Overexpression of monomeric and multimeric GIRK4 subunits in rat atrial myocytes removes fast desensitization and reduces inward rectification of muscarinic K(+) current (I(K(ACh))). Evidence for functional homomeric GIRK4 channels

K(+) channels composed of G-protein-coupled inwardly rectifying K(+) channel (GIRK) (Kir3.0) subunits are expressed in cardiac, neuronal, and various endocrine tissues. They are involved in inhibiting excitability and contribute to regulating important physiological functions such as cardiac frequency and secretion of hormones. The functional cardiac (K((ACh))) channel activated by G(i)/G(o)-coupled receptors such as muscarinic M(2) or purinergic A(1) receptors is supposed to be composed of the subunits GIRK1 and GIRK4 in a heterotetrameric (2:2) fashion. In the present study, we have manipulated the subunit composition of the K((ACh)) channels in cultured atrial myocytes from hearts of adult rats by transient transfection of vectors encoding for GIRK1 or GIRK4 subunits or GIRK4 concatemeric constructs and investigated the effects on properties of macroscopic I(K(ACh)). Transfection with a GIRK1 vector did not cause any measurable effect on properties of I(K(ACh)), whereas transfection with a GIRK4 vector resulted in a complete loss in desensitization, a reduction of inward rectification, and a slowing of activation. Transfection of myocytes with a construct encoding for a concatemeric GIRK4(2) subunit had similar effects on desensitization and inward rectification. Following transfection of a tetrameric construct (GIRK4(4)), these changes in properties of I(K(ACh)) were still observed but were less pronounced. Heterologous expression in Chinese hamster ovary cells and human embryonic kidney 293 cells of monomeric, dimeric, and tetrameric GIRK4 resulted in robust currents activated by co-expressed A(1) and M(2) receptors, respectively. These data provide strong evidence that homomeric GIRK4 complexes form functional G(beta)gamma gated ion channels and that kinetic properties of GIRK channels, such as activation rate, desensitization, and inward rectification, depend on subunit composition.     

A Kir2.1 gain-of-function mutation underlies familial atrial fibrillation

The inward rectifier K(+) channel Kir2.1 mediates the potassium I(K1) current in the heart. It is encoded by KCNJ2 gene that has been linked to Andersen's syndrome. Recently, strong evidences showed that Kir2.1 channels were associated with mouse atrial fibrillation (AF), therefore we hypothesized that KCNJ2 was associated with familial AF. Thirty Chinese AF kindreds were evaluated for mutations in KCNJ2 gene. A valine-to-isoleucine mutation at position 93 (V93I) of Kir2.1 was found in all affected members in one kindred. This valine and its flanking sequence is highly conserved in Kir2.1 proteins among different species. Functional analysis of the V93I mutant demonstrated a gain-of-function consequence on the Kir2.1 current. This effect is opposed to the loss-of-function effect of previously reported mutations in Andersen's syndrome. Kir2.1 V93I mutation may play a role in initiating and/or maintaining AF by increasing the activity of the inward rectifier K(+) channel.     

Short variable sequence acquired in evolution enables selective inhibition of various inward-rectifier K+ channels

Tertiapin (TPN), a small protein toxin originally isolated from honey bee venom, inhibits only certain eukaryotic inward-rectifier K(+) (Kir) channels with high affinity. We found that a short ( approximately 10 residues) sequence in Kir channels, located in the N-terminal part of the linker between the two transmembrane segments, is essential for high-affinity inhibition by TPN and that variability in the region underlies the great variation of TPN affinities among eukaryotic Kir channels. This short variable region is however not present in a bacterial Kir channel (KirBac1.1) or in many other types of prokaryotic and eukaryotic K(+) channels. Thus, the acquisition in evolution of the variable region in eukaryotic Kir channels has created the opportunity to selectively target the numerous types of Kir channel that play important physiological roles. We also show that TPN sensitivity can be readily conferred onto some Kir channels that currently have no known inhibitors by replacing their variable region with that from a TPN-sensitive channel. In heterologous expression systems, such acquired toxin sensitivity will allow currents carried by mutant channels to be readily isolated from interfering background currents. Finally we show that, in the heteromeric GIRK1/4 channels, the GIRK4 and not GIRK1 subunit confers the high affinity for TPN.     

