Distant Cytosolic Residues Mediate a Two-way Molecular Switch That Controls the Modulation of Inwardly Rectifying Potassium (Kir) Channels by Cholesterol and Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2)

Inwardly rectifying potassium (Kir) channels play an important role in setting the resting membrane potential and modulating membrane excitability. An emerging feature of several Kir channels is that they are regulated by cholesterol. However, the mechanism by which cholesterol affects channel function is unclear. Here we show that mutations of two distant Kir2.1 cytosolic residues, Leu-222 and Asn-251, form a two-way molecular switch that controls channel modulation by cholesterol and affects critical hydrogen bonding. Notably, these two residues are linked by a residue chain that continues from Asn-251 to connect adjacent subunits. Furthermore, our data indicate that the same switch also regulates the sensitivity of the channels to phosphatidylinositol 4,5-bisphosphate, a phosphoinositide that is required for activation of Kir channels. Thus, although cholesterol and phosphatidylinositol 4,5-bisphosphate do not interact with the same region of Kir2.1, these different modulators induce a common gating pathway of the channel.     

Cholesterol sensitivity of KIR2.1 is controlled by a belt of residues around the cytosolic pore

Kir channels play an important role in setting the resting membrane potential and modulating membrane excitability. A common feature of several Kir channels is that they are regulated by cholesterol. Yet, the mechanism by which cholesterol affects channel function is unclear. We recently showed that the cholesterol sensitivity of Kir2 channels depends on several CD-loop residues. Here we show that this cytosolic loop is part of a regulatory site that also includes residues in the G-loop, the N-terminus, and the connecting segment between the C-terminus and the inner transmembrane helix. Together, these residues form a cytosolic belt that surrounds the pore of the channel close to its interface with the transmembrane domain, and modulate the cholesterol sensitivity of the channel. Furthermore, we show that residues in this cluster are correlated with residues located in the most flexible region of the G-loop, the major cytosolic gate of Kir2.1, implying that the importance of these residues extends beyond their effect on the channel's cholesterol sensitivity. We suggest that the residues of the cholesterol sensitivity belt are critical for channel gating.     

Decomposition of Slide Helix Contributions to ATP-dependent Inhibition of Kir6.2 Channels

Regulation of inwardly rectifying potassium channels by intracellular ligands couples cell membrane excitability to important signaling cascades and metabolic pathways. We investigated the molecular mechanisms that link ligand binding to the channel gate in ATP-sensitive Kir6.2 channels. In these channels, the “slide helix” forms an interface between the cytoplasmic (ligand-binding) domain and the transmembrane pore, and many slide helix mutations cause loss of function. Using a novel approach to rescue electrically silent channels, we decomposed the contribution of each interface residue to ATP-dependent gating. We demonstrate that effective inhibition by ATP relies on an essential aspartate at residue 58. Characterization of the functional importance of this conserved aspartate, relative to other residues in the slide helix, has been impossible because of loss-of-function of Asp-58 mutant channels. The Asp-58 position exhibits an extremely stringent requirement for aspartate because even a highly conservative mutation to glutamate is insufficient to restore normal channel function. These findings reveal unrecognized slide helix elements that are required for functional channel expression and control of Kir6.2 gating by intracellular ATP.     

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.     

Cholesterol sensitivity of KIR2.1 depends on functional inter-links between the N and C termini

In recent years, cholesterol has been emerging as a major regulator of ion channel function. We have previously shown that cholesterol suppresses Kir2 channels, a subfamily of constitutively active strongly rectifying K⁺ channels. Furthermore, our earlier studies have shown that cholesterol sensitivity of Kir2 channels depends on a group of residues that form a belt-like structure around the cytosolic pore of the channel in proximity to the transmembrane domain. In this study, we focus on the contributions of different structural domains of Kir2 channels in the regulation of their cholesterol sensitivity. Focusing on the mildest mutation in the sensitivity belt, L222I, we show that the sensitivity of the channel to cholesterol can be restored by crosstalk between three distinct cytosolic regions: the C-terminal CD loop, the EF and GA loops of the C-terminus, and the βA sheet of the N-terminus. Thus, in addition to the importance of residues that affect the cytosolic G-loop gate in the sensitivity of Kir2 channels to cholesterol, our data suggest an important role to the interactions at the interface between the channel’s N- and C- termini that couple the intracellular domains of its four subunits during gating.     

