Channelopathies of inwardly rectifying potassium channels.

Mutations in genes encoding ion channels have increasingly been identified to cause disease conditions collectively termed channelopathies. Recognizing the molecular basis of an ion channel disease has provided new opportunities for screening, early diagnosis, and therapy of such conditions. This synopsis provides an overview of progress in the identification of molecular defects in inwardly rectifying potassium (Kir) channels. Structurally and functionally distinct from other channel families, Kir channels are ubiquitously expressed and serve functions as diverse as regulation of resting membrane potential, maintenance of K(+) homeostasis, control of heart rate, and hormone secretion. In humans, persistent hyperinsulinemic hypoglycemia of infancy, a disorder affecting the function of pancreatic beta cells, and Bartter's syndrome, characterized by hypokalemic alkalosis, hypercalciuria, increased serum aldosterone, and plasma renin activity, are the two major diseases linked so far to mutations in a Kir channel or associated protein. In addition, the weaver phenotype, a neurological disorder in mice, has also been associated with mutations in a Kir channel subtype. Further genetic linkage analysis and full understanding of the consequence that a defect in a Kir channel would have on disease pathogenesis are among the priorities in this emerging field of molecular medicine.     

Transmembrane structure of an inwardly rectifying potassium channel

Inwardly rectifying potassium channels (K(ir)), comprising four subunits each with two transmembrane domains, M1 and M2, regulate many important physiological processes. We employed a yeast genetic screen to identify functional channels from libraries of K(ir) 2.1 containing mutagenized M1 or M2 domains. Patterns in the allowed sequences indicate that M1 and M2 are helices. Protein-lipid and protein-water interaction surfaces identified by the patterns were verified by sequence minimization experiments. Second-site suppressor analyses of helix packing indicate that the M2 pore-lining inner helices are surrounded by the M1 lipid-facing outer helices, arranged such that the M1 helices participate in subunit-subunit interactions. This arrangement is distinctly different from the structure of a bacterial potassium channel with the same topology and identifies helix-packing residues as hallmark sequences common to all K(ir) superfamily members.     

中枢神经系统中与G蛋白偶联的内向整流钾通道

G蛋白偶联的内向整流钾通道（GIRK）在中枢神经系统中具有广泛分布,并且与多种受体相偶联,在神经突触后抑制中具有重要作用。本文简要介绍了近年来G蛋白偶联钾通道在基因结构、脑内组织分布、细胞内调控,以及脑功能方面的研究结果。关于GIRK在中枢神经系统中的生理和病理意义还有待进一步研究。     

A recombinant inwardly rectifying potassium channel coupled to GTP- binding proteins

GTP-binding (G) proteins have been shown to mediate activation of inwardly rectifying potassium (K+) channels in cardiac, neuronal and neuroendocrine cells. Here, we report functional expression of a recombinant inwardly rectifying channel which we call KGP (or hpKir3.4), to signify that it is K+ selective, G-protein-gated and isolated from human pancreas. KGP expression in Xenopus oocytes resulted in sizeable basal (or agonist-independent) currents while coexpression with a G-protein-linked receptor, yielded additional agonist-induced currents. Coexpression of KGP and hGIRK1 (a human brain homolog of GIRK1/Kir3.1) produced much larger basal currents than those observed with KGP or hGIRK1 alone, and upon coexpression with receptor, similarly large agonist-induced currents could be obtained. Pertussis toxin treatment significantly diminished agonist-dependent currents due to either KGP or KGP/hGIRK1 expression. Interestingly, PTX also significantly reduced basal KGP or KGP/hGIRK1 currents, suggesting that basal activity is largely the result of G-protein gating as well. When the two channels were coexpressed with receptor, the relative increase in current elicited by agonist was similar whether KGP and hGIRK1 were expressed alone or together. When in vitro translated or when expressed in Xenopus oocytes or CHO mammalian cells, KGP gave rise to a nonglycosylated 45-kD protein. Antibodies directed against either KGP or hGIRK1 coprecipitated both proteins coexpressed in oocytes, providing evidence for the heteromeric assembly of the two channels and suggesting that the current potentiation seen with coexpression of the two channel subunits is due to specific interactions between them. An endogenous oocyte protein similar in size to KGP was also coprecipitated with hGIRK1.     

