RGS proteins reconstitute the rapid gating kinetics of G����-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 G��(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.

Human ��-aminobutyric acid type B receptors are differentially expressed and regulate inwardly rectifying K+ channels

��-Aminobutyric acid type B receptors (GABABRs) are involved in the fine tuning of inhibitory synaptic transmission. Presynaptic GABABRs inhibit neurotransmitter release by down-regulating high-voltage activated Ca2+ channels, whereas postsynaptic GABABRs decrease neuronal excitability by activating a prominent inwardly rectifying K+ (Kir) conductance that underlies the late inhibitory postsynaptic potentials. Here we report the cloning and functional characterization of two human GABABRs, hGABABR1a (hR1a) and hGABABR1b (hR1b). These receptors closely match the pharmacological properties and molecular weights of the most abundant native GABABRs. We show that in transfected mammalian cells hR1a and hR1b can modulate heteromeric Kir3.1/3.2 and Kir3.1/3.4 channels. Heterologous expression therefore supports the notion that Kir3 channels are the postsynaptic effectors of GABABRs. Our data further demonstrate that in principle either of the cloned receptors could mediate inhibitory postsynaptic potentials. We find that in the cerebellum hR1a and hR1b transcripts are largely confined to granule and Purkinje cells, respectively. This finding supports a selective association of hR1b, and not hR1a, with postsynaptic Kir3 channels. The mapping of the GABABR1 gene to human chromosome 6p21.3, in the vicinity of a susceptibility locus (EJM1) for idiopathic generalized epilepsies, identifies a candidate gene for inherited forms of epilepsy.

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���� 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 (PIP2) domain from IRK1, on the other hand, showed dramatically less inhibition with bupivacaine. Surprisingly, GIRK1/2 channels with high affinity for PIP2 were inhibited by ethanol, like IRK1 channels. We propose that membrane-permeant local anesthetics inhibit GIRK channels by antagonizing the interaction of PIP2 with the channel, which is essential for G���� and ethanol activation of GIRK channels.

Receptor stimulation causes slow inhibition of IRK1 inwardly rectifying K+ channels by direct protein kinase A-mediated phosphorylation.

Strongly rectifying IRK-type inwardly rectifying K+ channels are involved in the control of neuronal excitability in the mammalian brain. Whole-cell patch-clamp experiments show that cloned rat IRK1 (Kir 2.1) channels, when heterologously expressed in mammalian COS-7 cells, are inhibited following the activation of coexpressed serotonin (5-hydroxytryptamine) type 1A receptors by receptor agonists. Inhibition is mimicked by internal perfusion with GTP[gamma-S] and elevation of internal cAMP concentrations. Addition of the catalytic subunits of protein kinase A (PKA) to the internal recording solution causes complete inhibition of wild-type IRK1 channels, but not of mutant IRK1(S425N) channels in which a C-terminal PKA phosphorylation site has been removed. Our data suggest that in the nervous system serotonin may negatively control IRK1 channel activity by direct PKA-mediated phosphorylation.

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.

Defective ��-aminobutyric acid type B receptor-activated inwardly rectifying K+ currents in cerebellar granule cells isolated from weaver and Girk2 null mutant���mice

Stimulation of inhibitory neurotransmitter receptors, such as ��-aminobutyric acid type B (GABAB) receptors, activates G protein-gated inwardly rectifying K+ channels (GIRK) which, in turn, influence membrane excitability. Seizure activity has been reported in a Girk2 null mutant mouse lacking GIRK2 channels but showing normal cerebellar development as well as in the weaver mouse, which has mutated GIRK2 channels and shows abnormal development. To understand how the function of GIRK2 channels differs in these two mutant mice, we compared the G protein-activated inwardly rectifying K+ currents in cerebellar granule cells isolated from Girk2 null mutant and weaver mutant mice with those from wild-type mice. Activation of GABAB receptors in wild-type granule cells induced an inwardly rectifying K+ current, which was sensitive to pertussis toxin and inhibited by external Ba2+ ions. The amplitude of the GABAB receptor-activated current was severely attenuated in granule cells isolated from both weaver and Girk2 null mutant mice. By contrast, the G protein-gated inwardly rectifying current and possibly the agonist-independent basal current appeared to be less selective for K+ ions in weaver but not Girk2 null mutant granule cells. Our results support the hypothesis that a nonselective current leads to the weaver phenotype. The loss of GABAB receptor-activated GIRK current appears coincident with the absence of GIRK2 channel protein and the reduction of GIRK1 channel protein in the Girk2 null mutant mouse, suggesting that GABAB receptors couple to heteromultimers composed of GIRK1 and GIRK2 channel subunits.

