved region of the N-terminal interaction domain, which disrupts its interaction with the C terminus, severely compromised the ability of both Kir6.2 and Kir2.1 to form functional channels, suggesting that this interaction may be a feature common to all members of the Kir family of potassium channels.     

Golgi export of the Kir2.1 channel is driven by a trafficking signal located within its tertiary structure

Mechanisms that are responsible for sorting newly synthesized proteins for traffic to the cell surface from the Golgi are poorly understood. Here, we show that the potassium channel Kir2.1, mutations in which are associated with Andersen-Tawil syndrome, is selected as cargo into Golgi export carriers in an unusual signal-dependent manner. Unlike conventional trafficking signals, which are typically comprised of short linear peptide sequences, Golgi exit of Kir2.1 is dictated by residues that are embedded within the confluence of two separate domains. This signal patch forms a recognition site for interaction with the AP1 adaptor complex, thereby marking Kir2.1 for incorporation into clathrin-coated vesicles at the trans-Golgi. The identification of a trafficking signal in the tertiary structure of Kir2.1 reveals a quality control step that couples protein conformation to Golgi export and provides molecular insight into how mutations in Kir2.1 arrest the channels at the Golgi.     

C-Terminal Determinants of Kir4.2 Channel Expression

Inward rectifier potassium (Kir) channels serve important functional and modulatory roles in a wide variety of cells. While the activity of several members of this channel family are tightly regulated by intracellular messengers such as adenosine triphosphate, G proteins, protein kinases and pH, other members are tonically active and activity is controlled only by the expression level of the protein. In a number of Kir channels, sequence motifs have been identified which determine how effectively the channel is trafficked to and from the plasma membrane. In this report, we identify a number of trafficking determinants in the Kir4.2 channel. Using mutational analysis, we found that truncation of the C terminus of the protein increased current density in Xenopus oocytes, although multiple mutations of the C terminus had no effect on current density. Instead, mutation of a unique region of the channel significantly increased current density. Selective mutation of a putative tyrosine phosphorylation site within this region mimicked the increase in current, suggesting that tyrosine phosphorylation of the protein increases channel retrieval from the membrane (or prevents trafficking to the membrane). Mutation of a previously identified trafficking determinant, K110N, also caused an increase in current density, and combining these mutations caused a multiplicative increase in current, suggesting that these two mutations increase current by independent mechanisms. These data demonstrate novel determinants of Kir4.2 channel expression.     

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.     

Suppression of Kir2.3 activity by protein kinase C phosphorylation of the channel protein at threonine 53

Kir2.3 plays an important part in the maintenance of membrane potential in neurons and myocardium. Identification of intracellular signaling molecules controlling this channel thus may lead to an understanding of the regulation of membrane excitability. To determine whether Kir2.3 is modulated by direct phosphorylation of its channel protein and identify the phosphorylation site of protein kinase C (PKC), we performed experiments using several recombinant and mutant Kir2.3 channels. Whole-cell Kir2.3 currents were inhibited by phorbol 12-myristate 13-acetate (PMA) in Xenopus oocytes. When the N-terminal region of Kir2.3 was replaced with that of Kir2.1, another member in the Kir2 family that is insensitive to PMA, the chimerical channel lost its PMA sensitivity. However, substitution of the C terminus was ineffective. Four potential PKC phosphorylation sites in the N terminus were studied by comparing mutations of serine or threonine with their counterpart residues in Kir2.1. Whereas substitutions of serine residues at positions 5, 36, and 39 had no effect on the channel sensitivity to PMA, mutation of threonine 53 completely eliminated the channel response to PMA. Interestingly, creation of this threonine residue at the corresponding position (I79T) in Kir2.1 lent the mutant channel a PMA sensitivity almost identical to the wild-type Kir2.3. These results therefore indicate that Kir2.3 is directly modulated by PKC phosphorylation of its channel protein and threonine 53 is the PKC phosphorylation site in Kir2.3.     