Distinct specificities of inwardly rectifying K(+) channels for phosphoinositides

Activation of several inwardly rectifying K(+) channels (Kir) requires the presence of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). The constitutively active Kir2.1 (IRK1) channels interact with PtdIns(4,5)P(2) strongly, whereas the G-protein activated Kir3.1/3.4 channels (GIRK1/GIRK4), show only weak interactions with PtdIns(4,5)P(2). We investigated whether these inwardly rectifying K(+) channels displayed distinct specificities for different phosphoinositides. IRK1, but not GIRK1/GIRK4 channels, showed a marked specificity toward phosphates in the 4,5 head group positions. GIRK1/GIRK4 channels were activated with a similar efficacy by PtdIns(3,4)P(2), PtdIns(3,5)P(2), PtdIns(4,5)P(2), and PtdIns(3,4,5)P(3). In contrast, IRK1 channels were not activated by PtdIns(3,4)P(2) and only marginally by high concentrations of PtdIns(3,5)P(2). Similarly, high concentrations of PtdIns(3,4,5)P(3) were required to activate IRK1 channels. For either channel, PtdIns(4)P was much less effective than PtdIns(4,5)P(2), whereas PtdIns was inactive. In contrast to the dependence on the position of phosphates of the phospholipid head group, GIRK1/GIRK4, but not IRK1 channel activation, showed a remarkable dependence on the phospholipid acyl chains. GIRK1/GIRK4 channels were activated most effectively by the natural arachidonyl stearyl PtdIns(4,5)P(2) and much less by the synthetic dipalmitoyl analog, whereas IRK1 channels were activated equally by dipalmitoyl and arachidonyl stearyl PtdIns(4,5)P(2). Incorporation of PtdInsP(2) into the membrane is necessary for activation, as the short chain water soluble diC(4) PtdIns(4,5)P(2) did not activate either channel, whereas activation by diC(8) PtdIns(4, 5)P(2) required high concentrations.     

Mechanosensitivity of GIRK channels is mediated by protein kinase C-dependent channel-phosphatidylinositol 4,5-bisphosphate interaction

Gprotein-activated inwardly rectifying K+ channel (GIRK or Kir3) currents are inhibited by mechanical stretch of the cell membrane, but the underlying mechanisms are not understood. In Xenopus oocytes heterologously expressing GIRK channels, membrane stretch induced by 50% reduction of osmotic pressure caused a prompt reduction of GIRK1/4, GIRK1, and GIRK4 currents by 16.6-42.6%. Comparable GIRK current reduction was produced by protein kinase C (PKC) activation (phorbol 12-myristate 13-acetate). The mechanosensitivity of the GIRK4 current was abolished by pretreatment with PKC inhibitors (staurosporine or calphostin C). Neither hypo-osmotic challenge nor PKC activation affected IRK1 currents. GIRK4 chimera (GIRK4-IRK1-(Lys207-Leu245)) and single point mutant (GIRK4(I229L)), in which the phosphatidylinositol 4,5-bisphosphate (PIP2) binding domain or residue was replaced by the corresponding region of IRK1 to strengthen the channel-PIP2 interaction, showed no mechanosensitivity and minimal PKC sensitivity. IRK1 gained mechanosensitivity and PKC sensitivity by reverse double point mutation of the PIP2 binding domain (L222I/R213Q). Overexpression of Gbetagamma, which is known to strengthen the channel-PIP2 interaction, attenuated the mechanosensitivity of GIRK4 channels. In oocytes expressing a pleckstrin homology domain of PLC-delta tagged with green fluorescent protein, hypo-osmotic challenge or PKC activation caused a translocation of the fluorescence signal from the cell membrane to the cytosol, reflecting PIP2 hydrolysis. The translocation was prevented by pretreatment with PKC inhibitors. Involvement of PKC activation in the mechanosensitivity of muscarinic K+ channels was confirmed in native rabbit atrial myocytes. These results suggest that the mechanosensitivity of GIRK channels is mediated primarily by channel-PIP2 interaction, with PKC playing an important role in modulating the interaction probably through PIP2 hydrolysis.     

