IRK1 Channel Research Articles

Prediction of ion channel transport from Grote—Hynes and Kramers theories

Generalized Langevin equation based Grote—Hynes (GH) theory and Langevin equation based Kramers theory are used to calculate the transmission coefficient for K+ diffusion through a model of the biological potassium ion channel IRK1, which contains a high potential barrier in the selectivity filter. The ion friction kernel is determined from a molecular dynamics (MD) simulation of the force on a stationary ion at the barrier top. The GH and Kramers estimates of the transmission coefficient are compared with those obtained from MD simulations of ion diffusion at the barrier top of the IRK1 channel. It is found that the GH estimate agrees with the value determined by rigorous MD, but the Kramers estimate is about 40% too small. The success or failure of GH and Kramers theories for various other systems is discussed and compared with these results.

Modulation of the Inward Rectifier Potassium Channel IRK1 by the Ras Signaling Pathway

In this study, we investigated the role of Ras and the mitogen-activated protein kinase (MAPK) pathway in the modulation of the inward rectifier potassium channel IRK1. We show that although expression of IRK1 in HEK 293 cells leads to the appearance of a potassium current with strong inward rectifying properties, coexpression of the constitutively active form of Ras (Ras-L61) results in a significant reduction of the mean current density without altering the biophysical properties of the channel. The inhibitory effect of Ras-L61 is not due to a decreased expression of IRK1 since Northern analysis indicates that IRK1 mRNA level is not affected by Ras-L61 co-expression. Moreover, the inhibition can be relieved by treatment with the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor PD98059. Confocal microscopy analysis of cells transfected with the fusion construct green fluorescent protein-IRK1 shows that the channel is mainly localized at the plasma membrane. Coexpression of Ras-L61 delocalizes fluorescence to the cytoplasm, whereas treatment with PD98059 partially restores the membrane localization. In conclusion, our data indicate that the Ras-MAPK pathway modulates IRK1 current by affecting the subcellular localization of the channel. This suggests a role for Ras signaling in regulating the intracellular trafficking of this channel.

Phasic and tonic attenuation of EPSPs by inward rectifier K+ channels in rat hippocampal pyramidal cells

We made whole-cell recordings from CA1 pyramidal cells of hippocampal slices in combination with brief dendritic glutamate pulses to study the role of constitutive inwardly rectifying K+ channels (IRK, Kir2.0) and G-protein-activated inwardly rectifying K+ channels (GIRK, Kir3.0) in the processing of excitatory inputs. Phasic activation of GIRK channels by baclofen (20 microM) produced a reversible reduction of glutamate-evoked postsynaptic potentials (GPSPs), our equivalent of EPSPs, by about one-third. Conversely, tertiapin (30 nM), a selective inhibitor of GIRK channels, and Ba2+ (200 microM), a non-selective blocker of inwardly rectifying K+ channels, enhanced GPSPs and, in voltage-clamp experiments, reduced the underlying K+ conductances, indicating a functionally significant background GIRK conductance, in addition to constitutive IRK channel activity. When examined after suppression of endogenous adenosinergic inhibition, using either adenosine deaminase or the selective A1 receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine, tertiapin failed to influence either the GPSPs or the inwardly rectifying K+ conductance. Voltage-clamp recordings from acutely isolated CA1 pyramidal cells not exposed to ambient adenosine exhibited no response to tertiapin, whereas Ba2+ was still capable of reducing hyperpolarizing inward rectification. Our data indicate that in hippocampal pyramidal cells, two components of the inwardly rectifying K+ conductance can be identified, which together exert a tonic modulation of excitatory synaptic input: one arises from constitutive putative IRK channels, the other is mediated by the background activity of GIRK channels that results from the tonic activation of A1 receptors by ambient adenosine.

The complex and intriguing lives of PIP2 with ion channels and transporters.