Regulation of cardiac inwardly rectifying potassium channels by membrane lipid metabolism

Types and distributions of inwardly rectifying potassium (Kir) channels are one of the major determinants of the electrophysiological properties of cardiac myocytes. Kir2.1 (classical inward rectifier K(+) channel), Kir6.2/SUR2A (ATP-sensitive K(+) channel) and Kir3.1/3.4 (muscarinic K(+) channels) in cardiac myocytes are commonly upregulated by a membrane lipid, phosphatidylinositol 4,5-bisphosphates (PIP(2)). PIP(2) interaction sites appear to be conserved by positively charged amino acid residues and the putative alpha-helix in the C-terminals of Kir channels. PIP(2) level in the plasma membrane is regulated by the agonist stimulation. Kir channels in the cardiac myocytes seem to be actively regulated by means of the change in PIP(2) level rather than by downstream signal transduction pathways.     

Multiple PIP2 binding sites in Kir2.1 inwardly rectifying potassium channels

Inwardly rectifying potassium channels require binding of phosphatidylinositol-4,5-bisphosphate (PIP2) for channel activity. Three independent sites (aa 175-206, aa 207-246, aa 324-365) were located in the C-terminal domain of Kir2.1 channels by assaying the binding of overlapping fragments to PIP2 containing liposomes. Mutations in the first site, which abolished channel activity, reduced PIP2 binding of this fragment but not of the complete C-terminus. Point mutations in the third site also reduced both, channel activity and PIP2 binding of this segment. The relevance of the third PIP2 binding site provides a basis for the understanding of constitutively active Kir2 channels.     

Characterization of heteromultimeric G protein-coupled inwardly rectifying potassium channels of the tunicate tadpole with a unique pore property

Two cDNAs that encode the G protein-coupled inwardly rectifying K(+) channel (GIRK, Kir3) of tunicate tadpoles (tunicate G protein-coupled inwardly rectifying K(+) channel-A and -B; TuGIRK-A and -B) have been isolated. The deduced amino acid sequences showed approximately 60% identity with the mammalian Kir3 family. Detected by whole mount in situ hybridization, both TuGIRK-A and -B were expressed similarly in the neural cells of the head and neck region from the tail bud stage to the young tadpole stage. By co-injecting cRNAs of TuGIRK-A and G protein beta(1)/gamma(2) subunits (Gbetagamma) in Xenopus oocytes, an inwardly rectifying K(+) current was expressed. In contrast, coinjection of TuGIRK-B with Gbetagamma did not express any current. When both TuGIRK-A and -B were coexpressed together with Gbetagamma, an inwardly rectifying K(+) current was also detected. The properties of this current clearly differed from those of TuGIRK-A current, since it displayed a characteristic decline of the macroscopic conductance at strongly hyperpolarized potentials. TuGIRK-A/B current also differed from TuGIRK-A current in terms of the lower sensitivity to the Ba(2+) block, the higher sensitivity to the Cs(+) block, and the smaller single channel conductance. Taken together, we concluded that TuGIRK-A and -B form functional heteromultimeric G protein-coupled inwardly rectifying K(+) channels in the neural cells of the tunicate tadpole. By introducing a mutation of Lys(161) to Thr in TuGIRK-B, TuGIRK-A/B channels acquired a higher sensitivity to the Ba(2+) block and a slightly lower sensitivity to the Cs(+) block, and the decrease in the macroscopic conductance at hyperpolarized potentials was no longer observed. Thus, the differences in the electrophysiological properties between TuGIRK-A and TuGIRK-A/B channels were shown to be, at least partly, due to the presence of Lys(161) at the external mouth of the pore of the TuGIRK-B subunit.     

Interactions between GABA-B1 receptors and Kir 3 inwardly rectifying potassium channels

γ-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the mammalian brain. It acts via both ionotropic GABA-A and metabotropic GABA-B receptors. We evaluated the interaction of receptors with members of the inwardly rectifying potassium (Kir 3) channel family, which also play an important role in neuronal transmission and membrane excitability. These channels are functionally regulated by GABA-B receptors. Possible physical interactions between GABA-B receptor and Kir 3 channels expressed in HEK cells were evaluated using Bioluminescence Resonance Energy Transfer (BRET) experiments, co-immunoprecipitation and confocal microscopy. Our data indicate that Kir 3 channels and Gβγ subunits can interact with the GABA-B₁ subunits independently of the GABA-B₂ subunit or Kir 3.4 which are ultimately responsible for their targetting to the cell surface. Thus signalling complexes containing GABA-B receptors, G proteins and Kir channels are formed shortly after biosynthesis most likely in the endoplasmic reticulum.     