Functional and biochemical evidence for G-protein-gated inwardly rectifying K+ (GIRK) channels composed of GIRK2 and GIRK3

G-protein-gated inwardly rectifying K(+) (GIRK) channels are widely expressed in the brain and are activated by at least eight different neurotransmitters. As K(+) channels, they drive the transmembrane potential toward E(K) when open and thus dampen neuronal excitability. There are four mammalian GIRK subunits (GIRK1-4 or Kir 3.1-4), with GIRK1 being the most unique of the four by possessing a long carboxyl-terminal tail. Early studies suggested that GIRK1 was an integral component of native GIRK channels. However, more recent data indicate that native channels can be either homo- or heterotetrameric complexes composed of several GIRK subunit combinations. The functional implications of subunit composition are poorly understood at present. The purpose of this study was to examine the functional and biochemical properties of GIRK channels formed by the co-assembly of GIRK2 and GIRK3, the most abundant GIRK subunits found in the mammalian brain. To examine the properties of a channel composed of these two subunits, we co-transfected GIRK2 and GIRK3 in CHO-K1 cells and assayed the cells for channel activity by patch clamp. The most significant difference between the putative GIRK2/GIRK3 heteromultimeric channel and GIRK1/GIRKx channels at the single channel level was an approximately 5-fold lower sensitivity to activation by Gbetagamma. Complexes containing only GIRK2 and GIRK3 could be immunoprecipitated from transfected cells and could be purified from native brain tissue. These data indicate that functional GIRK channels composed of GIRK2 and GIRK3 subunits exist in brain.     

The Stoichiometry of Gbeta gamma binding to G-protein-regulated inwardly rectifying K+ channels (GIRKs)

G-protein-coupled inwardly rectifying K(+) (GIRK; Kir3.x) channels are the primary effectors of numerous G-protein-coupled receptors. GIRK channels decrease cellular excitability by hyperpolarizing the membrane potential in cardiac cells, neurons, and secretory cells. Although direct regulation of GIRKs by the heterotrimeric G-protein subunit Gbetagamma has been extensively studied, little is known about the number of Gbetagamma binding sites per channel. Here we demonstrate that purified GIRK (Kir 3.x) tetramers can be chemically cross-linked to exogenously purified Gbetagamma subunits. The observed laddering pattern of Gbetagamma attachment to GIRK4 homotetramers was consistent with the binding of one, two, three, or four Gbetagamma molecules per channel tetramer. The fraction of channels chemically cross-linked to four Gbetagamma molecules increased with increasing Gbetagamma concentrations and approached saturation. These results suggest that GIRK tetrameric channels have four Gbetagamma binding sites. Thus, GIRK (Kir 3.x) channels, like the distantly related cyclic nucleotide-gated channels, are tetramers and exhibit a 1:1 subunit/ligand binding stoichiometry.     

Inhibition of G protein-activated inwardly rectifying K+ channels by different classes of antidepressants

Various antidepressants are commonly used for the treatment of depression and several other neuropsychiatric disorders. In addition to their primary effects on serotonergic or noradrenergic neurotransmitter systems, antidepressants have been shown to interact with several receptors and ion channels. However, the molecular mechanisms that underlie the effects of antidepressants have not yet been sufficiently clarified. G protein-activated inwardly rectifying K(+) (GIRK, Kir3) channels play an important role in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to have therapeutic potential for several neuropsychiatric disorders and cardiac arrhythmias. In the present study, we investigated the effects of various classes of antidepressants on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 subunits, extracellular application of sertraline, duloxetine, and amoxapine effectively reduced GIRK currents, whereas nefazodone, venlafaxine, mianserin, and mirtazapine weakly inhibited GIRK currents even at toxic levels. The inhibitory effects were concentration-dependent, with various degrees of potency and effectiveness. Furthermore, the effects of sertraline were voltage-independent and time-independent during each voltage pulse, whereas the effects of duloxetine were voltage-dependent with weaker inhibition with negative membrane potentials and time-dependent with a gradual decrease in each voltage pulse. However, Kir2.1 channels were insensitive to all of the drugs. Moreover, the GIRK currents induced by ethanol were inhibited by sertraline but not by intracellularly applied sertraline. The present results suggest that GIRK channel inhibition may reveal a novel characteristic of the commonly used antidepressants, particularly sertraline, and contributes to some of the therapeutic effects and adverse effects.     

