Scanning mutagenesis of the putative transmembrane segments of Kir2.1, an inward rectifier potassium channel

Structural models of inward rectifier K+ channels incorporate four identical or homologous subunits, each of which has two hydrophobic segments (M1 and M2) which are predicted to span the membrane as α helices. Since hydrophobic interactions between proteins and membrane lipids are thought to be generally of a nonspecific nature, we attempted to identify lipid-contacting residues in Kir2.1 as those which tolerate mutation to tryptophan, which has a large hydrophobic side chain. Tolerated mutations were defined as those which produced measurable inwardly rectifying currents in Xenopus oocytes. To distinguish between water-accessible positions and positions adjacent to membrane lipids or within the protein interior we also mutated residues in M1 and M2 individually to aspartate, since an amino acid with a charged side chain should not be tolerated at lipid-facing or interior positions, due to the energy cost of burying a charge in a hydrophobic environment. Surprisingly, 17 out of 20 and 17 out of 22 non-tryptophan residues in M1 and M2, respectively, tolerated being mutated to tryptophan. Moreover, aspartate was tolerated at 15 out of 22 and 15 out of 21 non-aspartate M1 and M2 positions respectively. Periodicity in the pattern of tolerated vs. nontolerated mutations consistent with α helices or β strands did not emerge convincingly from these data. We consider the possibility that parts of M1 and M2 may be in contact with water.

Flexibility of the Kir6.2 inward rectifier K+ channel pore

Interactions of sulfhydryl reagents with introduced cysteines in the pore-forming (Kir6.2) subunits of the KATP channel were examined. 2-Aminoethyl methanethiosulfonate (MTSEA+) failed to modify Cd2+-insensitive control-Kir6.2 channels, but rapidly and irreversibly modified Kir6.2[L164C] (L164C) channels. Although a single Cd2+ ion is coordinated by L164C, four MTSEA+ “hits” can occur, each sequentially reducing the single-channel current. A dimeric fusion of control-Kir6.2 and L164C subunits generates Cd2+-insensitive channels, confirming that at least three cysteines are required for coordination, but MTSEA+ modification of the dimer occurs in two hits. L164C channels were not modified by bromotrimethyl ammoniumbimane (qBBr+), even though qBBr+ caused voltage-dependent block (as opposed to modification) that was comparable to that of MTSEA+ or 3-(triethylammonium)propyl methanethiosulfonate (MTSPTrEA+), implying that qBBr+ can also enter the inner cavity but does not modify L164C residues. The Kir channel pore structure was modeled by homology with the KcsA crystal structure. A stable conformation optimally places the four L164C side chains for coordination of a single Cd2+ ion. Modification of these cysteines by up to four MTSEA+ (or three MTSPTrEA+, or two qBBr+) does not require widening of the cavity to accommodate the derivatives within it. However, like the KcsA crystal structure, the energy-minimized model shows a narrowing at the inner entrance, and in the Kir6.2 model this narrowing excludes all ions. To allow entry of ions as large as MTSPTrEA+ or qBBr+, the entrance must widen to >8 Å, but this widening is readily accomplished by minimal M2 helix motion and side-chain rearrangement.

Carbon monoxide inhibits inward rectifier potassium channels in cardiomyocytes

Reperfusion-induced ventricular fibrillation (VF) severely threatens the lives of post-myocardial infarction patients. Carbon monoxide (CO) - produced by haem oxygenase in cardiomyocytes - has been reported to prevent VF through an unknown mechanism of action. Here, we report that CO prolongs action potential duration (APD) by inhibiting a subset of inward-rectifying potassium (Kir) channels. We show that CO blocks Kir2.2 and Kir2.3 but not Kir2.1 channels in both cardiomyocytes and HEK-293 cells transfected with Kir. CO directly inhibits Kir2.3 by interfering with its interaction with the second messenger phosphatidylinositol (4,5)-bisphosphate (PIP 2). As the inhibition of Kir2.2 and Kir2.3 by CO prolongs APD in myocytes, cardiac Kir2.2 and Kir2.3 are promising targets for the prevention of reperfusion-induced VF. © 2014 Macmillan Publishers Limited. All rights reserved.