A Steep Dependence of Inward-Rectifying Potassium Channels on Cytosolic Free Calcium Concentration Increase Evoked by Hyperpolarization in Guard Cells1

Inactivation of inward-rectifying K+ channels (IK,in) by a rise in cytosolic free [Ca2+] ([Ca2+]i) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca2+]i action on IK,in has never been quantified, nor are its origins well understood. We used membrane voltage to manipulate [Ca2+]i (A. Grabov and M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778���4783) while recording IK,in under a voltage clamp and [Ca2+]i by Fura-2 fluorescence ratiophotometry. IK,in inactivation correlated positively with [Ca2+]i and indicated a Ki of 329 �� 31 nm with cooperative binding of four Ca2+ ions per channel. IK,in was promoted by the Ca2+ channel antagonists Gd3+ and calcicludine, both of which suppressed the [Ca2+]i rise, but the [Ca2+]i rise was unaffected by the K+ channel blocker Cs+. We also found that ryanodine, an antagonist of intracellular Ca2+ channels that mediate Ca2+-induced Ca2+ release, blocked the [Ca2+]i rise, and Mn2+ quenching of Fura-2 fluorescence showed that membrane hyperpolarization triggered divalent release from intracellular stores. These and additional results point to a high signal gain in [Ca2+]i control of IK,in and to roles for discrete Ca2+ flux pathways in feedback control of the K+ channels by membrane voltage.

Sensitivity to abscisic acid of guard-cell K+ channels is suppressed by abi1-1, a mutant Arabidopsis gene encoding a putative protein phosphatase.

Abscisic acid (ABA) modulates the activities of three major classes of ion channels--inward- and outward-rectifying K+ channels (IK,in and IK,out, respectively) and anion channels--at the guard-cell plasma membrane to achieve a net efflux of osmotica and stomatal closure. Disruption of ABA sensitivity in wilty abi1-1 mutants of Arabidopsis and evidence that this gene encodes a protein phosphatase suggest that protein (de)-phosphorylation contributes to guard-cell transport control by ABA. To pinpoint the role of ABI1, the abi1-1 dominant mutant allele was stably transformed into Nicotiana benthamiana and its influence on IK,in, IK,out, and the anion channels was monitored in guard cells under voltage clamp. Compared with guard cells from wild-type and vector-transformed control plants, expression of the abi1-1 gene was associated with 2- to 6-fold reductions in IK,out and an insensitivity of both IK,in and IK,out to 20 microM ABA. In contrast, no differences between control and abi1-1 transgenic plants were observed in the anion current or its response to ABA. Parallel measurements of intracellular pH (pHi) using the fluorescent dye 2',7'-bis(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF) in every case showed a 0.15- to 0.2-pH-unit alkalinization in ABA, demonstrating that the transgene was without effect on the pHi signal that mediates in ABA-evoked K+ channel control. In guard cells from the abi1-1 transformants, normal sensitivity of both K+ channels to and stomatal closure in ABA was recovered in the presence of 100 microM H7 and 0.5 microM staurosporine, both broad-range protein kinase antagonists. These results demonstrate an aberrant K+ channel behavior--including channel insensitivity to ABA-dependent alkalinization of pHi--as a major consequence of abi1-1 action and implicate AB11 as part of a phosphatase/kinase pathway that modulates the sensitivity of guard-cell K+ channels to ABA-evoked signal cascades.

Cloning and expression of two brain-specific inwardly rectifying potassium channels.