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K⁺ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue^1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (G_i and G_o) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K⁺ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders³. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations³.Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K⁺ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K⁺ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry⁴ or radiotracer analysis⁵, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.     

Kir4.1 K+ channels are regulated by external cations

The inwardly rectifying potassium channel (Kir), Kir4.1 mediates spatial K(+)-buffering in the CNS. In this process the channel is potentially exposed to a large range of extracellular K(+) concentrations ([K(+)]o). We found that Kir4.1 is regulated by K(+)o. Increased [K(+)]o leads to a slow (mins) increase in the whole-cell currents of Xenopus oocytes expressing Kir4.1. Conversely, removing K(+) from the bath solution results in a slow decrease of the currents. This regulation is not coupled to the pHi-sensitive gate of the channel, nor does it require the presence of K67, a residue necessary for K(+)o-dependent regulation of Kir1.1. The voltage-dependent blockers Cs(+) and Ba(2+) substitute for K(+) and prevent deactivation of the channel in the absence of K(+)o. Cs(+) blocks and regulates the channel with similar affinity, consistent with the regulatory sites being in the selectivity-filter of the channel. Although both Rb(+) and NH4(+) permeate Kir4.1, only Rb(+) is able to regulate the channel. We conclude that Kir4.1 is regulated by ions interacting with specific sites in the selectivity filter. Using a kinetic model of the permeation process we show the plausibility of the channel's sensing the extracellular ionic environment through changes in the selectivity occupancy pattern, and that it is feasible for an ion with the selectivity properties of NH4(+) to permeate the channel without inducing these changes.     

Transfection of a phosphatidyl-4-phosphate 5-kinase gene into rat atrial myocytes removes inhibition of GIRK current by endothelin and alpha-adrenergic agonists

GIRK (G protein-activated inward-rectifying K(+) channel) channels, important regulators of membrane excitability in the heart and in the central nervous, are activated by interaction with betagamma subunits from heterotrimeric G proteins upon receptor stimulation. For activation interaction of the channel with phosphatidylinositol 4,5-bisphosphate (PtIns(4,5)P(2)) is conditional. Previous studies have provided evidence that in myocytes PtIns(4,5)P(2) levels relevant to GIRK channel regulation are under regulatory control of receptors activating phospholipase C. In the present study a phosphatidyl-4-phosphate 5-kinase was expressed in atrial myocytes by transient transfection. This did not affect basal properties of GIRK current activated by acetylcholine via M(2) receptors but completely abolished inhibition of guanosine triphosphate-gamma-S activated current by endothelin-1 or alpha-adrenergic agonists. We conclude that though PtIns(4,5)P(2) is conditional for channel gating, its normal level in the membrane is not limiting basal function of GIRK channels. Moreover, our data provide further evidence for a regulation of GIRK channels by alpha(1A) receptors and endothelin-A receptors, endogenously expressed in atrial myocytes, via depletion of PtIns(4,5)P(2).     

Cytoplasmic unsaturated free fatty acids inhibit ATP-dependent gating of the G protein-gated K(+) channel

