Phosphoinositide-mediated gating of inwardly rectifying K+ channels

Phosphoinositides, such as phosphatidylinositol-bisphosphate (PIP₂), control the activity of many ion channels in yet undefined ways. Inwardly, rectifying potassium (Kir) channels were the first shown to be dependent on direct interactions with phosphoinositides. Alterations in channel-PIP₂ interactions affect Kir single-channel gating behavior. Aberrations in channel-PIP₂ interactions can lead to human disease. As the activity of all Kir channels depends on their interactions with phosphoinositides, future research will aim to understand the molecular events that occur from phosphoinositide binding to channel gating. The determination of atomic resolution structures for several mammalian and bacterial Kir channels provides great promise towards this goal. We have mapped onto the three-dimensional channel structure the position of basic residues identified through mutagenesis studies that contribute to the sensitivity of a Kir channel to PIP₂. The localization of these putative PIP₂-interacting residues relative to the channel’s permeation pathway has given rise to a testable model, which could account for channel activation by PIP₂.     

Ethanol opens G-protein-activated inwardly rectifying K+ channels

Ethanol affects many functions of the brain and peripheral organs. Here we show that ethanol opens G-protein-activated, inwardly rectifying K + (GIRK) channels, which has important implications for inhibitory regulation of neuronal excitability and heart rate. At pharmacologically relevant concentrations, ethanol activated both brain-type GIRK1/2 and cardiac-type GIRK1/4 channels without interaction with G proteins or second messengers. Moreover, weaver mutant mice, which have a missense mutation in the GIRK2 channel, showed a loss of ethanol-induced analgesia. These results suggest that the GIRK channels in the brain and heart are important target sites for ethanol.     

CO2-dependent opening of an inwardly rectifying K+ channel

CO(2) chemosensing is a vital function for the maintenance of life that helps to control acid-base balance. Most studies have reported that CO(2) is measured via its proxy, pH. Here we report an inwardly rectifying channel, in outside-out excised patches from HeLa cells that was sensitive to modest changes in PCO(2) under conditions of constant extracellular pH. As PCO(2) increased, the open probability of the channel increased. The single-channel currents had a conductance of 6.7 pS and a reversal potential of -70?mV, which lay between the K(+) and Cl(-) equilibrium potentials. This reversal potential was shifted by +61?mV following a tenfold increase in extracellular [K(+)] but was insensitive to variations of extracellular [Cl(-)]. The single-channel conductance increased with extracellular [K(+)]. We propose that this channel is a member of the Kir family. In addition to this K(+) channel, we found that many of the excised patches also contained a conductance carried via a Cl(-)-selective channel. This CO(2)-sensitive Kir channel may hyperpolarize excitable cells and provides a potential mechanism for CO(2)-dependent inhibition during hypercapnia.     

Characterisation of Ca2+-dependent inwardly rectifying K+ currents in HeLa cells

The whole-cell configuration of the patch-clamp technique was used to examine K⁺ currents in HeLa cells. Under quasi-physiological ionic gradients, using an intracellular solution containing 10^–7 mol/l free Ca²⁺, mainly outward currents were observed. Large inwardly rectifying currents were elicited in symmetrical 145 mmol/l KCl. Replacement of all extracellular K⁺ by isomolar Na⁺, greatly decreased inward currents and shifted the reversal potential as expected for K⁺ selectivity. The inwardly rectifying K⁺ currents exhibited little or no apparent voltage dependence within the range of from –120 mV to 120 mV. A square-root relationship between chord conductance and [K⁺]₀ at negative potentials could be established. The inwardly rectifying nature of the currents was unaltered after removal of intracellular Mg²⁺ and chelation with ATP and ethylenediaminetetraacetic acid (EDTA). Permeability ratios for other monovalent cations relative to K⁺ were: K⁺ (1.0)>Rb⁺ (0.86)>Cs⁺ (0.12)>Li⁺ (0.08)>Na⁺ (0.03). Slope conductance ratios measured at –100 mV were: Rb⁺ (1.66)>K⁺ (1.0)>Na⁺ (0.09)>Li⁺ (0.08)>Cs⁺ (0.06). K⁺ conductance was highly sensitive to intracellular free Ca²⁺ concentration. The relationship between conductance at 0 mV and Ca²⁺ concentration was well described by a Hill expression with a dissociation constant, K _D, of 70 nmol/l and a Hill coefficient, n, of 1.81. Extracellular Ba²⁺ blocked the currents in a concentration- and voltage-dependent manner. The dependence of the K _D for the blockade was analysed using a Woodhull-type treatment, locating the ion interaction site at 19 % of the distance across the electrical field of the membrane and a K _D (0 mV) of 7 mmol/l. Tetraethylammonium and 4-aminopyridine were without effect whilst quinine and quinidine blocked the currents with concentrations for half-maximum effects equal to 7 mol/l and 3.5 mol/l, respectively. The unfractionated venom of the scorpion Leiurus quinquestriatus (LQV) blocked the K⁺ currents of HeLa cells. The toxins apamin and scyllatoxin had no detectable effect whilst charybdotoxin, a component of LQV, blocked in a voltage-dependent manner with half-maximal concentrations of 40 nmol/l at –120 mV and 189 nmol/l at 60 mV; blockade by charybdotoxin accounts for the effect of LQV. Application of ionomycin (5–10 mol/l), histamine (1 mmol/l) or bradykinin (1–10 mol/l) to cells dialysed with low-buffered intracellular solutions induced K⁺ currents showing inward rectification and a lack of voltage dependence.     