Molecular basis of native inward rectifier currents : role of Kir2 subunits

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Molecular basis of native inward rectifier currents : role of Kir2 subunits

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Differential effects of glutamate transporter inhibitors on the global electrophysiological response of astrocytes to neuronal stimulation

Astrocytes are responsible for regulating extracellular levels of glutamate and potassium during neuronal activity. Glutamate clearance is handled by glutamate transporter subtypes glutamate transporter 1 and glutamate-aspartate transporter in astrocytes. DL-threo-beta-benzyloxyaspartate (TBOA) and dihydrokainate (DHK) are extensively used as inhibitors of glial glutamate transport activity. Using whole-cell recordings, we characterized the effects of both transporter inhibitors on afferent-evoked astrocyte currents in acute cortical slices of 3-week-old rats. When neuronal afferents were stimulated, passive astrocytes responded by a rapid inward current followed by a persistent tail current. The first current corresponded to a glutamate transporter current. This current was inhibited by both inhibitors and by tetrodotoxin. The tail current is an inward potassium current as it was blocked by barium. Besides inhibiting transporter currents, TBOA strongly enhanced the tail current. This effect was barium-sensitive and might be due to a rise in extracellular potassium level and increased glial potassium uptake. Unlike TBOA, DHK did not enhance the tail current but rather inhibited it. This result suggests that, in addition to inhibiting glutamate transport, DHK prevents astrocyte potassium uptake, possibly by blockade of inward-rectifier channels. This study revealed that, in brain slices, glutamate transporter inhibitors exert complex effects that cannot be attributed solely to glutamate transport inhibition.

L’effet de la surexpression du récepteur de type 1 à l’angiotensine II sur les courants potassiques et calciques au niveau des oreillettes

Le système rénine-angiotensine est impliqué dans le remodelage structurel et électrique caractérisant la fibrillation auriculaire (FA). L’angiotensine II (ANG II) induit le développement de fibrose et d’hypertrophie au niveau des oreillettes, prédisposant à la FA. Or, les mécanismes électrophysiologiques par lesquels l’ANG II pourrait promouvoir la FA sont peu connus. L’objectif de ce projet de recherche est d’évaluer l’effet de l’ANG II sur les courants potassiques et calciques au niveau auriculaire indépendamment du remodelage structurel. Pour ce faire, nous avons utilisé la technique de patch-clamp avec un modèle de souris surexprimant le récepteur de type 1 à l’angiotensine II (AT1R) spécifiquement au niveau cardiaque. Pour distinguer les effets directs de la surexpression d’AT1R des effets induits par le remodelage cardiaque, nous avons étudié des souris âgées de 180 jours, qui présentent du remodelage structurel, et des souris âgées de 50 jours, qui n’en présentent pas. Des études précédentes sur ce modèle ont montré qu’au niveau des myocytes ventriculaires, l’ANG II réduit le courant potassique global (Ipeak) et rectifiant entrant (IK1) ainsi que le courant calcique de type L (ICaL). Ainsi, notre hypothèse est que l’ANG II modulera aussi ces courants au niveau auriculaire, pouvant ainsi augmenter l’hétérogénéité de repolarisation auriculaire et de ce fait le risque de développer et maintenir la FA. Nous avons observé une diminution significative de la densité d’IK1 dans l’oreillette gauche des souris transgéniques sans changement d’Ipeak. De plus, la densité d’ ICaL n’est pas réduite chez les souris transgéniques âgées de 50 jours. En conclusion, l’effet de l’ANG II sur les courants potassiques et calciques semble dépendre de la chambre cardiaque. En effet, nous savions que l’ANGII réduisait Ipeak, IK1 et ICaL au niveau ventriculaire, mais nos résultats ont montré qu’il ne les affectait pas directement au niveau des oreillettes. Ceci suggère des mécanismes de régulation impliquant des voies de signalisation distinctes selon les chambres cardiaques. Enfin, nos résultats montrant l’absence de l’influence directe de la surexpression d’AT1R sur les canaux K+ et Ca2+ au niveau des myocytes auriculaires renforcent l’importance d’approfondir nos connaissances sur les effets de l’angiotensine II sur le développement de la fibrose, sur le remodelage structurel et sur la conduction électrique cardiaque.

