Dualistic behavior of ATP-sensitive K+ channels toward intracellular nucleoside diphosphates.

ATP-sensitive K+ (KATP) channels are intracellular ligand-gated channels which regulate diverse cellular functions. Intracellular nucleoside diphosphates (NDPs) are essential for the physiological opening of KATP channels which would otherwise be permanently closed by their overt sensitivity to intracellular ATP. We find that KATP channels exhibit dualistic behavior toward NDPs depending on their operative condition. When channels are in the spontaneous operative condition, NDPs antagonize channel inhibition by intracellular ATP. When channels have "run down", NDPs induce channel opening but no longer antagonize intracellular ATP. The switch of the KATP channel response to the same ligand, i.e., NDPs, is controlled by a Mg-ATP-dependent reaction. The condition of the target protein therefore determines the effect of the ligand. This property provides a novel basis to evaluate the dynamic regulation of ion channels by their ligands.

G proteins activate ATP-sensitive K+ channels by antagonizing ATP-dependent gating.

To determine whether G proteins activate cardiac ATP-sensitive K+ (KATP) channels by regulating intracellular ATP (ATPi)-dependent gating, currents were measured in inside-out patches. When ATPi closed KATP channels, activators of endogenous G proteins, GTP (plus adenosine or acetylcholine), GTP gamma S, or AlF-4 stimulated channels, an effect prevented by GDP beta S. In the absence of ATPi, G protein activators were ineffective. Intracellular nucleoside diphosphates restored KATP channel openings after the "rundown" of spontaneous activity. Only when ATPi suppressed nucleoside diphosphate-induced openings, GTP gamma S or AlF-4 enhanced KATP channel activity. Active forms of exogenous G protein subunits (G alpha i-1, G alpha i-2, or G alpha o) activated only KATP channels closed by ATPi. G proteins stimulate cardiac KATP channels apparently by antagonizing ATPi-dependent inhibitory gating. Regulation of ligand-dependent gating represents a distinct type of G protein modulation of ion channels.

Characterization of a novel ATP-sensitive K+ channel opener, A-251179, on urinary bladder relaxation and cystometric parameters.

BACKGROUND AND PURPOSE: ATP-sensitive K(+) channels (K(ATP)) play a pivotal role in contractility of urinary bladder smooth muscle. This study reports the characterization of 4-methyl-N-(2,2,2-trichloro-1-(3-pyridin-3-ylthioureido)ethyl)benzamide (A-251179) as a K(ATP) channel opener. EXPERIMENTAL APPROACH: Glyburide-sensitive membrane potential, patch clamp and tension assays were employed to study the effect of A-251179 in vitro. The in vivo efficacy of A-251179 was characterized by suppression of spontaneous contractions in obstructed rat bladder and by measuring urodynamic function of urethane-anesthetized rat models. KEY RESULTS: A-251179 was about 4-fold more selective in activating SUR2B-Kir6.2 derived K(ATP) channels compared to those derived from SUR2A-Kir6.2. In pig bladder smooth muscle strips, A-251179 suppressed spontaneous contractions, about 27- and 71-fold more potently compared to suppression of contractions evoked by low-frequency electrical stimulation and carbachol, respectively. In vivo, A-251179 suppressed spontaneous non-voiding bladder contractions from partial outlet-obstructed rats. Interestingly, in the neurogenic model where isovolumetric contractions were measured by continuous transvesical cystometry, A-251179 at a dose of 0.3 micromol kg(-1), but not higher, was found to increase bladder capacity without affecting either the voiding efficiency or changes in mean arterial blood pressure. CONCLUSIONS AND IMPLICATIONS: The thioureabenzamide analog, A-251179 is a potent novel K(ATP) channel opener with selectivity for SUR2B/Kir6.2 containing K(ATP) channels relative to pinacidil. The pharmacological profile of A-251179 is to increase bladder capacity and to prolong the time between voids without affecting voiding efficiency and represents an interesting characteristic to be explored for further investigations of K(ATP) channel openers for the treatment of overactive bladder.

Ca(2+) elevation evoked by membrane depolarization regulates G protein cycle via RGS proteins in the heart.

Regulators of G protein signaling (RGS), which act as GTPase activators, are a family of cytosolic proteins emerging rapidly as an important means of controlling G protein-mediated cell signals. The importance of RGS action has been verified in vitro for various kinds of cell function. Their in situ modes of action in intact cells are, however, poorly understood. Here we show that an increase in intracellular Ca(2+) evoked by membrane depolarization controls the RGS action on G protein activation of muscarinic K(+) (K(G)) channel in the heart. Acetylcholine-induced K(G) current exhibits a slow time-dependent increase during hyperpolarizing voltage steps, referred to as "relaxation." This reflects the relief from the decrease in available K(G) channel number induced by cell depolarization. This phenomenon is abolished when an increase in intracellular Ca(2+) is prevented. It is also abolished when a calmodulin inhibitor or a mutant RGS4 is applied that can bind to calmodulin but that does not accelerate GTPase activity. Therefore, an increase in intracellular Ca(2+) and the resultant formation of Ca(2+)/calmodulin facilitate GTPase activity of RGS and thus decrease the available channel number on depolarization. These results indicate a novel and probably general pathway that Ca(2+)-dependent signaling regulates the G protein cycle via RGS proteins.