Postsynaptic inhibition by adenosine in hippocampal CA3 neurons: Co2+-sensitive activation of an inwardly rectifying K+ conductance

The properties of the current underlying the membrane hyperpolarization evoked by adenosine (50–100 m) were investigated in hippocampal CA3 neurons in vitro using current-clamp and single-electrode voltage-clamp techniques. In voltage-clamp measurements, the adenosine-induced current (I _Ado) was outward at rest and reversed at membrane potentials close to the equilibrium potential of K⁺ (E _K), indicating that I _Ado was carried by K⁺ ions. Determination of I _Ado at several membrane potentials revealed a nonlinear current/voltage (I/V) relationship of the current displaying inward rectification in the hyperpolarizing direction. Similarly, adenosine increased the membrane slope conductance only at membrane potentials negative to rest, whereas the slope of the neuronal I/V curve remained unchanged when determined at potentials positive to rest. Since the electrophysiological properties of I _Ado were very similar to those described for K⁺ conductances activated by other neuroactive substances like serotonin, opioid peptides and -aminobutyric acid B receptor (GABA_B) agonists, we conclude that I _Ado belongs to a family of ligand-operated, inwardly rectifying K⁺ currents which apparently share a common mechanism to reduce postsynaptic excitability. As an additional feature, the postsynaptic adenosine response was reduced by bath application of Co²⁺ or Ni²⁺. The adenosine-induced membrane hyperpolarization was not affected by low-Ca²⁺ or low-Mg²⁺ solutions, nor by buffering of intra-cellular Ca²⁺, but a gradual decline of I _Ado was observed following superfusion with Co²⁺ or Ni²⁺. In contrast, Mn²⁺ caused only a weak attenuation of the adenosine response.     

Rb+, Cs+ ions and the inwardly rectifying K+ channels in guinea-pig ventricular cells

Rb⁺ and Cs⁺ ion permeability and the effects of these ions from the inside on the inwardly rectifying K⁺ channel were studied in guinea-pig ventricular cells. A total substitution of either Rb⁺ or Cs⁺ for external K⁺ in the outside-out configuration of the patch-clamp technique abolished the inward current. The outward current carried by K⁺ was recorded. The unitary amplitude was reduced to about half of the control value with Rb⁺ but was not changed with Cs⁺. Internal Rb⁺ and Cs⁺, at a concentration of 10–40 mM, reduced the unitary amplitude of the outward current. No substate behaviour was observed. The reversal potential was +18 mV after replacing 105 mM internal K⁺ with Rb⁺ at 150 mM external K⁺. This value gives a permeability ratio of Rb⁺ to K⁺ of 0.27. Under a total substitution of Rb⁺ or Cs⁺ for internal K⁺, the outward currents were not measurable. Cs⁺ induced flickering in the inward current carried by K⁺. It is thus concluded that Rb⁺ and Cs⁺ ions are not measurably permeant at the single-channel level but permit K⁺ permeation in place of external K⁺ and that internal Rb⁺ and Cs⁺ produce a voltage-dependent block of the channel with fast kinetics. Received: 9 March 1995/Received after revision: 27 September 1995/Accepted: 2 January 1996     

Somatostatin activates an inwardly rectifying K+ channel in neonatal rat atrial cells

Somatostatin, localized throughout the central and peripheral nervous systems has been found in neurons of the vagal inhibitory pathway of the heart and has been shown to have negative inotropic effects in cardiac tissue. Using patch clamp techniques we show that somatostatin activates an inwardly rectifying K⁺ channel in rat atrial cells. Loss of somatostatin-induced K⁺ channel activity in excised inside-out patches is restored by the addition of GTP to the bath. Pertussis toxin pretreatment blocked GTP-dependent somatostatin activation of the inwardly rectifying K+ channel. This K+ channel has a conductance of 34 pS and a mean open time of approximately 1 ms. It is apparently the same K+ channel activated by muscarinic and adenosine receptors in atrial and cardiac pacemaker cells. Thus, atrial cells have at least three receptors which act via pertussis toxin-sensitive G proteins to activate the same class of K+ channels.     

