Inhibition of G-protein-coupled inward rectifying K+ channels by intracellular acidosis

G-protein-coupled inward rectification K(+) (GIRK) channels play an important role in modulation of synaptic transmission and cellular excitability. The GIRK channels are regulated by diverse intra- and extracellular signaling molecules. Previously, we have shown that GIRK1/GIRK4 channels are activated by extracellular protons. The channel activation depends on a histidine residue in the M1-H5 linker and may play a role in neurotransmission. Here, we show evidence that the heteromeric GIRK1/GIRK4 channels are inhibited by intracellular acidification. This inhibition was produced by selective decrease in the channel open probability with a modest drop in the single-channel conductance. The inhibition does not seem to require G-proteins as it was seen in two G-protein coupling-defective GIRK mutants and in excised patches in the absence of exogenous G-proteins. Three histidine residues in intracellular domains were critical for the inhibition. Individual mutation of His-64, His-228, or His-352 in GIRK4 abolished or greatly diminished the inhibition in homomeric GIRK4. Mutations of any of these histidine residues in GIRK4 or their counterparts in GIRK1 were sufficient to eliminate the pH(i) sensitivity of the heteromeric GIRK1/GIRK4 channels. Thus, the molecular and biophysical bases for the inhibition of GIRK channels by intracellular protons are illustrated. Because of the inequality of the pH(i) and pH(o) in most cells and their relatively independent controls by cellular versus systemic mechanisms, such pH(i) sensitivity may allow these channels to regulate cellular excitability in certain physiological and pathophysiological conditions when intracellular acidosis occurs.     

Molecular determinants for sodium-dependent activation of G protein-gated K+ channels

G protein-gated inwardly rectifying K+ channels (GIRKs) are activated by a direct interaction with Gbetagamma subunits and also by raised internal [Na+]. Both processes require the presence of phosphatidylinositol bisphosphate (PIP2). Here we show that the proximal C-terminal region of GIRK2 mediates the Na+-dependent activation of both the GIRK2 homomeric channels and the GIRK1/GIRK2 heteromeric channels. Within this region, GIRK2 has an aspartate at position 226, whereas GIRK1 has an asparagine at the equivalent position (217). A single point mutation, D226N, in GIRK2, abolished the Na+-dependent activation of both the homomeric and heteromeric channels. Neutralizing a nearby negative charge, E234S had no effect. The reverse mutation in GIRK1, N217D, was sufficient to restore Na+-dependent activation to the GIRK1N217D/GIRK2D226N heteromeric channels. The D226N mutation did not alter either the single channel properties or the ability of these channels to be activated via the m2-muscarinic receptor. PIP2 dramatically increased the open probability of GIRK1/GIRK2 channels in the absence of Na+ or Gbetagamma but did not preclude further activation by Na+, suggesting that Na+ is not acting simply to promote PIP2 binding to GIRKs. We conclude that aspartate 226 in GIRK2 plays a crucial role in Na+-dependent gating of GIRK1/GIRK2 channels.     

The polyamine binding site in inward rectifier K+ channels

Strongly inwardly rectifying potassium channels exhibit potent and steeply voltage-dependent block by intracellular polyamines. To locate the polyamine binding site, we have examined the effects of polyamine blockade on the rate of MTSEA modification of cysteine residues strategically substituted in the pore of a strongly rectifying Kir channel (Kir6.2[N160D]). Spermine only protected cysteines substituted at a deep location in the pore, between the "rectification controller" residue (N160D in Kir6.2, D172 in Kir2.1) and the selectivity filter, against MTSEA modification. In contrast, blockade with a longer synthetic polyamine (CGC-11179) also protected cysteines substituted at sites closer to the cytoplasmic entrance of the channel. Modification of a cysteine at the entrance to the inner cavity (169C) was unaffected by either spermine or CGC-11179, and spermine was clearly "locked" into the inner cavity (i.e., exhibited a dramatically slower exit rate) following modification of this residue. These data provide physical constraints on the spermine binding site, demonstrating that spermine stably binds at a deep site beyond the "rectification controller" residue, near the extracellular entrance to the channel.     