Multiple cholesterol recognition/interaction amino acid consensus (CRAC) motifs in cytosolic C tail of Slo1 subunit determine cholesterol sensitivity of Ca2+- and voltage-gated K+ (BK) channels

Large conductance, Ca(2+)- and voltage-gated K(+) (BK) channel proteins are ubiquitously expressed in cell membranes and control a wide variety of biological processes. Membrane cholesterol regulates the activity of membrane-associated proteins, including BK channels. Cholesterol modulation of BK channels alters action potential firing, colonic ion transport, smooth muscle contractility, endothelial function, and the channel alcohol response. The structural bases underlying cholesterol-BK channel interaction are unknown. Such interaction is determined by strict chemical requirements for the sterol molecule, suggesting cholesterol recognition by a protein surface. Here, we demonstrate that cholesterol action on BK channel-forming Cbv1 proteins is mediated by their cytosolic C tail domain, where we identified seven cholesterol recognition/interaction amino acid consensus motifs (CRAC4 to 10), a distinct feature of BK proteins. Cholesterol sensitivity is provided by the membrane-adjacent CRAC4, where Val-444, Tyr-450, and Lys-453 are required for cholesterol sensing, with hydrogen bonding and hydrophobic interactions participating in cholesterol location and recognition. However, cumulative truncations or Tyr-to-Phe substitutions in CRAC5 to 10 progressively blunt cholesterol sensitivity, documenting involvement of multiple CRACs in cholesterol-BK channel interaction. In conclusion, our study provides for the first time the structural bases of BK channel cholesterol sensitivity; the presence of membrane-adjacent CRAC4 and the long cytosolic C tail domain with several other CRAC motifs, which are not found in other members of the TM6 superfamily of ion channels, very likely explains the unique cholesterol sensitivity of BK channels.     

Gating of a G protein-sensitive mammalian Kir3.1 prokaryotic Kir channel chimera in planar lipid bilayers

Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the βγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K(+) channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP(2) and Mg(2+)-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gβγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels.     

Energetics and Location of Phosphoinositide Binding in Human Kir2.1 Channels

Kir2.1 channels are uniquely activated by phosphoinositide 4,5-bisphosphate (PI(4,5)P₂) and can be inhibited by other phosphoinositides (PIPs). Using biochemical and computational approaches, we assess PIP-channel interactions and distinguish residues that are energetically critical for binding from those that alter PIP sensitivity by shifting the open-closed equilibrium. Intriguingly, binding of each PIP is disrupted by a different subset of mutations. In silico ligand docking indicates that PIPs bind to two sites. The second minor site may correspond to the secondary anionic phospholipid site required for channel activation. However, 96–99% of PIP binding localizes to the first cluster, which corresponds to the general PI(4,5)P₂ binding location in recent Kir crystal structures. PIPs can encompass multiple orientations; each di- and triphosphorylated species binds with comparable energies and is favored over monophosphorylated PIPs. The data suggest that selective activation by PI(4,5)P₂ involves orientational specificity and that other PIPs inhibit this activation through direct competition.     

Functional Consequences of the Interactions among the Neural Cell Adhesion Molecule NCAM,the Receptor Tyrosine Kinase TrkB,and the Inwardly Rectifying K+ Channel KIR3.3

Cell adhesion molecules and neurotrophin receptors are crucial for the development and the function of the nervous system. Among downstream effectors of neurotrophin receptors and recognition molecules are ion channels. Here, we provide evidence that G protein-coupled inwardly rectifying K⁺ channel Kir3.3 directly binds to the neural cell adhesion molecule (NCAM) and neurotrophin receptor TrkB. We identified the binding sites for NCAM and TrkB at the C-terminal intracellular domain of Kir3.3. The interaction between NCAM, TrkB, and Kir3.3 was supported by immunocytochemical co-localization of Kir3.3, NCAM, and/or TrkB at the surface of hippocampal neurons. Co-expression of TrkB and Kir3.1/3.3 in Xenopus oocytes increased the K⁺ currents evoked by Kir3.1/3.3 channels. This current enhancement was reduced by the concomitant co-expression with NCAM. Both surface fluorescence measurements of microinjected oocytes and cell surface biotinylation of transfected CHO cells indicated that the cell membrane localization of Kir3.3 is regulated by TrkB and NCAM. Furthermore, the level of Kir3.3, but not of Kir3.2, at the plasma membranes was reduced in TrkB-deficient mice, supporting the notion that TrkB regulates the cell surface expression of Kir3.3. The premature expression of developmentally late appearing Kir3.1/3.3 in hippocampal neurons led to a reduction of NCAM-induced neurite outgrowth. Our observations indicate a decisive role for the neuronal K⁺ channel in regulating NCAM-dependent neurite outgrowth and attribute a physiologically meaningful role to the functional interplay of Kir3.3, NCAM, and TrkB in ontogeny.     