RGS proteins reconstitute the rapid gating kinetics of Gβγ-activated inwardly rectifying K+ channels

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Gα(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20–40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of “regulators of G protein signaling” (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor → GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent “basal” GIRK current was suppressed by ≈40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of “slow” postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.

pH gating of ROMK (Kir1.1) channels: Control by an Arg-Lys-Arg triad disrupted in antenatal Bartter syndrome

Inward-rectifier K+ channels of the ROMK (Kir1.1) subtype are responsible for K+ secretion and control of NaCl absorption in the kidney. A hallmark of these channels is their gating by intracellular pH in the neutral range. Here we show that a lysine residue close to TM1, identified previously as a structural element required for pH-induced gating, is protonated at neutral pH and that this protonation drives pH gating in ROMK and other Kir channels. Such anomalous titration of this lysine residue (Lys-80 in Kir1.1) is accomplished by the tertiary structure of the Kir protein: two arginines in the distant N and C termini of the same subunit (Arg-41 and Arg-311 in Kir1.1) are located in close spatial proximity to the lysine allowing for electrostatic interactions that shift its pKa into the neutral pH range. Structural disturbance of this triad as a result from a number of point mutations found in patients with antenatal Bartter syndrome shifts the pKa of the lysine residue off the neutral pH range and results in channels permanently inactivated under physiological conditions. Thus, the results provide molecular understanding for normal pH gating of Kir channels as well as for the channel defects found in patients with antenatal Bartter syndrome.

A regenerative link in the ionic fluxes through the weaver potassium channel underlies the pathophysiology of the mutation

The homozygous weaver mouse displays neuronal degeneration in several brain regions. Previous experiments in heterologous expression systems showed that the G protein-gated inward rectifier K+ channel (GIRK2) bearing the weaver pore-region GYG-to-SYG mutation (i) is not activated by Gβγ subunits, but instead shows constitutive activation, and (ii) is no longer a K+-selective channel but conducts Na+ as well. The present experiments on weaverGIRK2 (wvGIRK2) expressed in Xenopus oocytes show that the level of constitutive activation depends on intracellular Na+ concentration. In particular, manipulations that decrease intracellular Na+ produce a component of Na+-permeable current activated via a G protein pathway. Therefore, constitutive activation may not arise because the weaver mutation directly alters the gating transitions of the channel protein. Instead, there may be a regenerative cycle of Na+ influx through the wvGIRK2 channel, leading to additional Na+ activation. We also show that the wvGIRK2 channel is permeable to Ca2+, providing an additional mechanism for the degeneration that characterizes the weaver phenotype. We further demonstrate that the GIRK4 channel bearing the analogous weaver mutation has properties similar to those of the wvGIRK2 channel, providing a glimpse of the selective pressures that have maintained the GYG sequence in nearly all known K+ channels.

γ-Aminobutyric acid type B receptors are expressed and functional in mammalian cardiomyocytes

γ-Hydroxybutyrate (GHB), an anesthetic adjuvant analog of γ-aminobutyrate (GABA), depresses cell excitability in hippocampal neurons by inducing hyperpolarization through the activation of a prominent inwardly rectifying K+ (Kir3) conductance. These GABA type B (GABAB)-like effects are clearly shown at high concentrations of GHB corresponding to blood levels usually reached during anesthesia and are mimicked by the GABAB agonist baclofen. Recent studies of native GABAB receptors (GABABRs) have favored the concept that GHB is also a selective agonist. Furthermore, cloning has demonstrated that GABABRs assemble heteromeric complexes from the GABABR1 and GABABR2 subtypes and that these assemblies are activated by GHB. The surprisingly high tissue content, together with anti-ischemic and protective effects of GHB in the heart, raises the question of a possible influence of GABAB agonists on excitable cardiac cells. In the present study, we provide electrophysiological evidence that GHB activates an inwardly rectifying K+ current in rat ventricular myocytes. This effect is mimicked by baclofen, reversibly inhibited by GABAB antagonists, and prevented by pertussis toxin pretreatment. Both GABABR1 and GABABR2 are detected in cardiomyocytes by Western blotting and are shown to coimmunoprecipitate. Laser scanning confocal microscopy discloses an even distribution of the two receptors in the sarcolemma and along the transverse tubular system. Hence, we conclude that GABABRs are distributed not only in neuronal tissues but also in the heart, where they can be activated and induce electrophysiological alterations through G-protein-coupled inward rectifier potassium channels.