This study reports the identification of an endogenous inhibitor of the G protein-gated (K(ACh)) channel and its effect on the K(ACh) channel kinetics. In the presence of acetylcholine in the pipette, K(ACh) channels in inside-out atrial patches were activated by applying GTP to the cytoplasmic side of the membrane. In these patches, addition of physiological concentration of intracellular ATP (4 mM) upregulated K(ACh) channel activity approximately fivefold and induced long-lived openings. However, such ATP-dependent gating is normally not observed in cell-attached patches, indicating that an endogenous substance that inhibits the ATP effect is present in the cell. We searched for such an inhibitor in the cell. ATP-dependent gating of the K(ACh) channel was inhibited by the addition of the cytosolic fraction of rat atrial or brain tissues. The lipid component of the cytosolic fraction was found to contain the inhibitory activity. To identify the lipid inhibitor, we tested the effect of approximately 40 different lipid molecules. Among the lipids tested, only unsaturated free fatty acids such as oleic, linoleic, and arachidonic acids (0.2-2 microM) reversibly inhibited the ATP-dependent gating of native K(ACh) channels in atrial cells and hippocampal neurons, and of recombinant K(ACh) channels (GIRK1/4 and GIRK1/2) expressed in oocytes. Unsaturated free fatty acids also inhibited phosphatidylinositol-4, 5-bisphosphate (PIP(2))-induced changes in K(ACh) channel kinetics but were ineffective against ATP-activated background K(1) channels and PIP(2)-activated K(ATP) channels. These results show that during agonist-induced activation, unsaturated free fatty acids in the cytoplasm help to keep the cardiac and neuronal K(ACh) channels downregulated by antagonizing their ATP-dependent gating. The opposing effects of ATP and free fatty acids represent a novel regulatory mechanism for the G protein-gated K(+) channel.     

Functional properties of a truncated recombinant GIRK5 potassium channel

Xenopus laevis oocytes codify a G-protein-activated inward rectifier potassium channel (GIRK5 or Kir3.5). Coinjection of other GIRKs, the muscarinic m2 receptor, or Gbetagamma protein cRNAs is required to observe functional GIRKx-GIRK5 heteromultimers in oocytes. Studies with GIRK2 isoforms have shown that the size of the amino or carboxyl terminus plays a crucial role on giving functional K(+) channels. In this work we studied the properties of a GIRK5 with 25 amino acids deleted toward its amino-terminal domain. Injection of GIRK5-Delta25 cRNA alone displayed large basal and transient inward rectifying currents in oocytes. The instantaneous currents reached a stationary level after a long duration voltage pulse (10 s). For this relaxation, fast (tau(1)) and slow (tau(2)) time constants were estimated at different voltages. Recovery from inactivation followed a monoexponential function (tau=0.95+/-0.07 s). By contrast with other inward rectifier channels, blockade of GIRK5-Delta25 by extracellular Ba(2+) was voltage-independent (K(d)=102+/-2 microM), suggesting the presence of a Ba(2+) site at the external channel vestibule. To confirm this hypothesis, the Ba(2+) sensitivity of two charged mutants GIRK5-Delta25(N129E) and GIRK5-Delta25(K157E) at each of the external loops was determined. GIRK5-Delta25(N129E) and GIRK5-Delta25(K157E) showed a 100-fold and 2-fold higher affinity to Ba(2+), respectively, supporting the existence of this Ba(2+) binding site.     

Regulation of cell proliferation of human induced pluripotent stem cell-derived mesenchymal stem cells via ether-à-go-go 1 (hEAG1) potassium channel

The successful generation of a high yield of mesenchymal stem cells (MSCs) from human induced pluripotent stem cells (iPSCs) may represent an unlimited cell source with superior therapeutic benefits for tissue regeneration to bone marrow (BM)-derived MSCs. We investigated whether the differential expression of ion channels in iPSC-MSCs was responsible for their higher proliferation capacity than BM-MSCs. The expression of ion channels for K(+), Na(+), Ca(2+), and Cl(-) was examined by RT-PCR. The electrophysiological properties of iPSC-MSCs and BM-MSCs were then compared by patch-clamp experiments to verify their functional roles. Significant mRNA expression of ion channel genes including KCa1.1, KCa3.1, KCNH1, Kir2.1, SCN9A, CACNA1C, and Clcn3 was observed in both human iPSC-MSCs and BM-MSCs, whereas Kir2.2 and Kir2.3 were only detected in human iPSC-MSCs. Five types of currents [big-conductance Ca(2+)-activated K(+) current (BK(Ca)), delayed rectifier K(+) current (IK(DR)), inwardly rectifying K(+) current (I(Kir)), Ca(2+)-activated K(+) current (IK(Ca)), and chloride current (I(Cl))] were found in iPSC-MSCs (83%, 47%, 11%, 5%, and 4%, respectively) but only four of them (BK(Ca), IK(DR), I(Kir), and IK(Ca)) were identified in BM-MSCs (76%, 25%, 22%, and 11%, respectively). Cell proliferation was examined with MTT or bromodeoxyuridine assay, and doubling times were 2.66 and 3.72 days for iPSC-MSCs and BM-MSCs, respectively, showing a 1.4-fold discrepancy. Blockade of IK(DR) with short hairpin RNA or human ether-à-go-go 1 (hEAG1) channel blockers, 4-AP and astemizole, significantly reduced the rate of proliferation of human iPSC-MSCs. These treatments also decreased the rate of proliferation of human BM-MSCs albeit to a lesser extent. These findings demonstrate that the hEAG1 channel plays a crucial role in controlling the proliferation rate of human iPSC-MSCs and to a lesser extent in BM-MSCs.     