Regulation of inwardly rectifying K+ channels by intracellular pH in opossum kidney cells

The effects of intracellular pH on an inwardly rectifying K⁺ channel (K_in channel) in opossum kidney (OK) cells were examined using the patch-clamp technique. Experiments with inside-out patches were first carried out in Mg²⁺-and adenosine triphosphate (ATP)-free conditions, where Mg²⁺-induced inactivation and ATP-induced reactivation of K_in channels were suppressed. When the bath (cytoplasmic side) pH was decreased from 7.3 to either 6.8 or 6.3, K_in channels were markedly inhibited. The effect of acid pH was not fully reversible. When the bath pH was increased from 7.3 to 7.8, 8.3 or 8.8, the channels were activated reversibly. The channel activity exhibited a sigmoidal pH dependence with a maximum sensitivity at pH 7.5. Inside-out experiments were also carried out with a solution containing 3 mM Mg-ATP and a similar pH sensitivity was observed. However, in contrast with the results obtained in the absence of Mg²⁺ and ATP, the effect of acid pH was fully reversible. Experiments with cell-attached patches demonstrated that changes in intracellular pH, which were induced by changing extracellular pH in the presence of an H⁺ ionophore, could influence the channel activity reversibly. It is concluded that the activity of K_in channels can be controlled by the intracellular pH under physiological conditions.     

Galanin activates an inwardly rectifying potassium conductance in mudpuppy atrial myocytes

Galanin- and bethanechol-activated K⁺ currents have been studied in mudpuppy atrial myocytes. The galanin and bethanechol K⁺ currents were time-dependent and inwardly rectifying. In GTPS, the galanin and bethanechol currents were reduced progressively as G-protein gated K⁺ channels became activated. GDPS inhibited agonist-induced outward currents. We conclude that galanin and bethanechol activate the same or a very similar inwardly rectifying K⁺ conductance and that activation of a G protein is required.     

Effect of the inactivating "ball" peptide of Shaker B on intermediate conductance Ca2+-dependent inwardly rectifying K+ channels of HeLa cells

The patch-clamp technique was used to study the effect of intracellularly added inactivating "ball" peptide (BP) of the Shaker B K+ channel upon Ca(2+)-dependent inwardly rectifying K+ channels of the intermediate conductance type expressed in HeLa cells. Intracellular BP caused only moderate inhibition of outward K+ currents when assayed at an intracellular Ca2+ concentration of 100 nmol/l. Increasing intracellular Ca2+ levels led in itself to some voltage-dependent blockade of K+ currents, which was absent when high extracellular K+ was used. An additional strong blockade by intracellular BP was nevertheless observed both in Na(+)- and K(+)-rich extracellular solutions. A non-inactivating BP analogue had no effect. At this higher intracellular Ca2+ concentration the inhibition of these intermediate conductance Ca(2+)-dependent channels by BP was voltage-dependent, being absent at hyperpolarizing potentials, and could be relieved by increasing extracellular K+. These data suggest that BP acts at an internal pore site in Ca(2+)-dependent intermediate conductance K+ channels of HeLa cells, and that these might possess a receptor site for the peptide similar to that of other K+ channels such as Ca(2+)-activated maxi-K+ channels.     

Mammalian retinal bipolar cells express inwardly rectifying K+ currents (IKir) with a different distribution than that of Ih

Retinal bipolar cells comprise multiple subtypes that are well known for the diversity of their physiological properties. We investigated the properties and functional roles of the hyperpolarization-activated currents in mammalian retinal bipolar cells using whole cell patch-clamp recording techniques. We report that bipolar cells express inwardly rectifying K+ currents (IKir) in addition to the hyperpolarization-activated cationic currents (Ih) previously reported. Furthermore, these two currents are differentially expressed among different subtypes of bipolar cells. One group of cone bipolar cells in particular displayed mainly IKir. A second group of cone bipolar cells displayed both currents but with a much larger Ih. Rod bipolar cells, on the other hand, showed primarily Ih. Moreover, we showed that IKir and Ih differentially influence the voltage responses of bipolar cells: Ih facilitates and/or accelerates the membrane potential rebound, whereas IKir counteracts or prevents such rebound. The findings of the expression of IKir and the differential expression of Ih and IKir in bipolar cells may provide new insights into an understanding of the physiological properties of bipolar cells.     