Characterization and Functional Restoration of a Potassium Channel Kir6.2 Pore Mutation Identified in Congenital Hyperinsulinism

The inwardly rectifying potassium channel Kir6.2 assembles with sulfonylurea receptor 1 to form the ATP-sensitive potassium (K_ATP) channels that regulate insulin secretion in pancreatic β-cells. Mutations in K_ATP channels underlie insulin secretion disease. Here, we report the characterization of a heterozygous missense Kir6.2 mutation, G156R, identified in congenital hyperinsulinism. Homomeric mutant channels reconstituted in COS cells show similar surface expression as wild-type channels but fail to conduct potassium currents. The mutated glycine is in the pore-lining transmembrane helix of Kir6.2; an equivalent glycine in other potassium channels has been proposed to serve as a hinge to allow helix bending during gating. We found that mutation of an adjacent asparagine, Asn-160, to aspartate, which converts the channel from a weak to a strong inward rectifier, on the G156R background restored ion conduction in the mutant channel. Unlike N160D channels, however, G156R/N160D channels are not blocked by intracellular polyamines at positive membrane potential and exhibit wild-type-like nucleotide sensitivities, suggesting the aspartate introduced at position 160 interacts with arginine at 156 to restore ion conduction and gating. Using tandem Kir6.2 tetramers containing G156R and/or N160D in designated positions, we show that one mutant subunit in the tetramer is insufficient to abolish conductance and that G156R and N160D can interact in the same or adjacent subunits to restore conduction. We conclude that the glycine at 156 is not essential for K_ATP channel gating and that the Kir6.2 gating defect caused by the G156R mutation could be rescued by manipulating chemical interactions between pore residues.     

Cholesterol regulates prokaryotic Kir channel by direct binding to channel protein

Cholesterol is a major regulator of a variety of ion channels but the mechanisms underlying cholesterol sensitivity of ion channels are still poorly understood. The key question is whether cholesterol regulates ion channels by direct binding to the channel protein or by altering the physical environment of lipid bilayer. In this study, we provide the first direct evidence that cholesterol binds to prokaryotic Kir channels, KirBac1.1, and that cholesterol binding is essential for its regulatory effect. Specifically, we show that cholesterol is eluted together with the KirBac1.1 protein when separated on an affinity column and that the amount of bound cholesterol is proportional to the amount of the protein. We also show that cholesterol binding to KirBac1.1 is saturable with a K(D) of 390μM. Moreover, there is clear competition between radioactive and non-radioactive cholesterol for the binding site. There is no competition, however, between cholesterol and 5-Androsten 3β-17 β-diol, a sterol that we showed previously to have no effect on KirBac1.1 function. Finally, we show that cholesterol-KirBac1.1 binding is significantly inhibited by trifluoperazine, known to inhibit cholesterol binding to other proteins, and that inhibition of cholesterol-KirBac1.1 binding results in full recovery of the channel activity. Collectively, results from this study indicate that cholesterol-induced suppression of KirBac1.1 activity is mediated by direct interaction between cholesterol and the channel protein.     

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.     

Enantioselective protein-sterol interactions mediate regulation of both prokaryotic and eukaryotic inward rectifier K+ channels by cholesterol

Cholesterol is the major sterol component of all mammalian cell plasma membranes and plays a critical role in cell function and growth. Previous studies have shown that cholesterol inhibits inward rectifier K(+) (Kir) channels, but have not distinguished whether this is due directly to protein-sterol interactions or indirectly to changes in the physical properties of the lipid bilayer. Using purified bacterial and eukaryotic Kir channels reconstituted into liposomes of controlled lipid composition, we demonstrate by (86)Rb(+) influx assays that bacterial Kir channels (KirBac1.1 and KirBac3.1) and human Kir2.1 are all inhibited by cholesterol, most likely by locking the channels into prolonged closed states, whereas the enantiomer, ent-cholesterol, does not inhibit these channels. These data indicate that cholesterol regulates Kir channels through direct protein-sterol interactions likely taking advantage of an evolutionarily conserved binding pocket.     

Identification of a Cholesterol-Binding Pocket in Inward Rectifier K+ (Kir) Channels

Cholesterol is the major sterol component of all mammalian plasma membranes. Recent studies have shown that cholesterol inhibits both bacterial (KirBac1.1 and KirBac3.1) and eukaryotic (Kir2.1) inward rectifier K⁺ (Kir) channels. Lipid-sterol interactions are not enantioselective, and the enantiomer of cholesterol (ent-cholesterol) does not inhibit Kir channel activity, suggesting that inhibition results from direct enantiospecific binding to the channel, and not indirect effects of changes to the bilayer. Furthermore, conservation of the effect of cholesterol among prokaryotic and eukaryotic Kir channels suggests an evolutionary conserved cholesterol-binding pocket, which we aimed to identify. Computational experiments were performed by docking cholesterol to the atomic structures of Kir2.2 (PDB: 3SPI) and KirBac1.1 (PDB: 2WLL) using Autodock 4.2. Poses were assessed to ensure biologically relevant orientation and then clustered according to location and orientation. The stability of cholesterol in each of these poses was then confirmed by molecular dynamics simulations. Finally, mutation of key residues (S95H and I171L) in this putative binding pocket found within the transmembrane domain of Kir2.1 channels were shown to lead to a loss of inhibition by cholesterol. Together, these data provide support for this location as a biologically relevant pocket.     