CO(2) inhibits specific inward rectifier K(+) channels by decreases in intra- and extracellular pH

Hypercapnia has been shown to affect cellular excitability by modulating K(+) channels. To understand the mechanisms for this modulation, four cloned K(+) channels were studied by expressing them in Xenopus oocytes. Exposures of the oocytes to CO(2) for 4-6 min produced reversible and concentration-dependent inhibitions of Kir1.1 and Kir2.3 currents, but had no effect on Kir2.1 and Kir6.1 currents. Intra- and extracellular pH (pH(i), pH(o)) dropped during CO(2) exposures. The inhibition of Kir2.3 currents was mediated by reductions in both intra- and extracellular pH, whereas the suppression of Kir1.1 resulted from intracellular acidification. In cell-free excised inside-out patches with cytosolic-soluble factors washed out, a decrease in pH(i) produced a fast and reversible inhibition of macroscopic Kir2.3 currents. The degree of this inhibition was similar to that produced by hypercapnia when compared at the same pH(i) level. Exposure of cytosolic surface of patch membranes to a perfusate bubbled with 15% CO(2) without changing pH failed to inhibit the Kir2.3 currents. These results therefore indicate that (1) hypercapnia inhibits specific K(+) channels, (2) these inhibitions are caused by intra- and extracellular protons rather than molecular CO(2), and (3) these effects are independent of cytosol-soluble factors.     

Regulation of inward rectifier K+ channels by shift of intracellular pH dependence

The mechanistic link between mitochondrial metabolism and inward rectifier K+ channel activity was investigated by studying the effects of a mitochondrial inhibitor, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) on inward rectifiers of the Kir2 subfamily expressed in Xenopus oocytes, using two-electrode voltage-clamp, patch-clamp, and intracellular pH recording. FCCP inhibited Kir2.2 and Kir2.3 currents and decreased intracellular pH, but the pH change was too small to account for the inhibitory effect by itself. However, pre-incubation of oocytes with imidazole prevented both the pH decrease and the inhibition of Kir2.2 and Kir2.3 currents by FCCP. The pH dependence of Kir2.2 was shifted to higher pH in membrane patches from FCCP-treated oocytes compared to control oocytes. Therefore, the inhibition of Kir2.2 by FCCP may involve a combination of intracellular acidification and a shift in the intracellular pH dependence of these channels. To investigate the sensitivity of heteromeric channels to FCCP, we studied its effect on currents expressed by heteromeric tandem dimer constructs. While Kir2.1 homomeric channels were insensitive to FCCP, both Kir2.1-Kir2.2 and Kir2.1-Kir2.3 heterotetrameric channels were inhibited. These data support the notion that mitochondrial dysfunction causes inhibition of heteromeric inward rectifier K+ channels. The reduction of inward rectifier K+ channel activity observed in heart failure and ischemia may result from the mitochondrial dysfunction that occurs in these conditions.     

Differential sensitivity of inward rectifier K+ channels to metabolic inhibitors

Inhibition of inward rectifier K(+) channels under ischemic conditions may contribute to electrophysiological consequences of ischemia such as cardiac arrhythmia. Ischemia causes metabolic inhibition, and the use of metabolic inhibitors is one experimental method of simulating ischemia. The effects of metabolic inhibitors on the activity of inward rectifier K(+) channels K(ir)2.1, K(ir)2.2, and K(ir)2.3 were studied by heterologous expression in Xenopus oocytes and two-electrode voltage clamp. 10 microm carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) inhibited K(ir)2.2 and K(ir)2.3 currents but was without effect on K(ir)2.1 currents. The rate of decline of current in FCCP was faster for K(ir)2.3 than for K(ir)2.2. K(ir)2.3 was inhibited by 3 mm sodium azide (NaN(3)), whereas K(ir)2.1 and K(ir)2.2 were not. K(ir)2.2 was inhibited by 10 mm NaN(3). All three of these inward rectifiers were inhibited by lowering the pH of the solution perfusing inside-out membrane patches. K(ir)2.3 was most sensitive to pH (pK = 6.9), whereas K(ir)2.1 was least sensitive (pK = 5.9). For K(ir)2.2 the pK was 6.2. These results demonstrate the differential sensitivity of these inward rectifiers to metabolic inhibition and internal pH. The electrophysiological response of a particular cell type to ischemia may depend on the relative expression levels of different inward rectifier genes.     