Phosphatidylinositol-4,5-bisphosphate (PIP(2)), the precursor of several signaling molecules in eukayotic cells, is itself also used by cells to signal to membrane-associated proteins. PIP(2) anchors numerous signaling molecules and cytoskeleton at the cell membrane, and the metabolism of PIP(2) is closely connected to membrane trafficking. Recently, ion transporters and channels have been discovered to be regulated by PIP(2). Systems reported to be activated by PIP(2) include (i) plasmalemmal calcium pumps (PMCA), (ii) cardiac sodium-calcium exchangers (NCX1), (iii) sodium-proton exchangers (NHE1-4), (iv) a sodium-magnesium exchanger of unknown identity, (v) all inward rectifier potassium channels (KATP, IRK, GIRK, and ROMK channels), (vi) epithelial sodium channels (ENaC), and (vii) ryanodine-sensitive calcium release channels (RyR). Systems reported to be inhibited by PIP(2) include (i) cyclic nucleotide-gated channels of the rod (CNG), (ii) transient receptor potential-like (TRPL) Drosophila phototransduction channels, (iii) capsaicin-activated transient receptor potential (TRP) channels (VR1), and (iv) IP(3)-gated calcium release channels (IP3R). Systems that appear to be completely insensitive to PIP(2) include (i) voltage-gated sodium channels, (ii) most voltage-gated potassium channels, (iii) sodium-potassium pumps, (iv) several neurotransmitter transporters, and (v) cystic fibrosis transmembrane receptor (CFTR)-type chloride channels. Presumably, local changes of the concentration of PIP(2) in the plasma membrane represent cell signals to those mechanisms sensitive to PIP(2) changes. Unfortunately, our understanding of how local PIP(2) concentrations are regulated remains very limited. One important complexity is the probable existence of phospholipid microdomains, or lipid rafts. Such domains may serve to localize PIP(2) and thereby PIP(2) signaling, as well as to organize PIP(2) binding partners into signaling complexes. A related biological role of PIP(2) may be to control the activity of ion transporters and channels during biosynthesis or vesicle trafficking. Low PIP(2) concentrations in the secretory pathway would inactivate all of the systems that are stimulated by PIP(2). How, in detail, is PIP(2) used by cells to control ion channel and transporter activities? Further progress requires an improved understanding of lipid kinases and phosphatases, how they are regulated, where they are localized in cells, and with which ion channels and transporters they might localize.

Differential effects of general anesthetics on G protein-coupled inwardly rectifying and other potassium channels.

General anesthetics differentially affect various families of potassium channels, and some potassium channels are suggested to be potential targets for anesthetics and alcohols. The voltage-gated (ERG1, ELK1, and KCNQ2/3) and inwardly rectifying (GIRK1/2, GIRK1/4, GIRK2, IRK1, and ROMK1) potassium channels were expressed in Xenopus oocytes. Effects of volatile agents [halothane, isoflurane, enflurane, F3 (1-chloro-1,2,2-trifluorocyclobutane), and the structurally related nonimmobilizer F6 (1,2-dichlorohexafluorocyclobutane)], as well as intravenous (pentobarbital, propofol, etomidate, alphaxalone, ketamine), and gaseous (nitrous oxide) anesthetics and alcohols (ethanol and hexanol) on channel function were studied using a two-electrode voltage clamp. ERG1, ELK1, and KCNQ2/3 channels were either inhibited slightly or unaffected by concentrations corresponding to twice the minimum alveolar concentrations or twice the anesthetic EC50 of volatile and intravenous anesthetics and alcohols. In contrast, G protein-coupled inwardly rectifying potassium (GIRK) channels were inhibited by volatile anesthetics but not by intravenous anesthetics. The neuronal-type GIRK1/2 channels were inhibited by 2 minimum alveolar concentrations of halothane or F3 by 45 and 81%, respectively, whereas the cardiac-type GIRK1/4 channels were inhibited only by F3. Conversely, IRK1 and ROMK1 channels were completely resistant to all anesthetics tested. Current responses of GIRK2 channels activated by mu-opioid receptors were also inhibited by halothane. Nitrous oxide (approximately 0.6 atmosphere) slightly but selectively potentiated GIRK channels. Results of chimeric and multiple amino acid mutations suggest that the region containing the transmembrane domains, but not the pore-forming domain, may be involved in determining differences in anesthetic sensitivity between GIRK and IRK channels. G protein-coupled inwardly rectifying potassium channels, especially those composed of GIRK2 subunits, were inhibited by clinical concentrations of volatile anesthetics. This action may be related to some side effects of these agents.