Molecular mechanisms of centipede toxin SsTx-4 inhibition of inwardly rectifying potassium channels

Inwardly rectifying potassium channels (Kirs) are important drug targets, with antagonists for the Kir1.1, Kir4.1, and pancreatic Kir6.2/SUR1 channels being potential drug candidates for treating hypertension, depression, and diabetes, respectively. However, few peptide toxins acting on Kirs are identified and their interacting mechanisms remain largely elusive yet. Herein, we showed that the centipede toxin SsTx-4 potently inhibited the Kir1.1, Kir4.1, and Kir6.2/SUR1 channels with nanomolar to submicromolar affinities and intensively studied the molecular bases for toxin–channel interactions using patch-clamp analysis and site-directed mutations. Other Kirs including Kir2.1 to 2.4, Kir4.2, and Kir7.1 were resistant to SsTx-4 treatment. Moreover, SsTx-4 inhibited the inward and outward currents of Kirs with different potencies, possibly caused by a K⁺ “knock-off” effect, suggesting the toxin functions as an out pore blocker physically occluding the K⁺-conducting pathway. This conclusion was further supported by a mutation analysis showing that M137 located in the outer vestibule of the Kir6.2/ΔC26 channel was the key residue mediating interaction with SsTx-4. On the other hand, the molecular determinants within SsTx-4 for binding these Kir channels only partially overlapped, with K13 and F44 being the common key residues. Most importantly, K11A, P15A, and Y16A mutant toxins showed improved affinity and/or selectivity toward Kir6.2, while R12A mutant toxin had increased affinity for Kir4.1. To our knowledge, SsTx-4 is the first characterized peptide toxin with Kir4.1 inhibitory activity. This study provides useful insights for engineering a Kir6.2/SUR1 channel–specific antagonist based on the SsTx-4 template molecule and may be useful in developing new antidiabetic drugs.     

The role of inwardly rectifying potassium channels in the relaxation of rat hind-limb arteries

An increase in the extracellular K⁺ concentration, which causes relaxation of arteries due to the activation of inwardly rectifying potassium channels, can occur in some organs under intensive metabolism, as well as endothelium-dependent hyperpolarization. The aim of this work was a comparison of the contribution of these channels in the regulation of the tone of arteries that supply skeletal muscles and the skin. The reactions of skin-region arteries (a subcutaneous artery and its branch) and gastrocnemius muscle arteries were recorded in the isometric mode. During the contraction caused by α₁-adrenoceptor agonist, the relaxation reactions upon an increase in extracellular K⁺ concentration and on acetylcholine in the presence of inhibitors of NO-synthase and cyclooxygenase were recorded (to detect the effects of endothelium-dependent hyperpolarization). The muscle arteries at both effects showed a pronounced relaxation, which was strongly suppressed by Ba²⁺ ions (blockers of inwardly rectifying potassium channels); both reactions did not exceed 20% in the skin arteries. Thus, the regulatory effect of inwardly rectifying potassium channels in the muscle arteries is much higher than in the skin arteries which is consistent with the idea about the functioning of these arteries in the organism.     