A novel,radiolabel-free pulse chase strategy to study Kir3 channel ontogeny

Abstract

Kir3 channels are essential regulators of cellular excitability, maintaining cells at resting membrane potentials. While much research has been dedicated to elucidating the mechanisms regulating Kir3 channel gating, little is known regarding the channel’s early associations with signaling partners, its stability at the plasma membrane or mechanisms regulating its internalization and degradation. To address these issues we have established an inducible Kir3.1 cell line that allows monitoring of a discrete “pulse” of channel as it progresses along the biosynthetic pathway. Using this system, we have been able to track Kir3 maturation and the influence of partner subunits on Kir3 lifetime and stability. Of note, we show that Kir3.1, in the absence of trafficking partner subunits, can exit the endoplasmic reticulum (ER) and reach the Golgi (though not the plasma membrane), and that expression of Kir3.3 subunits drastically reduced levels of Kir3.1 in the cell. We also show that interfering with trafficking from the ER to Golgi has a pronounced inhibitory effect on Kir3.1-Kir3.2 interactions, suggesting that this complex is stabilized either en route to the Golgi or in the Golgi itself. Finally, we showed that the Kir3 channel can reach the cell surface as early as 6?h post-induction and that removal of cell surface-localized channel occurs within 48?h. This system can be adapted to study the life cycle of any cellular protein without the confounds associated with radioactive labeling or the complications noted with expressing supraphysiological levels of proteins.     

Topogenesis of two transmembrane type K+ channels,Kir 2.1 and KcsA

Potassium channels, which control the passage of K+ across cell membranes, have two transmembrane segments, M1 and M2, separated by a hydrophobic P region containing a highly conserved signature sequence. Here we analyzed the membrane topogenesis characteristics of the M1, M2, and P regions in two animal and bacterial two-transmembrane segment-type K+ channels, Kir 2.1 and KcsA, using an in vitro translation and translocation system. In contrast to the equivalent transmembrane segment, S5, in the voltage-dependent K+ channel, KAT1, the M1 segment in KcsA, was found to have a strong type II signal-anchor function, which favors the Ncyt/Cexo topology. The N-terminal cytoplasmic region was required for efficient, correctly orientated integration of M1 in Kir 2.1. Analysis of N-terminal modification by in vitro metabolic labeling showed that the N terminus in Kir 2.1 was acetylated. The hydrophobic P region showed no topogenic function, allowing it to form a loop, but not a transmembrane structure in the membrane; this region was transiently exposed in the endoplasmic reticulum lumen during the membrane integration process. M2 was found to possess a stop-transfer function and a type I signal-anchor function, enabling it to span the membrane. The C-terminal cytoplasmic region in KcsA was found to affect the efficiency with which the M2 achieved their final structure. Comparative topogenesis studies of Kir 2.1 and KcsA allowed quantification of the relative contributions of each segment and the cytoplasmic regions to the membrane topology of these two proteins. The membrane topogenesis of the pore-forming structure is discussed using results for Kir 2.1, KcsA, and KAT1.     

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.     

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.     

Identification of the PIP2-binding site on Kir6.2 by molecular modelling and functional analysis

ATP-sensitive potassium (K(ATP)) channels couple cell metabolism to electrical activity by regulating K(+) fluxes across the plasma membrane. Channel closure is facilitated by ATP, which binds to the pore-forming subunit (Kir6.2). Conversely, channel opening is potentiated by phosphoinositol bisphosphate (PIP(2)), which binds to Kir6.2 and reduces channel inhibition by ATP. Here, we use homology modelling and ligand docking to identify the PIP(2)-binding site on Kir6.2. The model is consistent with a large amount of functional data and was further tested by mutagenesis. The fatty acyl tails of PIP(2) lie within the membrane and the head group extends downwards to interact with residues in the N terminus (K39, N41, R54), transmembrane domains (K67) and C terminus (R176, R177, E179, R301) of Kir6.2. Our model suggests how PIP(2) increases channel opening and decreases ATP binding and channel inhibition. It is likely to be applicable to the PIP(2)-binding site of other Kir channels, as the residues identified are conserved and influence PIP(2) sensitivity in other Kir channel family members.     