Identification of G protein-coupled, inward rectifier potassium channel gene products from the rat anterior pituitary gland.

Dopamine (DA) is a physiological regulator of PRL secretion, exerting tonic inhibitory control. DA activates an inward rectifier K(+) (IRK) channel in rat lactotropes, causing membrane hyperpolarization and inhibition of Ca(2+)-dependent action potentials. Both the activation of this effector K(+) channel and the inhibition of PRL release are mediated by D(2)-type receptor activation and pertussis toxin- sensitive G proteins. To study the molecular basis of this physiologically relevant channel, a homology-based PCR approach was employed to identify members of the IRK channel family expressed in the anterior pituitary gland. Nondegenerate primers corresponding to regions specific for IRK channels known to be G protein activated (GIRKs; gene subfamily Kir 3.0) were synthesized and used in the PCR with reverse transcribed female rat anterior pituitary messenger RNA as the template. PCR products of predicted sizes for Kir 3.1, 3.2, and 3.4 were consistently observed by ethidium bromide staining after 16 amplification cycles. The identities of the products were confirmed by subcloning and sequencing. Expression of each of these gene products in anterior pituitary was confirmed by Northern blot analysis. Functional analysis of the GIRK proteins was performed in the heterologous expression system, Xenopus laevis oocytes. Macroscopic K(+) currents were examined in oocytes injected with different combinations of Kir 3.0 complementary RNA (cRNA) and G protein subunit (beta(1)gamma(2)) cRNA. The current-voltage relationships demonstrated strong inward rectification for each individual and pairwise combination of GIRK channel subunits. Oocytes coinjected with any pair of GIRK subunit cRNA exhibited significantly larger inward K(+) currents than oocytes injected with only one GIRK channel subtype. Ligand-dependent activation of only one of the GIRK combinations (GIRK1 and GIRK4) was observed when channel subunits were coexpressed with the D(2) receptor in Xenopus oocytes. Dose-response data fit to a Michaelis-Menten equation gave an apparent K(d) similar to that for DA binding in anterior pituitary tissue. GIRK1 and GIRK4 proteins were coimmunoprecipitated from anterior pituitary lysates, confirming the presence of native GIRK1/GIRK4 oligomers in this tissue. These data indicate that GIRK1 and GIRK4 are excellent candidate subunits for the D(2)-activated, G protein-gated channel in pituitary lactotropes, where they play a critical role in excitation-secretion coupling.

Mechanism underlying bupivacaine inhibition of G protein-gated inwardly rectifying K+ channels.

Local anesthetics, commonly used for treating cardiac arrhythmias, pain, and seizures, are best known for their inhibitory effects on voltage-gated Na(+) channels. Cardiovascular and central nervous system toxicity are unwanted side-effects from local anesthetics that cannot be attributed to the inhibition of only Na(+) channels. Here, we report that extracellular application of the membrane-permeant local anesthetic bupivacaine selectively inhibited G protein-gated inwardly rectifying K(+) channels (GIRK:Kir3) but not other families of inwardly rectifying K(+) channels (ROMK:Kir1 and IRK:Kir2). Bupivacaine inhibited GIRK channels within seconds of application, regardless of whether channels were activated through the muscarinic receptor or directly via coexpressed G protein G(beta)gamma subunits. Bupivacaine also inhibited alcohol-induced GIRK currents in the absence of functional pertussis toxin-sensitive G proteins. The mutated GIRK1 and GIRK2 (GIRK1/2) channels containing the high-affinity phosphatidylinositol 4,5-bisphosphate (PIP(2)) domain from IRK1, on the other hand, showed dramatically less inhibition with bupivacaine. Surprisingly, GIRK1/2 channels with high affinity for PIP(2) were inhibited by ethanol, like IRK1 channels. We propose that membrane-permeant local anesthetics inhibit GIRK channels by antagonizing the interaction of PIP(2) with the channel, which is essential for G(beta)gamma and ethanol activation of GIRK channels.