Susceptibility of cloned K+ channels to reactive oxygen species.

Free radical-induced oxidant stress has been implicated in a number of physiological and pathophysiological states including ischemia and reperfusion-induced dysrhythmia in the heart, apoptosis of T lymphocytes, phagocytosis, and neurodegeneration. We have studied the effects of oxidant stress on the native K+ channel from T lymphocytes and on K+ channels cloned from cardiac, brain, and T-lymphocyte cells and expressed in Xenopus oocytes. The activity of three Shaker K+ channels (Kv1.3, Kv1.4, and Kv1.5), one Shaw channel (Kv3.4), and one inward rectifier K+ channel (IRK3) was drastically inhibited by photoactivation of rose bengal, a classical generator of reactive oxygen species. Other channel types (such as Shaker K+ channel Kv1.2, Shab channels Kv2.1 and Kv2.2, Shal channel Kv4.1, inward rectifiers IRK1 and ROMK1, and hIsK) were completely resistant to this treatment. On the other hand tert-butyl hydroperoxide, another generator of reactive oxygen species, removed the fast inactivation processes of Kv1.4 and Kv3.4 but did not alter other channels. Xanthine/xanthine oxidase system had no effect on all channels studied. Thus, we show that different types of K+ channels are differently modified by reactive oxygen species, an observation that might be of importance in disease states.

Inhibition of function in Xenopus oocytes of the inwardly rectifying G-protein-activated atrial K channel (GIRK1) by overexpression of a membrane-attached form of the C-terminal tail.

Coexpression in Xenopus oocytes of the inwardly rectifying guanine nucleotide binding (G)-protein-gated K channel GIRK1 with a myristoylated modification of the (putative) cytosolic C-terminal tail [GIRK1 aa 183-501 fused in-frame to aa 1-15 of p60src and denoted src+ (183-501)] leads to a high degree of inhibition of the inward G-protein-gated K+ current. The nonmyristoylated segment, src- (183-501), is not active. Although some interference with assembly is not precluded, the evidence indicates that the main mechanism of inhibition is interference with functional activation of the channel by G proteins. In part, the tail functions as a blocking particle similar to a "Shaker ball"; it may also function by competing for the available supply of free G beta gamma liberated by hormone activation of a seven-helix receptor. The non-G-protein-gated weak inward rectifier ROMK1 is less effectively inhibited, and a Shaker K channel was not inhibited. Immunological assays show the presence of a high concentration of src+ (183-501) in the plasma membrane and the absence of any membrane forms for the nonmyristoylated segment.

Evidence that neuronal G-protein-gated inwardly rectifying K+ channels are activated by G beta gamma subunits and function as heteromultimers.

Guanine nucleotide-binding proteins (G proteins) activate K+ conductances in cardiac atrial cells to slow heart rate and in neurons to decrease excitability. cDNAs encoding three isoforms of a G-protein-coupled, inwardly rectifying K+ channel (GIRK) have recently been cloned from cardiac (GIRK1/Kir 3.1) and brain cDNA libraries (GIRK2/Kir 3.2 and GIRK3/Kir 3.3). Here we report that GIRK2 but not GIRK3 can be activated by G protein subunits G beta 1 and G gamma 2 in Xenopus oocytes. Furthermore, when either GIRK3 or GIRK2 was coexpressed with GIRK1 and activated either by muscarinic receptors or by G beta gamma subunits, G-protein-mediated inward currents were increased by 5- to 40-fold. The single-channel conductance for GIRK1 plus GIRK2 coexpression was intermediate between those for GIRK1 alone and for GIRK2 alone, and voltage-jump kinetics for the coexpressed channels displayed new kinetic properties. On the other hand, coexpression of GIRK3 with GIRK2 suppressed the GIRK2 alone response. These studies suggest that formation of heteromultimers involving the several GIRKs is an important mechanism for generating diversity in expression level and function of neurotransmitter-coupled, inward rectifier K+ channels.