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

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

Sodium azide dilates coronary arterioles via activation of inward rectifier K+ channels and Na+-K+-ATPase

Sodium azide (NaN(3)), a potent vasodilator, causes severe hypotension on accidental exposure. Although NaN(3) has been shown to increase coronary blood flow, the direct effect of NaN(3) on coronary resistance vessels and the mechanism of the NaN(3)-induced response remain to be established. To address these issues without confounding influences from systemic parameters, subepicardial coronary arterioles were isolated from porcine hearts for in vitro study. Arterioles developed basal tone at 60 cmH(2)O intraluminal pressure and dilated acutely, in a concentration-dependent manner, to NaN(3) (0.1 microM to 50 microM). The NaN(3) response was not altered by the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester or endothelial removal. Neither inhibition of phosphoinositol 3-kinase and tyrosine kinases nor blockade of ATP-sensitive, Ca(2+)-activated, and voltage-dependent K(+) channels affected NaN(3)-induced dilation. However, the vasomotor action of NaN(3) was significantly attenuated in a similar manner by the inward rectifier K(+) (K(IR)) channel inhibitor Ba(2+), the Na(+)-K(+) ATPase inhibitor ouabain, or the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ). Ba(2+), in combination with either ouabain or ODQ, nearly abolished the vasodilatory response. However, there was no additive inhibition by combining ouabain and ODQ. The NaN(3)-mediated vasodilation was also attenuated by morin, an inhibitor of phosphatidylinositolphosphate (PIP) kinase, which can regulate K(IR) channel activity. With the use of whole cell patch-clamp methods, NaN(3) acutely enhanced Ba(2+)-sensitive K(IR) current in isolated coronary arteriolar smooth muscle cells. Collectively, this study demonstrates that NaN(3), at clinically toxic concentrations, dilates coronary resistance vessels via activation of both K(IR) channels and guanylyl cyclase/Na(+)-K(+)-ATPase in the vascular smooth muscle. The K(IR) channels appear to be modulated by PIP kinase.     

Interaction mechanisms between polyamines and IRK1 inward rectifier K+ channels

Rectification of macroscopic current through inward-rectifier K+ (Kir) channels reflects strong voltage dependence of channel block by intracellular cations such as polyamines. The voltage dependence results primarily from the movement of K+ ions across the transmembrane electric field, which accompanies the binding-unbinding of a blocker. Residues D172, E224, and E299 in IRK1 are critical for high-affinity binding of blockers. D172 appears to be located somewhat internal to the narrow K+ selectivity filter, whereas E224 and E299 form a ring at a more intracellular site. Using a series of alkyl-bis-amines of varying length as calibration, we investigated how the acidic residues in IRK1 interact with amine groups in the natural polyamines (putrescine, spermidine, and spermine) that cause rectification in cells. To block the pore, the leading amine of bis-amines of increasing length penetrates ever deeper into the pore toward D172, while the trailing amine in every bis-amine binds near a more intracellular site and interacts with E224 and E299. The leading amine in nonamethylene-bis-amine (bis-C9) makes the closest approach to D172, displacing the maximal number of K+ ions and exhibiting the strongest voltage dependence. Cells do not synthesize bis-amines longer than putrescine (bis-C4) but generate the polyamines spermidine and spermine by attaching an amino-propyl group to one or both ends of putrescine. Voltage dependence of channel block by the tetra-amine spermine is comparable to that of block by the bis-amines bis-C9 (shorter) or bis-C12 (equally long), but spermine binds to IRK1 with much higher affinity than either bis-amine does. Thus, counterintuitively, the multiple amines in spermine primarily confer the high affinity but not the strong voltage dependence of channel block. Tetravalent spermine achieves a stronger interaction with the pore by effectively behaving like a pair of tethered divalent cations, two amine groups in its leading half interacting primarily with D172, whereas the other two in the trailing half interact primarily with E224 and E299. Thus, nature has optimized not only the blocker but also, in a complementary manner, the channel for producing rapid, high-affinity, and strongly voltage-dependent channel block, giving rise to exceedingly sharp rectification.     