Mutational analysis of ethanol interactions with G-protein-coupled inwardly rectifying potassium channels

G-protein-coupled inwardly rectifying potassium (GIRK) channels play a major role in regulating inhibitory responses in the nervous system. Previously, we showed that function of GIRK channels, but not that of the related inwardly rectifying potassium (IRK) channels, was enhanced by intoxicating concentrations of ethanol in native and recombinant systems. In addition, by using chimeras constructed between GIRK2 and IRK1 channels and truncation mutants of GIRK2 channel, a region of 43 amino acids in the carboxyl (C) terminus of GIRK2 channels was shown to be important for enhancement by ethanol. In the present study, we attempted to define the critical amino acids for ethanol actions within this 43 amino acid region. We made GIRK2 channel mutants with single or multiple amino acids changed to those corresponding to IRK1 channel by using site-directed mutagenesis and expressed them in Xenopus oocytes. Each of the amino acid residues not identical between GIRK2 and IRK1 channels was systematically changed. Ethanol (100 mM) enhanced function of wild type GIRK2 channel by 38%, as observed previously. However, none of the mutants showed any significant difference from wild type, indicating that none of the mutations tested were sufficient to abolish the enhancement by ethanol.

Kinetics of inward-rectifier K+ channel block by quaternary alkylammonium ions. dimension and properties of the inner pore.

We examined block of two inward-rectifier K+ channels, IRK1 and ROMK1, by a series of intracellular symmetric quaternary alkylammonium ions (QAs) whose side chains contain one to five methylene groups. As shown previously, the ROMK1 channels bind larger QAs with higher affinity. In contrast, the IRK1 channels strongly select TEA over smaller or larger QAs. This remarkable difference in QA selectivity between the two channels results primarily from differing QA unbinding kinetics. The apparent rate constant for binding (kon) of all examined QAs is significantly smaller than expected for a diffusion-limited process. Furthermore, a large ( approximately 30-fold) drop in kon occurs when the number of methylene groups in QAs increases from three to four. These observations argue that between the intracellular solution and the QA-binding locus, there exists a constricted pathway, whose dimension ( approximately 9 A) is comparable to that of a K+ ion with a single H2O shell.

GTP-binding protein Gq mediates muscarinic-receptor-induced inhibition of the inwardly rectifying potassium channel IRK1 (Kir 2.1)

The inwardly rectifying potassium channel IRK1, a member of the Kir 2.0 family, is inhibited by m1 muscarinic receptor stimulation. In this study the mechanism of action underlying the muscarinic response was investigated by identification of the subtype of heterotrimeric G-protein involved in transduction of the signal. tsA201 cells were simultaneously transfected with cDNAs encoding IRK1, m1 and the Gα subunit of either G q, G 12 or G 13. The whole-cell patch–clamp technique was used to study the effects of G-protein transfection. Antibodies generated against the C-terminal regions of Gα q/11 and Gα 12 were used to confirm G-protein expression by Western blot. When challenged with carbachol, IRK1 currents recorded from cells co-transfected with Gα q were potently inhibited compared with controls. Conversely, co-transfection with Gα 12 or Gα 13 subunits had no effect on muscarinic-receptor-induced inhibition of IRK1. Concentration response curves revealed that carbachol was 16 times more potent at inhibiting IRK1 currents in cells co-transfected with Gα q as compared with Gα 12 co-transfected cells. Immunoblotting illustrated low levels of endogenous Gα q/11 and Gα 12 in untransfected tsA cells. Transfection with Gα q or Gα 12 cDNAs greatly increased the levels of G-protein expression in both cell populations. G-protein expression did not interfere with m1 muscarinic receptor expression levels. These findings suggest that the m1 muscarinic-receptor-induced inhibition of IRK1 is mediated by the heterotrimeric G-protein, Gα q, in tsA cells.

Pore block versus intrinsic gating in the mechanism of inward rectification in strongly rectifying IRK1 channels.