Targeting of an A kinase-anchoring protein, AKAP79, to an inwardly rectifying potassium channel, Kir2.1

Protein kinase A (PKA) is targeted to discrete subcellular locations close to its intended substrates through interaction with A kinase-anchoring proteins (AKAPs). Ion channels represent a diverse and important group of kinase substrates, and it has been shown that membrane targeting of PKA through association with AKAPs facilitates PKA-mediated phosphorylation and regulation of several classes of ion channel. Here, we investigate the effect of AKAP79, a membrane-associated multivalent-anchoring protein, upon the function and modulation of the strong inwardly rectifying potassium channel, Kir2.1. Functionally, the presence of AKAP79 enhanced the response of Kir2.1 to elevated intracellular cAMP, suggesting a requirement for a pool of PKA anchored close to the channel. Antibodies directed against a hemagglutinin epitope tag on Kir2.1 coimmunoprecipitated AKAP79, indicating that the two proteins exist together in a complex within intact cells. In support of this, glutathione S-transferase fusion proteins of both the intracellular N and C domains of Kir2.1 isolated AKAP79 from cell lysates, while glutathione S-transferase alone failed to interact with AKAP79. Together, these findings suggest that AKAP79 associates directly with the Kir2.1 ion channel and may serve to anchor kinase enzymes in close proximity to key channel phosphorylation sites.     

A multiprotein trafficking complex composed of SAP97, CASK, Veli, and Mint1 is associated with inward rectifier Kir2 potassium 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.     

Specific Sorting and Post-Golgi Trafficking of Dendritic Potassium Channels in Living Neurons

Proper membrane localization of ion channels is essential for the function of neuronal cells. Particularly, the computational ability of dendrites depends on the localization of different ion channels in specific subcompartments. However, the molecular mechanisms that control ion channel localization in distinct dendritic subcompartments are largely unknown. Here, we developed a quantitative live cell imaging method to analyze protein sorting and post-Golgi vesicular trafficking. We focused on two dendritic voltage-gated potassium channels that exhibit distinct localizations: Kv2.1 in proximal dendrites and Kv4.2 in distal dendrites. Our results show that Kv2.1 and Kv4.2 channels are sorted into two distinct populations of vesicles at the Golgi apparatus. The targeting of Kv2.1 and Kv4.2 vesicles occurred by distinct mechanisms as evidenced by their requirement for specific peptide motifs, cytoskeletal elements, and motor proteins. By live cell and super-resolution imaging, we identified a novel trafficking machinery important for the localization of Kv2.1 channels. Particularly, we identified non-muscle myosin II as an important factor in Kv2.1 trafficking. These findings reveal that the sorting of ion channels at the Golgi apparatus and their subsequent trafficking by unique molecular mechanisms are crucial for their specific localizations within dendrites.     

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.     

Self-directed assembly and clustering of the cytoplasmic domains of inwardly rectifying Kir2.1 potassium channels on association with PSD-95

The interaction of the extra-membranous domain of tetrameric inwardly rectifying Kir2.1 ion channels (Kir2.1NC(4)) with the membrane associated guanylate kinase protein PSD-95 has been studied using Transmission Electron Microscopy in negative stain. Three types of complexes were observed in electron micrographs corresponding to a 1:1 complex, a large self-enclosed tetrad complex and extended chains of linked channel domains. Using models derived from small angle X-ray scattering experiments in which high resolution structures from X-ray crystallographic and Nuclear Magnetic Resonance studies are positioned, the envelopes from single particle analysis can be resolved as a Kir2.1NC(4):PSD-95 complex and a tetrad of this unit (Kir2.1NC(4):PSD-95)(4). The tetrad complex shows the close association of the Kir2.1 cytoplasmic domains and the influence of PSD-95 mediated self-assembly on the clustering of these channels.     