Regulation of the inward rectifying properties of G-protein-activated inwardly rectifying K+ (GIRK) channels by Gbeta gamma subunits

Gbetagamma subunits are known to bind to and activate G-protein-activated inwardly rectifying K(+) channels (GIRK) by regulating their open probability and bursting behavior. Studying G-protein regulation of either native GIRK (I(KACh)) channels in feline atrial myocytes or heterologously expressed GIRK1/4 channels in Chinese hamster ovary cells and HEK 293 cells uncovered a novel Gbetagamma subunit mediated regulation of the inwardly rectifying properties of these channels. I(KACh) activated by submaximal concentrations of acetylcholine exhibited a approximately 2.5-fold stronger inward rectification than I(KACh) activated by saturating concentrations of acetylcholine. Similarly, the inward rectification of currents through GIRK1/4 channels expressed in HEK cells was substantially weakened upon maximal stimulation with co-expressed Gbetagamma subunits. Analysis of the outward current block underlying inward rectification demonstrated that the fraction of instantaneously blocked channels was reduced when Gbetagamma was over-expressed. The Gbetagamma induced weakening of inward rectification was associated with reduced potencies for Ba(2+) and Cs(+) to block channels from the extracellular side. Based on these results we propose that saturation of the channel with Gbetagamma leads to a conformational change within the pore of the channel that reduced the potency of extracellular cations to block the pore and increased the fraction of channels inert to a pore block in outward direction.     

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.     

Basal activity of GIRK5 isoforms

G protein-coupled inwardly rectifying K(+) channels (GIRK or Kir3) form functional heterotetramers gated by Gbetagamma subunits. GIRK channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK5 (Kir3.5) is the oocyte homologue of the mammalian GIRK subunits that conform the K(ACh) channel. It has been claimed that even when the oocytes express GIRK5 proteins they do not form functional channels. However, the GIRK5 gene shows three initiation sites that suggest the existence of three isoforms. In a previous work we demonstrated the functionality of homomultimers of the shortest isoform overexpressed in the own oocytes. Remarkably, the basal GIRK5-Delta25 inward currents were not coupled to the activation of a G-protein receptor in the oocytes. These results encouraged us to study this channel in another expression system. In this work we show that Sf21 insect cells can be successfully transfected with this channel. GIRK5-Delta25 homomultimers produce time-dependent inward currents only with GTPgammaS in the recording pipette. Therefore, alternative modes of stimulus input to heterotrimeric G-proteins should be present in the oocytes to account for these results.     

Losartan prevents stretch-induced electrical remodeling in cultured atrial neonatal myocytes