Gating of inward rectifier K+ channels by proton-mediated interactions of N- and C-terminal domains

Ion channels play an important role in cellular functions, and specific cellular activity can be produced by gating them. One important gating mechanism is produced by intra- or extracellular ligands. Although the ligand-mediated channel gating is an important cellular process, the relationship between ligand binding and channel gating is not well understood. It is possible that ligands are involved in the interactions of different protein domains of the channel leading to opening or closing. To test this hypothesis, we studied the gating of Kir2.3 (HIR) by intracellular protons. Our results showed that hypercapnia or intracellular acidification strongly inhibited these channels. This effect relied on both the N and C termini. The CO(2)/pH sensitivities were abolished or compromised when one of the intracellular termini was replaced. Using purified N- and C-terminal peptides, we found that the N and C termini bound to each other in vitro. Although their binding was weak at pH 7.4, stronger binding was seen at pH 6.6. Two short sequences in the N and C termini were found to be critical for the N/C-terminal interaction. Interestingly, there was no titratable residue in these motifs. To identify the potential protonation sites, we systematically mutated most histidine residues in the intracellular N and C termini. We found that mutations of several histidine residues in the C but not the N terminus had a major effect on channel sensitivities to CO(2) and pH(i). These results suggest that at acidic pH, protons appear to interact with the C-terminal histidine residues and present the C terminus to the N terminus. Consequentially, these two intracellular termini bound to each other through two short motifs and closed the channel. Thus, a novel mechanism for K(+) channel gating is demonstrated, which involves the N- and C-terminal interaction with protons as the mediator.     

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

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

Direct activation of cloned K(atp) channels by intracellular acidosis

ATP-sensitive K(+) (K(ATP)) channels may be regulated by protons in addition to ATP, phospholipids, and other nucleotides. Such regulation allows a control of cellular excitability in conditions when pH is low but ATP concentration is normal. However, whether the K(ATP) changes its activity with pH alterations remains uncertain. In this study we showed that the reconstituted K(ATP) was strongly activated during hypercapnia and intracellular acidosis using whole-cell recordings. Further characterizations in excised patches indicated that channel activity increased with a moderate drop in intracellular pH and decreased with strong acidification. The channel activation was produced by a direct action of protons on the Kir6 subunit and relied on a histidine residue that is conserved in all K(ATP). The inhibition appeared to be a result of channel rundown and was not seen in whole-cell recordings. The biphasic response may explain the contradictory pH sensitivity observed in cell-endogenous K(ATP) in excised patches. Site-specific mutations of two residues showed that pH and ATP sensitivities were independent of each other. Thus, these results demonstrate that the proton is a potent activator of the K(ATP). The pH-dependent activation may enable the K(ATP) to control vascular tones, insulin secretion, and neuronal excitability in several pathophysiologic conditions.     

Activation of inward rectifier K+ channels by hypoxia in rabbit coronary arterial smooth muscle cells

We examined the effects of acute hypoxia on Ba2+-sensitive inward rectifier K+ (K(IR)) current in rabbit coronary arterial smooth muscle cells. The amplitudes of K(IR) current was definitely higher in the cells from small-diameter (<100 microm) coronary arterial smooth muscle cells (SCASMC, -12.8 +/- 1.3 pA/pF at -140 mV) than those in large-diameter coronary arterial smooth muscle cells (>200 microm, LCASMC, -1.5 +/- 0.1 pA pF(-1)). Western blot analysis confirmed that Kir2.1 protein was expressed in SCASMC but not LCASMC. Hypoxia activated much more KIR currents in symmetrical 140 K+. This effect was blocked by the adenylyl cyclase inhibitor SQ-22536 (10 microM) and mimicked by forskolin (10 microM) and dibutyryl-cAMP (500 microM). The production of cAMP in SCASMC increased 5.7-fold after 6 min of hypoxia. Hypoxia-induced increase in KIR currents was abolished by the PKA inhibitors, Rp-8-(4-chlorophenylthio)-cAMPs (10 microM) and KT-5720 (1 microM). The inhibition of G protein with GDPbetaS (1 mM) partially reduced (approximately 50%) the hypoxia-induced increase in KIR currents. In Langendorff-perfused rabbit hearts, hypoxia increased coronary blood flow, an effect that was inhibited by Ba2+. In summary, hypoxia augments the KIR currents in SCASMC via cAMP- and PKA-dependent signaling cascades, which might, at least partly, explain the hypoxia-induced coronary vasodilation.     