Hypercholesterolemia induces up-regulation of KACh cardiac currents via a mechanism independent of phosphatidylinositol 4,5-bisphosphate and Gβγ

Hypercholesterolemia is a well-known risk factor for cardiovascular disease. In the heart, activation of K(ACh) mediates the vagal (parasympathetic) negative chronotropic effect on heart rate. Yet, the effect of cholesterol on K(ACh) is unknown. Here we show that cholesterol plays a critical role in modulating K(ACh) currents (I(K,ACh)) in atrial cardiomyocytes. Specifically, cholesterol enrichment of rabbit atrial cardiomyocytes led to enhanced channel activity while cholesterol depletion suppressed I(K,ACh). Moreover, a high-cholesterol diet resulted in up to 3-fold increase in I(K,ACh) in rodents. In accordance, elevated currents were observed in Xenopus oocytes expressing the Kir3.1/Kir3.4 heteromer that underlies I(K,ACh). Furthermore, our data suggest that cholesterol affects I(K,ACh) via a mechanism which is independent of both PI(4,5)P(2) and Gβγ. Interestingly, the effect of cholesterol on I(K,ACh) is opposite to its effect on I(K1) in atrial myocytes. The latter are suppressed by cholesterol enrichment and by high-cholesterol diet, and facilitated following cholesterol depletion. These findings establish that cholesterol plays a critical role in modulating I(K,ACh) in atrial cardiomyocytes via a mechanism independent of the channel's major modulators.     

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.     

Book reviews

ATP sensitive potassium (K-ATP) channels are widely expressed in many cell types including neurons. K-ATP channels are heteromeric membrane proteins that consist of two very different subunits: the pore-forming, two-transmembrane spanning potassium channel subunit (Kir6) and the regulatory, 17 transmembrane spanning sulphonylurea receptor (SUR). This ensemble―joined together in a 4:4 stoichiometry―endows this channel with a unique combination of functional properties. The open probability of K-ATP channels directly depends on the intracellular ATP/ADP levels allowing the channels to directly couple the metabolic state of a cell to its electrical activity. Here, recent progress on the molecular composition and functional diversity of neuronal K-ATP channels is reviewed. One is particular concerned with single-cell mRNA expression studies that give insight to the coexpression patterns of Kir6 and SUR isoforms in identified neurons. In addition, the physiological roles of neuronal K-ATP channels in glucose sensing and adapting neuronal activity to metabolic demands are discussed, as well as their emerging pathophysiological functions in acute brain ischemia and chronic neurodegenerative diseases.     

Structural Diversity in the Cytoplasmic Region of G Protein-Gated Inward Rectifier K+ Channels

Inward rectifier K+ (Kir) channels can be functionally categorized into two groups: those that are constitutively active and those that are constitutively inactive, with examples such as Kir2.x and Kir3.x, respectively. Their cytoplasmic regions are thought to be critical for control of channel gating, but a structural basis for this hypothesis is not known. In this study, we report a structure for the cytoplasmic region of a G protein-gated Kir channel, Kir3.2, and compare it with those of Kir3.1 and Kir2.1 channels. The isolated cytoplasmic region of Kir3.2 forms a tetrameric assembly in solution and also in the crystal. While the secondary structure arrangement and the subunit interface of the Kir3.2 crystal structure are found to be nearly identical to those of Kir3.1 and Kir2.1, it is quite different at and around loops between βC- and βD-strands and between βH- and βI-strands. These structural elements are located at the interface with the plasma membrane. Therefore, these structural elements could associate with the Kir channel transmembrane helices and be involved in the regulation of Kir channel gating.     

Membrane anchoring and interaction between transmembrane domains are crucial for K+ channel function

The small viral channel Kcv is a Kir-like K(+) channel of only 94 amino acids. With this simple structure, the tetramer of Kcv represents the pore module of all complex K(+) channels. To examine the structural contribution of the transmembrane domains (TMDs) to channel function, we performed Ala scanning mutagenesis of the two domains and tested the functionality of the mutants in a yeast complementation assay. The data reveal, in combination with computational models, that the upper halves of both TMDs, which face toward the external medium, are rather rigid, whereas the inner parts are more flexible. The rigidity of the outer TMD is conferred by a number of essential aromatic amino acids that face the membrane and probably anchor this domain in the bilayer. The inner TMD is intimately connected with the rigid part of the outer TMD via π···π interactions between a pair of aromatic amino acids. This structural principle is conserved within the viral K(+) channels and also present in Kir2.2, implying a general importance of this architecture for K(+) channel function.