Phosphatidylinositol 4,5-bisphosphate (PIP2) modulation of ATP and pH sensitivity in Kir channels. A tale of an active and a silent PIP2 site in the N terminus

Phosphatidylinositol polyphosphates (PIPs) are potent modulators of Kir channels. Previous studies have implicated basic residues in the C terminus of Kir6.2 channels as interaction sites for the PIPs. Here we examined the role of the N terminus and identified an arginine (Arg-54) as a major determinant for PIP(2) modulation of ATP sensitivity in K(ATP) channels. Mutation of Arg-54 to the neutral glutamine (R54Q) and, in particular, to the negatively charged glutamate (R54E) impaired PIP(2) modulation of ATP inhibition, while mutation to lysine (R54K) had no effect. These data suggest that electrostatic interactions between PIP(2) and Arg-54 are an essential step for the modulation of ATP sensitivity. This N-terminal PIP(2) site is highly conserved in Kir channels with the exception of the pH-gated channels Kir1.1, Kir4.1, and Kir5.1 that contain a neutral residue at the corresponding positions. Introduction of an arginine at this position in Kir1.1 channels rendered the N-terminal PIP(2) site functional largely increasing the PIP(2) affinity. Moreover, Kir1.1 channels lose the ability to respond to physiological changes of the intracellular pH. These results explain the need of a silent N-terminal PIP(2) site in pH-gated channels and highlight the N terminus as an important region for PIP(2) modulation of Kir channel gating.     

N4WBP5, a potential target for ubiquitination by the Nedd4 family of proteins, is a novel Golgi-associated protein.

Nedd4 belongs to a family of ubiquitin-protein ligases that is characterized by 2--4 WW domains, a carboxyl-terminal Hect (homologous to E6-AP Carboxyl terminus)domain and in most cases an amino-terminal C2 domain. We had previously identified a series of proteins that associates with the WW domains of Nedd4. In this paper, we demonstrate that one of the Nedd4-binding proteins, N4WBP5, belongs to a small group of evolutionarily conserved proteins with three transmembrane domains. N4WBP5 binds Nedd4 WW domains via the two PPXY motifs present in the amino terminus of the protein. In addition to Nedd4, N4WBP5 can interact with the WW domains of a number of Nedd4 family members and is ubiquitinated. Endogenous N4WBP5 localizes to the Golgi complex. Ectopic expression of the protein disrupts the structure of the Golgi, suggesting that N4WBP5 forms part of a family of integral Golgi membrane proteins. Based on previous observations in yeast, we propose that N4WBP5 may act as an adaptor for Nedd4-like proteins and their putative targets to control ubiquitin-dependent protein sorting and trafficking.     

Identification of a critical motif responsible for gating of Kir2.3 channel by intracellular protons.

Protons are involved in gating Kir2.3. To identify the molecular motif in the Kir2.3 channel protein that is responsible for this process, experiments were performed using wild-type and mutated Kir2. 3 and Kir2.1. CO2 and low pHi strongly inhibited wild-type Kir2.3 but not Kir2.1 in whole cell voltage clamp and excised inside-out patches. This CO2/pH sensitivity was completely eliminated in a mutant Kir2.3 in which the N terminus was substituted with that in Kir2.1, whereas a similar replacement of its C terminus had no effect. Site-specific mutations of all titratable residues in the N terminus, however, did not change the CO2/pH sensitivity. Using several chimeras generated systematically in the N terminus, a 10-residue motif near the M1 region was identified in which only three amino acids are different between Kir2.3 and Kir2.1. Mutations of these residues, especially Thr53, dramatically reduced the pH sensitivity of Kir2.3. Introducing these residues or even a single threonine to the corresponding positions of Kir2.1 made the mutant channel pH-sensitive. Thus, a critical motif responsible for gating Kir2.3 by protons was identified in the N terminus, which contained about 10 residues centered by Thr53.     

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.