Structure and dynamics of the pore of inwardly rectifying K(ATP) channels

Inwardly rectifying K(+) currents are generated by a complex of four Kir (Kir1-6) subunits. Pore properties are conferred by the second transmembrane domain (M2) of each subunit. Using cadmium ions as a cysteine-interacting probe, we examined the accessibility of substituted cysteines in M2 of the Kir6.2 subunit of inwardly rectifying K(ATP) channels. The ability of Cd(2+) ions to inhibit channels was used as the estimate of accessibility. The distribution of Cd(2+) accessibility is consistent with an alpha-helical structure of M2. The apparent surface of reactivity is broad, and the most reactive residues correspond to the solvent-accessible residues in the bacterial KcsA channel crystal structure. In several mutants, single channel measurements indicated that inhibition occurred by a single transition from the open state to a zero-conductance state. Analysis of currents expressed from mixtures of control and L164C mutant subunits indicated that at least three cysteines are required for coordination of the Cd(2+) ion. Application of phosphatidylinositol 4,5-diphosphate to inside-out membrane patches stabilized the open state of all mutants and also reduced cadmium sensitivity. Moreover, the Cd(2+) sensitivity of several mutants was greatly reduced in the presence of inhibitory ATP concentrations. Taken together, these results are consistent with state-dependent accessibility of single Cd(2+) ions to coordination sites within a relatively narrow inner vestibule.     

An inwardly rectifying potassium channel in the basolateral membrane of sheep parotid secretory cells

Summary Using whole-cell patch-clamp techniques, we demonstrate that sheep parotid secretory cells have both inwardly and outwardly rectifying currents. The outwardly rectifying current, which is blocked by 10 mmol/liter tetraethylammonium (TEA) applied extracellularly, is probably carried by the 250 pS Ca²⁺-and voltage-activated K⁺ (BK) channel which has been described in previous studies. In contrast, the inwardly rectifying current, which is also carried by K⁺ ions, is not sensitive to TEA. It is similar to the inwardly rectifying currents observed in many excitable tissues in that (i) its conductance is dependent on the square root of the extracellular K⁺, (ii) the voltage range over which it is activated is influenced by the extracellular K⁺ concentration and (iii) it is blocked by the addition of Cs⁺ ions (670 µmol/liter) to the bathing solution. Our previously published cell-attached patch studies have shown that the channel type most commonly observed in the basolateral membrane of unstimulated sheep parotid secretory cells is a K⁺ channel with a conductance of 30 pS and, in this study, we find that its conductance also depends on the square root of the extracellular K⁺ concentration. It thus seems likely that it carries the inwardly rectifying K⁺ current seen in the whole-cell studies.     

Mitochondrial ATP-sensitive potassium channel activity and hypoxic preconditioning are independent of an inwardly rectifying potassium channel subunit in Caenorhabditis elegans

Hypoxic preconditioning (HP) is an evolutionarily-conserved mechanism that protects an organism against stress. The mitochondrial ATP-sensitive K(+) channel (mK(ATP)) plays an essential role in the protective signaling, but remains molecularly undefined. Several lines of evidence suggest that mK(ATP) may arise from an inward rectifying K(+) channel (Kir). The genetic model organism Caenorhabditis elegans exhibits HP and displays mK(ATP) activity. Here, we investigate the tissue expression profile of the three C. elegans Kir genes and demonstrate that mutant strains where the irk genes have been deleted either individually or in combination can be protected by HP and exhibit robust mK(ATP) channel activity in purified mitochondria. These data suggest that the mK(ATP) in C. elegans does not arise from a Kir derived channel.     

G protein-coupled receptors form stable complexes with inwardly rectifying potassium channels and adenylyl cyclase

A large number of studies have demonstrated co-purification or co-immunoprecipitation of receptors with G proteins. We have begun to look for the presence of effector molecules in these receptor complexes. Co-expression of different channel and receptor permutations in COS-7 and HEK 293 cells in combination with co-immunoprecipitation experiments established that the dopamine D(2) and D(4), and beta(2)-adrenergic receptors (beta(2)-AR) form stable complexes with Kir3 channels. The D(4)/Kir3 and D(2) receptor/Kir3 interaction does not occur when the channel and receptor are expressed separately and mixed prior to immunoprecipitation, indicating that the interaction is not an artifact of the experimental protocol and reflects a biosynthetic event. The observed complexes are stable in that they are not disrupted by receptor activation or modulation of G protein alpha subunit function. However, using a peptide that binds Gbetagamma (betaARKct), we show that Gbetagamma is critical for dopamine receptor-Kir3 complex formation, but not for maintenance of the complex. We also provide evidence that Kir3 channels and another effector, adenylyl cyclase, are stably associated with the beta(2)-adrenergic receptor and can be co-immunoprecipitated by anti-receptor antibodies. Using bioluminescence resonance energy transfer, we have shown that in living cells under physiological conditions, beta(2)AR interacts directly with Kir3.1/3.4 and Kir3.1/3.2c heterotetramers as well as with adenylyl cyclase. All of these interactions are stable in the presence of receptor agonists, suggesting that these signaling complexes persist during signal transduction. In addition, we provide evidence that the receptor-effector complexes are also found in vivo. The observation that several G protein-coupled receptors form stable complexes with their effectors suggests that this arrangement might be a general feature of G protein-coupled signal transduction.     