Dual control by ATP and acetylcholine of inwardly rectifying K+ channels in bovine atrial cells

Whole-cell and single-channel recordings were used to study an ionic current activated by extracellular adenosine 5′-triphosphate (ATP) applied to calf atrial cells. ATP (K _d ≈ 10μM) elicited an inwardly rectifying current that reversed nearE _K and was blocked by external Cs⁺ (10 mM). Under identical conditions, adenosine had no effect. Cell-attached patch recordings revealed an ATP-activated channel with a slope conductance of about 30 pS. At both the whole-cell and single-channel levels, the channels activated by ATP seemed nearly identical to the potassium channels activated by acetylcholine (ACh) in the same cells. However, the effects of ATP were not affected by atropine, suggesting that ATP does not interact with the same receptors as ACh. In some cells, whole-cell currents of similar magnitude were activated by ACh alone, ATP alone, or ACh and ATP applied together. These results suggest that calf atrial cells possess a population of inwardly rectifying potassium channels that are controlled jointly by two populations of receptors selective for ACh and ATP.     

Activation of inwardly rectifying K+ channel in OK proximal tubule cells involves cGMP-dependent phosphorylation process

The inwardly rectifying K+ channel with an inward conductance of about 90 pS in the surface membrane of cultured opossum kidney proximal tubule (OKP) cell is activated by cyclic AMP-dependent protein kinase (PKA). In this study, we further examined the involvement of the guanosine 3',5'-cyclic monophosphate (cGMP)-dependent process in modulation of this K+ channel by using the patch-clamp technique. In cell-attached patches, channel activity was increased by the application of either N2, 2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate (DBcGMP, 100 microM) or 8-bromoguanosine 3',5'-cyclic monophosphate (8BrcGMP, 100 microM), and it was inhibited by KT5823 (10 microM), a membrane-permeable specific inhibitor of cGMP-dependent protein kinase (PKG). The effect of DBcGMP on channel activity was abolished by the pretreatment of cells with KT5823 (10 microM), but it was observed in the presence of KT5720 (200 nM), a specific inhibitor of PKA. Furthermore, atrial natriuretic peptide (ANP, 10 nM) increased channel activity, which was also prevented by the application of KT5823 (10 microM). In inside-out patches, ATP (3 mM) was required to maintain channel activity, which was inhibited by KT5823 (10 microM), but it was not increased by cGMP (100 microM) alone. The channel activity was increased by the coapplication of PKG (500 U/ml) and cGMP (100 microM). These results suggest that cGMP activates the inwardly rectifying K+ channel in OKP cells through PKG-mediated phosphorylation processes independent of PKA-mediated processes, and that ANP is an agonist which stimulates PKG-mediated processes in the proximal tubule cell. Furthermore, it is suggested that the ATP-dependent channel activity in inside-out patches is maintained at least in part by PKG, which is the membrane-bound catalytic domain.     

m4 muscarinic receptor subtype activates an inwardly rectifying potassium conductance in AtT20 cells.

The modulation of an inwardly rectifying potassium conductance by muscarinic receptor stimulation was studied in the AtT-20 pituitary cell line, using the whole-cell patch-clamp technique. Only m4 mRNA was detected in these cells, thus, it is assumed that the actions of muscarinic receptor stimulation are mediated by the m4 receptor. AtT-20 cells express a slowly activating inwardly rectifying potassium conductance. Application of acetylcholine (ACh), resulted in an atropine sensitive, reversible increase in inwardly rectifying current. The ACh-induced current differed from the current recorded in control, in that it was fast activating, while the control current was slowly activating. Inclusion of GTP gamma S in the patch pipette activated an inward current with characteristics similar to the ACh-induced current, and the ACh-induced current response could be inhibited by pre-incubation with pertussis toxin (PTX). It is concluded that the m4 muscarinic receptor is coupled to an inwardly rectifying potassium conductance via a PTX sensitive G-protein.     

Quickly flickering inwardly rectifying K channels in goldfish hair cell membrane

The kinetics of the quickly flickering inwardly rectifying K channel were studied with the cell-attached patch clamp method in goldfish hair cells. The activity of the channel was very unique in repeating continuously open and closed events at an ultrafast rate. Moreover, open-close events of the channel showed a marked dependence on membrane potential; a shift toward hyperpolarized levels brought about an elongation of closed events resulting in a decrease in the open state probability, although no marked change was produced in open event duration itself. The unitary conductance of the channel was 109 pS as 123 mM KCl solution was used.