We have cloned two inwardly rectifying K+ channels that occur selectively in neurons in the brain and are designated BIRK (brain inwardly rectifying K+) channels. BIRK1 mRNA is extremely abundant and is enriched in specific brainstem nuclei, BIRK1 displays a consensus phosphate-binding loop, and expression in Xenopus oocytes has shown that its conductance is inhibited by ATP and adenosine 5'-[gamma-thio]triphosphate. BIRK2 is far less abundant and is selectively localized in telencephalic neurons. BIRK2 has a consensus sequence for cAMP-dependent phosphorylation.

Maxi-K channels contribute to urinary potassium excretion in the ROMK-deficient mouse model of Type II Bartter's syndrome and in adaptation to a high-K diet

Type II Bartter's syndrome is a hereditary hypokalemic renal salt-wasting disorder caused by mutations in the ROMK channel (Kir1.1; Kcnj1), mediating potassium recycling in the thick ascending limb of Henle's loop (TAL) and potassium secretion in the distal tubule and cortical collecting duct (CCT). Newborns with Type II Bartter are transiently hyperkalemic, consistent with loss of ROMK channel function in potassium secretion in distal convoluted tubule and CCT. Yet, these infants rapidly develop persistent hypokalemia owing to increased renal potassium excretion mediated by unknown mechanisms. Here, we used free-flow micropuncture and stationary microperfusion of the late distal tubule to explore the mechanism of renal potassium wasting in the Romk-deficient, Type II Bartter's mouse. We show that potassium absorption in the loop of Henle is reduced in Romk-deficient mice and can account for a significant fraction of renal potassium loss. In addition, we show that iberiotoxin (IBTX)-sensitive, flow-stimulated maxi-K channels account for sustained potassium secretion in the late distal tubule, despite loss of ROMK function. IBTX-sensitive potassium secretion is also increased in high-potassium-adapted wild-type mice. Thus, renal potassium wasting in Type II Bartter is due to both reduced reabsorption in the TAL and K secretion by max-K channels in the late distal tubule. �� 2006 International Society of Nephrology.

Outward Rectification of Voltage-Gated K+ Channels Evolved at Least Twice in Life History

Voltage-gated potassium (K+) channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD), this K+ channel type segregates into at least two main functional categories���hyperpolarization-activated, inward-rectifying (Kin) and depolarization-activated, outward-rectifying (Kout) channels. Voltage-gated K+ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in Kin and Kout channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K+ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between Kin and Kout channels. Among them is a loop between TMD S5 and the pore that is very short in animal Kout, longer in plant and animal Kin and the longest in plant Kout channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom.

Regulation of K+ Channels in Maize Roots by Water Stress and Abscisic Acid1

Root cortical and stelar protoplasts were isolated from maize (Zea mays L.) plants that were either well watered or water stressed, and the patch-clamp technique was used to investigate their plasma membrane K+ channel activity. In the root cortex water stress did not significantly affect inward- or outward-rectifying K+ conductances relative to those observed in well-watered plants. In contrast, water stress significantly reduced the magnitude of the outward-rectifying K+ current in the root stele but had little effect on the inward-rectifying K+ current. Pretreating well-watered plants with abscisic acid also significantly affected K+ currents in a way that was consistent with abscisic acid mediating, at least in part, the response of roots to water stress. It is proposed that the K+ channels underlying the K+ currents in the root stelar cells represent pathways that allow K+ exchange between the root symplasm and xylem apoplast. It is suggested that the regulation of K+ channel activity in the root in response to water stress could be part of an important adaptation of the plant to survive drying soils.

Tertiapin-Q blocks recombinant and native large conductance K+ channels in a use-dependent manner