The IRK1 channel is inhibited by intracellular cations such as Mg(2+) and polyamines in a voltage-dependent manner, which renders its I-V curve strongly inwardly rectifying. However, even in excised patches exhaustively perfused with a commonly used artificial intracellular solution nominally free of Mg(2+) and polyamines, the macroscopic I-V curve of the channels displays modest rectification. This observation forms the basis of a hypothesis, alternative to the pore-blocking hypothesis, that inward rectification reflects the enhancement of intrinsic channel gating by intracellular cations. We find, however, that residual rectification is caused primarily by the commonly used pH buffer HEPES and/or some accompanying impurity. Therefore, inward rectification in the strong rectifier IRK1, as in the weak rectifier ROMK1, can be accounted for by voltage-dependent block of its ion conduction pore by intracellular cations.

Mechanism of IRK1 channel block by intracellular polyamines.

Intracellular polyamines inhibit the strongly rectifying IRK1 potassium channel by a mechanism different from that of a typical ionic pore blocker such as tetraethylammonium. As in other K(+) channels, in the presence of intracellular TEA, the IRK1 channel current decreases with increasing membrane voltage and eventually approaches zero. However, in the presence of intracellular polyamines, the channel current varies with membrane voltage in a complex manner: when membrane voltage is increased, the current decreases in two phases separated by a hump. Furthermore, contrary to the expectation for a nonpermeant ionic pore blocker, a significant residual IRK1 current persists at very positive membrane voltages; the amplitude of the residual current decreases with increasing polyamine concentration. This complex blocking behavior of polyamines can be accounted for by a minimal model whereby intracellular polyamines inhibit the IRK1 channel by inducing two blocked channel states. In each of the blocked states, a polyamine is bound with characteristic affinity and probability of traversing the pore. The proposal that polyamines traverse the pore at finite rates is supported by the observation that philanthotoxin-343 (spermine with a bulky chemical group attached to one end) acts as a nonpermeant ionic blocker in the IRK1 channel.

Calculation of the conductance and selectivity of an ion-selective potassium channel (IRK1) from simulation of atomic scale models

We present the equations and methodology for the theoretical prediction of the conductance, permeability and selectivity of a K+ channel on the basis of atomic scale models for it. The methodology involves the use of Langevin dynamics and activated trajectories in order to obtain translocation free energies, rate constants and transmission coefficients for an ion going through the channel. The models are for the Inward Rectifier K+ channel (IRK1) which is a member of a family of ion-selective K+ channels. The IRK1 channel is biologically important because of its role in cardiac pacemaker function. The models we use for the IRK1 channel are developed from a model of the Shaker voltage-gated K+ channel. We find that the theoretically predicted conductance is too low by three orders of magnitude. We attribute this underestimate to a specific structural defect in the model used. Perhaps our most significant result is that the computed conductance is tremendously sensitive to the structural details of the so-called ‘P-loop’ that lines the outer half of the permeation pathway of the channel. This sensitivity may be useful in future studies on ion channel proteins for which the structure is not known from X-ray crystallography. In addition, this sensitivity may help determine whether X-ray structures of these proteins correspond to open or closed conformations.

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.

Expression of Ras-GRF in the SK-N-BE neuroblastoma accelerates retinoic-acid-induced neuronal differentiation and increases the functional expression of the IRK1 potassium channel.

Ras-GRF, a neuron-specific Ras exchange factor of the central nervous system, was transfected in the SK-N-BE neuroblastoma cell line and stable clones were obtained. When exposed to retinoic acid, these clones showed a remarkable enhancement of Ras-GRF expression with a concomitant high increase in the level of active (GTP-bound) Ras already after 24 h of treatment. In the presence of retinoic acid, the transfected cells stopped growing and acquired a differentiated neuronal-like phenotype more rapidly than the parental ones. Cells expressing Ras-GRF also exhibited a more hyperpolarized membrane potential. Moreover, treatment with retinoic acid led to the appearance of an inward rectifying potassium channel with electrophysiological properties similar to IRK1. This current was present in a large number of cells expressing Ras-GRF, while only a small percentage of parental cells exhibited this current. However, Northern analysis with a murine cDNA probe indicated that IRK1 mRNA was induced by retinoic acid at a similar level in both kinds of cells. Brief treatment with a specific inhibitor of the mitogen-activated protein kinase (MAPK) pathway reduced the number of transfected cells showing IRK1 activity. These findings suggest that activation of the Ras pathway accelerates neuronal differentiation of this cell line. In addition, our results suggest that Ras-GRF and/or Ras-pathway may have a modulatory effect on IRK1 channel activity.