Atrial fibrillation (AF) is the most frequent arrhythmia found in clinical practice. In recent studies, a decrease in the development or recurrence of AF was found in hypertensive patients treated with angiotensin-converting enzyme inhibitors or angiotensin receptor-blocking agents. Hypertension is related to an increased wall tension in the atria, resulting in increased stretch of the individual myocyte, which is one of the major stimuli for the remodeling process. In the present study, we used a model of cultured atrial neonatal rat cardiomyocytes under conditions of stretch to provide insight into the mechanisms of the preventive effect of the angiotensin receptor-blocking agent losartan against AF on a molecular level. Stretch significantly increased protein-to-DNA ratio and atrial natriuretic factor mRNA expression, indicating hypertrophy. Expression of genes encoding for the inward rectifier K(+) current (I(K1)), Kir 2.1, and Kir 2.3, as well as the gene encoding for the ultrarapid delayed rectifier K(+) current (I(Kur)), Kv 1.5, was significantly increased. In contrast, mRNA expression of Kv 4.2 was significantly reduced in stretched myocytes. Alterations of gene expression correlated with the corresponding current densities: I(K1) and I(Kur) densities were significantly increased in stretched myocytes, whereas transient outward K(+) current (I(to)) density was reduced. These alterations resulted in a significant abbreviation of the action potential duration. Losartan (1 microM) prevented stretch-induced increases in the protein-to-DNA ratio and atrial natriuretic peptide mRNA expression in stretched myocytes. Concomitantly, losartan attenuated stretch-induced alterations in I(K1), I(Kur), and I(to) density and gene expression. This prevented the stretch-induced abbreviation of action potential duration. Prevention of stretch-induced electrical remodeling might contribute to the clinical effects of losartan against AF.     

Molecular basis and structural insight of vascular K(ATP) channel gating by S-glutathionylation

The vascular ATP-sensitive K(+) (K(ATP)) channel is targeted by a variety of vasoactive substances, playing an important role in vascular tone regulation. Our recent studies indicate that the vascular K(ATP) channel is inhibited in oxidative stress via S-glutathionylation. Here we show evidence for the molecular basis of the S-glutathionylation and its structural impact on channel gating. By comparing the oxidant responses of the Kir6.1/SUR2B channel with the Kir6.2/SUR2B channel, we found that the Kir6.1 subunit was responsible for oxidant sensitivity. Oxidant screening of Kir6.1-Kir6.2 chimeras demonstrated that the N terminus and transmembrane domains of Kir6.1 were crucial. Systematic mutational analysis revealed three cysteine residues in these domains: Cys(43), Cys(120), and Cys(176). Among them, Cys(176) was prominent, contributing to >80% of the oxidant sensitivity. The Kir6.1-C176A/SUR2B mutant channel, however, remained sensitive to both channel opener and inhibitor, which indicated that Cys(176) is not a general gating site in Kir6.1, in contrast to its counterpart (Cys(166)) in Kir6.2. A protein pull-down assay with biotinylated glutathione ethyl ester showed that mutation of Cys(176) impaired oxidant-induced incorporation of glutathione (GSH) into the Kir6.1 subunit. In contrast to Cys(176), Cys(43) had only a modest contribution to S-glutathionylation, and Cys(120) was modulated by extracellular oxidants but not intracellular GSSG. Simulation modeling of Kir6.1 S-glutathionylation suggested that after incorporation to residue 176, the GSH moiety occupied a space between the slide helix and two transmembrane helices. This prevented the inner transmembrane helix from undergoing conformational changes necessary for channel gating, retaining the channel in its closed state.     

Development of recombinant cell line co-expressing mutated Nav1.5, Kir2.1, and hERG for the safety assay of drug candidates

To provide a high-throughput screening method for human ether-a-go-go-gene-related gene (hERG) K(+) channel inhibition, a new recombinant cell line, in which single action potential (AP)-induced cell death was produced by gene transfection. Mutated human cardiac Na(+) channel Nav1.5 (IFM/Q3), which shows extremely slow inactivation, and wild-type inward rectifier K(+) channel, Kir2.1, were stably co-expressed in HEK293 cells (IFM/Q3+Kir2.1). In IFM/Q3+Kir2.1, application of single electrical stimulation (ES) elicited a long AP lasting more than 30 s and led cells to die by more than 70%, whereas HEK293 co-transfected with wild-type Nav1.5 and Kir2.1 fully survived. The additional expression of hERG K(+) channels in IFM/Q3+Kir2.1 shortened the duration of evoked AP and thereby markedly reduced the cell death. The treatment of the cells with hERG channel inhibitors such as nifekalant, E-4031, cisapride, terfenadine, and verapamil, recovered the prolonged AP and dose-dependently facilitated cell death upon ES. The EC(50) values to induce the cell death were 3 μM, 19 nM, 17 nM, 74 nM, and 3 μM, respectively, whereas 10 μM nifedipine did not induce cell death. Results indicate the high utility of this cell system for hERG K(+) channel safety assay.