Tertiapin, a short peptide from honey bee venom, has been reported to specifically block the inwardly rectifying K+ (Kir) channels, including G protein-coupled inwardly rectifying potassium channel (GIRK) 1 + GIRK4 heteromultimers and ROMK1 homomultimers. In the present study, the effects of a stable and functionally similar derivative of tertiapin, tertiapin-Q, were examined on recombinant human voltage-dependent Ca2+-activated large conductance K+ channel (BK or MaxiK; alpha-subunit or hSlo1 homomultimers) and mouse inwardly rectifying GIRK1 + GIRK2 (i.e., Kir3.1 and Kir3.2) heteromultimeric K+ channels expressed in Xenopus oocytes and in cultured newborn mouse dorsal root ganglion (DRG) neurons. In two-electrode voltage-clamped oocytes, tertiapin-Q (1-100 nM) inhibited BK-type K+ channels in a use- and concentration-dependent manner. We also confirmed the inhibition of recombinant GIRK1 + GIRK2 heteromultimers by tertiapin-Q, which had no effect on endogenous depolarization- and hyperpolarization-activated currents sensitive to extracellular divalent cations (Ca2+, Mg2+, Zn2+, and Ba2+) in defolliculated oocytes. In voltage-clamped DRG neurons, tertiapin-Q voltage- and use-dependently inhibited outwardly rectifying K+ currents, but Cs+-blocked hyperpolarization-activated inward currents including I-H were insensitive to tertiapin-Q, baclofen, barium, and zinc, suggesting absence of functional GIRK channels in the newborn. Under current-clamp conditions, tertiapin-Q blocked the action potential after hyperpolarization (AHP) and increased action potential duration in DRG neurons. Taken together, these results demonstrate that the blocking actions of tertiapin-Q are not specific to Kir channels and that the blockade of recombinant BK channels and native neuronal AHP currents is use-dependent. Inhibition of specific types of Kir and voltage-dependent Ca2+-activated K+ channels by tertiapin-Q at nanomolar range via different mechanisms may have implications in pain physiology and therapy.

Ca2+-activated K+ channels in rat otic ganglion cells: Role of Ca2+ entry via Ca2+ channels and nicotinic receptors

1. Intracellular recordings were made from neurones in the rat otic ganglion in vitro in order to investigate their morphological, physiological and synaptic properties. We took advantage of the simple structure of these cells to test for a possible role of calcium influx via nicotinic acetylcholine receptors during synaptic transmission. 2. Cells filled with biocytin comprised a homogeneous population with ovoid somata and sparse dendritic trees. Neurones had resting membrane potentials of -53 +/- 0.7 mV (n = 69), input resistances of 112 + 7 M Omega, and membrane time constants of 14 +/- 0.9 ms (n = 60). Upon depolarization, all cells fired overshooting action potentials which mere followed by an apamin-sensitive after-hyperpolarization (AHP). In response to a prolonged current injection, all neurones fired tonically. 3. The repolarization phase of action potentials had a calcium component which was mediated by N-type calcium channels. Application of omega-conotoxin abolished both the repolarizing hump and the after-hgrperpolarization suggesting that calcium influx via N-type channels activates SK-type calcium-activated potassium channels which underlie the AHP. 4. The majority (70%) of neurones received innervation from a single preganglionic fibre which generated a suprathreshold excitatory postsynaptic potential mediated by nicotinic acetylcholine receptors. The other 30% of neurones also had one or more subthreshold nicotinic inputs. 5. Calcium influx via synaptic nicotinic receptors contributed to the AHP current, indicating that this calcium has access to the calcium-activated potassium channels and therefore plays a role in regulating cell excitability.

Properties and function of KCNQ1 K+ channels isolated from the rectal gland of Squalus acanthias

KCNQ1 (K(V)LQT1) K+ channels play an important role during electrolyte secretion in airways and colon. KCNQ1 was cloned recently from NaCl-secreting shark rectal glands. Here we study. the properties and regulation of the cloned sK(V)LQT1 expressed in Xenopus oocytes and Chinese hamster ovary (CHO) cells and compare the results with those obtained from in vitro perfused rectal gland tubules (RGT). The expression of sKCNQ1 induced voltage-dependent, delayed activated K+ currents, which were augmented by an increase in intracellular cAMP and Ca2+. The chromanol derivatives 293B and 526B potently inhibited sKCNQ1 expressed in oocytes and CHO cells, but had little effect on RGT electrolyte transport. Short-circuit currents in RGT were activated by alkalinization and were decreased by acidification. In CHO cells an alkaline pH activated and an acidic pH inhibited 293B-sensitive KCNQ1 currents. Noise analysis of the cell-attached basolateral membrane of RGT indicated the presence of low-conductance (