Molecular characterization of an inwardly rectifying K+ channel from HeLa cells.

Previous patch-clamp studies have shown that the potassium permeability of the plasma membrane in HeLa cells, a cell line derived from an epidermoid carcinoma of the cervix, is controlled by various K+-selective pores including an IRK1 type inwardly rectifying K+ channel. We used the sequence previously reported for the human heart Kir2.1 channel to design a RT-PCR strategy for cloning the IRK1 channel in HeLa cells. A full-length clone of 1.3 kb was obtained that was identical to the human cardiac Kir2.1 inward rectifier. The nature of the cloned channel was also confirmed in a Northern blot analysis where a signal of 5.3 kb corresponding to the molecular weight expected for a Kir2.1 channel transcript was identified not only in HeLa cells, but also in WI-38, ECV304 and bovine aortic endothelial cells. The HeLa IRK1 channel cDNA was subcloned in an expression vector (pMT21) and injected into Xenopus oocytes. Cell-attached and inside-out single channel recordings obtained from injected oocytes provided evidence for a voltage-independent K+-selective channel with current/voltage characteristics typical of a strong inward rectifier. The single channel conductance for inward currents measured in 200 mm K2SO4 conditions was estimated at 40 +/- 1 pS (n = 3), for applied voltages ranging from -100 to -160 mV, in agreement with the unitary conductance for the IRK1 channel identified in HeLa cells. In addition, the single channel conductance for inward currents, Gamma, was found to vary as a function of alphaK, the external K+ ion activity, according to Gamma = Gamma0 [alphaK]delta with Gamma0 = 3.3 pS and delta = 0.5. Single channel recordings from injected oocytes also provided evidence of a voltage-dependent block by external Cs+ and Ba2+. The presence of 500 micron Cs+ caused a voltage-dependent flickering, typical of a fast channel blocking process which resulted in a reduction of the channel open probability at increasingly negative membrane potential values. The fractional electrical distance computed for the Cs+ blocking site was greater than 1 indicating a multiple ion channel occupation. In contrast, external Ba2+ at concentrations ranging from 25 to 100 micron caused a slow channel block, consistent with the binding of a single Ba2+ ion at a site located at half the membrane span. It is concluded on the basis of these observations that HeLa cells expressed a Kir2.1 type inwardly rectifying channel likely to be involved in maintaining and regulating the cell resting potential.

Nonindependent K+ movement through the pore in IRK1 potassium channels.

We measured unidirectional K+ in- and efflux through an inward rectifier K channel (IRK1) expressed in Xenopus oocytes. The ratio of these unidirectional fluxes differed significantly from expectations based on independent ion movement. In an extracellular solution with a K+ concentration of 25 mM, the data were described by a Ussing flux-ratio exponent, n', of approximately 2.2 and was constant over a voltage range from -50 to -25 mV. This result indicates that the pore of IRK1 channels may be simultaneously occupied by at least three ions. The IRK1 n' value of 2.2 is significantly smaller than the value of 3.5 obtained for Shaker K channels under identical conditions. To determine if other permeation properties that reflect multi-ion behavior differed between these two channel types, we measured the conductance (at 0 mV) of single IRK1 channels as a function of symmetrical K+ concentration. The conductance could be fit by a saturating hyperbola with a half-saturation K+ activity of 40 mM, substantially less than the reported value of 300 mM for Shaker K channels. We investigated the ability of simple permeation models based on absolute reaction rate theory to simulate IRK1 current-voltage, conductance, and flux-ratio data. Certain classes of four-barrier, three-site permeation models are inconsistent with the data, but models with high lateral barriers and a deep central well were able to account for the flux-ratio and single channel data. We conclude that while the pore in IRK1 and Shaker channels share important similarities, including K+ selectivity and multi-ion occupancy, they differ in other properties, including the sensitivity of pore conductance to K+ concentration, and may differ in the number of K+ ions that can simultaneously occupy the pore: IRK1 channels may contain three ions, but the pore in Shaker channels can accommodate four or more ions.