Enantioselective protein-sterol interactions mediate regulation of both prokaryotic and eukaryotic inward rectifier K+ channels by cholesterol

Cholesterol is the major sterol component of all mammalian cell plasma membranes and plays a critical role in cell function and growth. Previous studies have shown that cholesterol inhibits inward rectifier K(+) (Kir) channels, but have not distinguished whether this is due directly to protein-sterol interactions or indirectly to changes in the physical properties of the lipid bilayer. Using purified bacterial and eukaryotic Kir channels reconstituted into liposomes of controlled lipid composition, we demonstrate by (86)Rb(+) influx assays that bacterial Kir channels (KirBac1.1 and KirBac3.1) and human Kir2.1 are all inhibited by cholesterol, most likely by locking the channels into prolonged closed states, whereas the enantiomer, ent-cholesterol, does not inhibit these channels. These data indicate that cholesterol regulates Kir channels through direct protein-sterol interactions likely taking advantage of an evolutionarily conserved binding pocket.     

C-terminus determinants for Mg2+ and polyamine block of the inward rectifier K+ channel IRK1.

Critical loci for ion conduction in inward rectifier K+ channels are only now being discovered. The C-terminal region of IRK1 plays a crucial role in Mg2+i blockade and single-channel K+ conductance. A negatively charged aspartate in the putative second transmembrane domain (position 172) is essential for time-dependent block by the cytoplasmic polyamines spermine and spermidine. We have now localized the C-terminus effect in IRK1 to a single, negatively charged residue (E224). Mutation of E224 to G, Q and S drastically reduced rectification. Furthermore, the IRK1 E224G mutation decreased block by Mg2+i and spermidine and, like the E224Q mutation, caused a dramatic reduction in the apparent single-channel K+ conductance. The double mutation IRK1 D172N+ E224G was markedly insensitive to spermidine block, displaying an affinity similar to ROMK1. The results are compatible with a model in which the negatively charged residue at position 224, E224, is a major determinant of pore properties in IRK1. By means of a specific interaction with the negatively charged residue at position 172, D172, E224 contributes to the formation of the binding pocket for Mg2+ and polyamines, a characteristic of strong inward rectifiers.     

Effects of external Rb+ on inward rectifier K+ channels of bovine pulmonary artery endothelial cells

Inward rectifier (IR) K+ channels of bovine pulmonary artery endothelial cells were studied using the whole-cell, cell-attached, and outside-out patch-clamp configurations. The effects of Rb+ on the voltage dependence and kinetics of IR gating were explored, with [Rb+]o + [K+]o = 160 mM. Partial substitution of Rb+ for K+ resulted in voltage-dependent reduction of inward currents, consistent with Rb+ being a weakly permeant blocker of the IR. In cells studied with a K(+)- free pipette solution, external Rb+ reduced inward IR currents to a similar extent at large negative potentials but block at more positive potentials was enhanced. In outside-out patches, the single-channel i-V relationship was approximately linear in symmetrical K+, but rectified strongly outwardly in high [Rb+]o due to a reduced conductance for inward current. The permeability of Rb+ based on reversal potential, Vrev, was 0.45 that of K+, whereas the Rb+ conductance was much lower, 0.034 that of K+, measured at Vrev-80 mV. The steady state voltage- dependence of IR gating was determined in Rb(+)-containing solutions by applying variable prepulses, followed by a test pulse to a potential at which outward current deactivation was observed. As [Rb+]o was increased, the half-activation potential, V1/2, changed less than Vrev. In high [K+]o solutions V1/2 was Vrev-6 mV, while in high [Rb+]o V1/2 was Vrev + 7 mV. This behavior contrasts with the classical parallel shift of V1/2 with Vrev in K+ solutions. Steady state IR gating was less steeply voltage-dependent in high [Rb+]o than in K+ solutions, with Boltzmann slope factors of 6.4 and 4.4 mV, respectively. Rb+ decreased (slowed) both activation and deactivation rate constants defined at V1/2, and decreased the steepness of the voltage dependence of the activation rate constant by 42%. Deactivation of IR channels in outside-out patches was also slowed by Rb+. In summary, Rb+ can replace K+ in setting the voltage-dependence of IR gating, but in doing so alters the kinetics.     