The pore helix is involved in stabilizing the open state of inwardly rectifying K+ channels

Ion channels can be gated by various extrinsic cues, such as voltage, pH, and second messengers. However, most ion channels display extrinsic cue-independent transitions as well. These events represent spontaneous conformational changes of the channel protein. The molecular basis for spontaneous gating and its relation to the mechanism by which channels undergo activation gating by extrinsic cue stimulation is not well understood. Here we show that the proximal pore helix of inwardly rectifying (Kir) channels is partially responsible for determining spontaneous gating characteristics, affecting the open state of the channel by stabilizing intraburst openings as well as the bursting state itself without affecting K(+) ion-channel interactions. The effect of the pore helix on the open state of the channel is qualitatively similar to that of two well-characterized mutations at the second transmembrane domain (TM2), which stabilize the channel in its activated state. However, the effects of the pore helix and the TM2 mutations on gating were additive and independent of each other. Moreover, in sharp contrast to the two TM2 mutations, the pore helix mutation did not affect the functionality of the agonist-responsive gate. Our results suggest that in Kir channels, the bottom of the pore helix and agonist-induced conformational transitions at the TM2 ultimately stabilize via different pathways the open conformation of the same gate.     

Protein expression of G-protein inwardly rectifying potassium channels (GIRK) in breast cancer cells

Background  

Previous data from our laboratory has indicated that a functional link exists between the G-protein-coupled inwardly rectifying potassium (GIRK) channel and the beta-adrenergic receptor pathway in breast cancer cell lines, and these pathways were involved in growth regulation of these cells. Alcohol is an established risk factor for breast cancer and has been found to open GIRK. In order to further investigate GIRK channels in breast cancer and possible alteration by ethanol, we identified GIRK channel protein expression in breast cancer cells.     

Coupling between voltage sensor activation,Ca2+ binding and channel opening in large conductance (BK) potassium channels

To determine how intracellular Ca(2+) and membrane voltage regulate the gating of large conductance Ca(2+)-activated K(+) (BK) channels, we examined the steady-state and kinetic properties of mSlo1 ionic and gating currents in the presence and absence of Ca(2+) over a wide range of voltage. The activation of unliganded mSlo1 channels can be accounted for by allosteric coupling between voltage sensor activation and the closed (C) to open (O) conformational change (Horrigan, F.T., and R.W. Aldrich. 1999. J. Gen. Physiol. 114:305-336; Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). In 0 Ca(2+), the steady-state gating charge-voltage (Q(SS)-V) relationship is shallower and shifted to more negative voltages than the conductance-voltage (G(K)-V) relationship. Calcium alters the relationship between Q-V and G-V, shifting both to more negative voltages such that they almost superimpose in 70 microM Ca(2+). This change reflects a differential effect of Ca(2+) on voltage sensor activation and channel opening. Ca(2+) has only a small effect on the fast component of ON gating current, indicating that Ca(2+) binding has little effect on voltage sensor activation when channels are closed. In contrast, open probability measured at very negative voltages (less than -80 mV) increases more than 1,000-fold in 70 microM Ca(2+), demonstrating that Ca(2+) increases the C-O equilibrium constant under conditions where voltage sensors are not activated. Thus, Ca(2+) binding and voltage sensor activation act almost independently, to enhance channel opening. This dual-allosteric mechanism can reproduce the steady-state behavior of mSlo1 over a wide range of conditions, with the assumption that activation of individual Ca(2+) sensors or voltage sensors additively affect the energy of the C-O transition and that a weak interaction between Ca(2+) sensors and voltage sensors occurs independent of channel opening. By contrast, macroscopic I(K) kinetics indicate that Ca(2+) and voltage dependencies of C-O transition rates are complex, leading us to propose that the C-O conformational change may be described by a complex energy landscape.