Neutralization of aspartate residues in the murine inwardly rectifying K+ channel IRK1 affects the substate behaviour in Mg2+ block.

1. To investigate the molecular basis of the sublevels induced in the outward current during block by intracellular Mg2+, single-channel currents through inwardly rectifying K+ (IRK1) channels were studied. 2. cDNA encoding a functional murine IRK1 channel was transfected into COS-1 cells (a Green Monkey kidney cell line) using the liposome method, and voltage clamp experiments were done after 48-72 h. 3. Intracellular Mg2+ at micromolar concentrations induced sublevels in the outward current at one-third and two-thirds of the unitary amplitude seen in wild-type channels. Replacing Asp 172 with Asn (D172N) and Gln (D172Q) abolished these sublevels, i.e. the channel showed only the fully open and fully blocked states. 4. Both mutations reduced the Mg2+ sensitivity of the channel at 2 microM Mg2+. However, the Mg2+ sensitivity did not differ significantly at higher concentrations (10 microM) and voltages (+70 mV). 5. Channels expressed from D172E showed the sublevels, indicating that a negative charge is indispensable to the substate behaviour. 6. Channels from tandem tetramers of IRK1 with one and two D172N mutant subunits mainly showed sublevels with two-thirds amplitude, while those from tetramers with three D172N mutant subunits showed no sublevels. 7. These findings suggest that differences in Mg2+ binding patterns lead to different conductive states in a single-barrelled channel.

Gβγ Binding to GIRK4 Subunit Is Critical for G Protein-gated K+ Channel Activation

The cardiac G protein-gated K+ channel, IKACh, is directly activated by G protein beta gamma subunits (Gbeta gamma). IKAChis composed of two inward rectifier K+ channel subunits, GIRK1 and GIRK4. Gbeta gamma binds to both GIRK1 and GIRK4 subunits of the heteromultimeric IKACh. Here we delineate the Gbeta gamma binding regions of IKACh by studying direct Gbeta gamma interaction with native purified IKACh, competition of this interaction with peptides derived from GIRK1 or GIRK4 amino acid sequences, mutational analysis of regions implicated in Gbeta gamma binding, and functional expression of mutated subunits in mammalian cells. Only two GIRK4 peptides, containing amino acids 209-225 or 226-245, effectively competed for Gbeta gamma binding. A single point mutation introduced into GIRK4 at position 216 (C216T) dramatically reduced the potency of the peptide in inhibiting Gbeta gamma binding and Gbeta gamma activation of expressed GIRK1/GIRK4(C216T) channels. Conversion of 5 amino acids in GIRK4 (226-245) to the corresponding amino acids found in the G protein-insensitive IRK1 channel, completely abolished peptide inhibition of Gbeta gamma binding to IKACh and Gbeta gamma activation of GIRK1/mutant GIRK4 channels. We conclude from this data that Gbeta gamma binding to GIRK4 is critical for IKACh activation.

Long-term exposure to retinoic acid induces the expression of IRK1 channels in HERG channel-endowed neuroblastoma cells.

The modulation of inward K+ conductances was studied during neuronal differentiation of human SH-SY5Y neuroblastoma cells. Under standard culture conditions, these cells express the herg gene, and the HERG current is the main inward K+ current regulating their Vrest. After 10-20 days exposure to Retinoic Acid (RA), SH-SY5Y cells showed, in addition to HERG currents, a novel current characterized by inward rectification, dependence on the extracellular K+ concentration, and blockade by Cs+ and Ba2+, the main features of the IRK1 current. The appearance of this current is accompanied by a strong hyperpolarisation of Vrest. RT-PCR experiments confirmed that a transcript of the IRK1 (Kir 2.1) gene actually appears in SH-SY5Y cells treated for 10-20 days with RA. On the whole, data here presented demonstrate that RA-induced neuronal differentiation of neuroblastoma cells is accompanied by the switch from a HERG-driven to a IRK1-driven control of Vrest, similarly to what happens in normal differentiating neurons; however, in tumor cells, this switch does not imply the abolition of HERG channel expression.