ATP-sensitive inward rectifier and voltage- and calcium-activated K+ channels in cultured pancreatic islet cells

Summary K⁺ channels in cultured rat pancreatic islet cells have been studied using patch-clamp single-channel recording techniques in cell-attached and excised inside-out and outside-out membrane patches. Three different K⁺-selective channels have been found. Two inward rectifier K⁺ channels with slope conductances of about 4 and 17 pS recorded under quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) and maximal conductances recorded in symmetrical K⁺-rich solutions of about 30 and 75 pS, respectively. A voltage- and calcium-activated K^– channel was recorded with a slope conductance of about 90 pS under the same conditions and a maximal conductance recorded in symmetrical K⁺-rich solutions of about 250 pS. Single-channel current recording in the cell-attached conformation revealed a continuous low level of activity in an apparently small number of both the inward rectifier K⁺ channels. But when membrane patches were excised from the intact cell a much larger number of inward rectifier K⁺ channels became transiently activated before showing an irreversible decline. In excised patches opening and closing of both the inward rectifier K⁺ channels were unaffected by voltage, internal Ca²⁺ or externally applied tetraethyl-ammonium (TEA) but the probability of opening of both inward rectifier K⁺ channels was reduced by internally applied 1–5mm adenosine-5-triphosphate (ATP). The large K⁺ channel was not operational in cell-attached membrane patches, but in excised patches it could be activated at negative membrane potentials by 10^–7 to 10^–6 m internal Ca²⁺ and blocked by 5–10mm external TEA.     

K+ channels of stomatal guard cells. Characteristics of the inward rectifier and its control by pH

Intracellular microelectrode recordings and a two-electrode voltage clamp have been used to characterize the current carried by inward rectifying K+ channels of stomatal guard cells from the broadbean, Vicia faba L. Superficially, the current displayed many features common to inward rectifiers of neuromuscular and egg cell membranes. In millimolar external K+ concentrations (Ko+), it activated on hyperpolarization with half-times of 100-200 ms, showed no evidence of time- or voltage-dependent inactivation, and deactivated rapidly (tau approximately 10 ms) on clamping to 0 mV. Steady-state conductance-voltage characteristics indicated an apparent gating charge of 1.3-1.6. Current reversal showed a Nernstian dependence on Ko+ over the range 3-30 mM, and the inward rectifier was found to be highly selective for K+ over other monovalent cations (K+ greater than Rb+ greater than Cs+ much greater than Na+). Unlike the inward rectifiers of animal membranes, the current was blocked by charybdotoxin and alpha-dendrotoxin (Kd much less than 50 nM), as well as by tetraethylammonium chloride (K1/2 = 9.1 mM); gating of the guard cell K+ current was fixed to voltages near -120 mV, independent of Ko+, and the current activated only with supramillimolar K+ outside (EK+ greater than -120 mV). Most striking, however, was inward rectifier sensitivity to [H+] with the K+ current activated reversibly by mild acid external pH. Current through the K+ inward rectifier was found to be largely independent of intracellular pH and the current reversal (equilibrium) potential was unaffected by pHo from 7.4 to 5.5. By contrast, current through the K+ outward rectifier previously characterized in these cells (1988. J. Membr. Biol. 102:235) was largely insensitive to pHo, but was blocked reversibly by acid-going intracellular pH. The action of pHo on the K+ inward rectifier could not be mimicked by extracellular Ca2+ for which changes in activation, deactivation, and conductance were consonant with an effect on surface charge ([Ca2+] less than or equal to 1 mM). Rather, extracellular pH affected activation and deactivation kinetics disproportionately, with acid-going pHo raising the K+ conductance and shifting the conductance-voltage profile positive-going along the voltage axis and into the physiological voltage range. Voltage and pH dependencies for gating were consistent with a single, titratable group (pKa approximately 7 at -200 mV) residing deep within the membrane electric field and accessible from the outside.(ABSTRACT TRUNCATED AT 400 WORDS)