C-terminal tails of sulfonylurea receptors control ADP-induced activation and diazoxide modulation of ATP-sensitive K(+) channels.

The ATP-sensitive K(+) (K(ATP)) channels are composed of the pore-forming K(+) channel Kir6.0 and different sulfonylurea receptors (SURs). SUR1, SUR2A, and SUR2B are sulfonylurea receptors that are characteristic for pancreatic, cardiac, and vascular smooth muscle-type K(ATP) channels, respectively. The structural elements of SURs that are responsible for their different characteristics have not been entirely determined. Here we report that the 42 amino acid segment at the C-terminal tail of SURs plays a critical role in the differential activation of different SUR-K(ATP) channels by ADP and diazoxide. In inside-out patches of human embryonic kidney 293T cells coexpressing distinct SURs and Kir6.2, much higher concentrations of ADP were needed to activate channels that contained SUR2A than SUR1 or SUR2B. In all types of K(ATP) channels, diazoxide increased potency but not efficacy of ADP to evoke channel activation. Replacement of the C-terminal segment of SUR1 with that of SUR2A inhibited ADP-mediated channel activation and reduced diazoxide modulation. Point mutations of the second nucleotide-binding domains (NBD2) of SUR1 and SUR2B, which would prevent ADP binding or ATP hydrolysis, showed similar effects. It is therefore suggested that the C-terminal segment of SUR2A possesses an inhibitory effect on NBD2-mediated ADP-induced channel activation, which underlies the differential effects of ADP and diazoxide on K(ATP) channels containing different SURs.

Identification and functional analysis of sulfonylurea receptor 1 variants in Japanese patients with NIDDM.

The sulfonylurea receptor 1 (SUR1) is an essential regulatory subunit of the beta-cell ATP-sensitive K+ channel (K[ATP]). The possible role of SUR1 gene mutation(s) in the development of NIDDM remains controversial as both a positive association and negative linkage results have been reported. Therefore, we examined the SUR1 gene at the single nucleotide level with single strand conformation polymorphism analysis in 100 Japanese NIDDM patients. We identified a total of five amino acid substitutions and 17 silent mutations by examining all 39 exons of this gene. Two rare novel mutations, D811N in exon 20 and R835C in exon 21, were identified in the first nucleotide-binding fold (NBF), a functionally important region of SUR1, in one patient each, both heterozygotes. To analyze possible functional alterations, we reconstituted the mutant K(ATP) by coexpressing beta-cell inward rectifier (BIR) (Kir 6.2), a channel subunit of K(ATP), and mutant SUR1 in HEK293T and COS-7 cells. As demonstrated by the patch clamp technique and rubidium (Rb+) efflux studies, neither mutation alters the properties of channel activities. Two other rare missense mutations, R275Q in exon 6 and V560M in exon 12, were also identified. The R275Q substitution was not found in 67 control subjects, and V560M was present in three control subjects. Neither of these substitutions appeared to cosegregate with NIDDM in the probands' families. A previously reported S1370A substitution located in the second NBF was also common in the Japanese subjects (allelic frequency 0.37), and was found at an equal frequency in nondiabetic control subjects. In conclusion, SUR1 mutations impairing K(ATP) function do not appear to be major determinants of NIDDM susceptibility in Japanese.

Effects of anions on the G protein-mediated activation of the muscarinic K+ channel in the cardiac atrial cell membrane. Intracellular chloride inhibition of the GTPase activity of GK.

The effects of various intracellular anions on the G protein (GK)-mediated activation of the muscarinic K+ (KACh) channel were examined in single atrial myocytes isolated from guinea pig hearts. The patch clamp technique was used in the inside-out patch configuration. With acetylcholine (ACh, 0.5 microM) in the pipette, 1 microM GTP caused different magnitudes of KACh channel activation in internal solutions containing different anions. The order of potency of anions to induce the KACh channel activity at 0.5 microM ACh and 1 microM GTP was Cl- greater than or equal to Br- greater than 1-. In the SO4(2-) or aspartic acid internal solution, no channel openings were induced by 1 microM GTP with 0.5 microM ACh. In both the Cl- and SO4(2-) internal solutions (with 0.5 microM ACh) the relationship between the concentration of GTP and the channel activity was fit by the Hill equation with a Hill coefficient of approximately 3-4. However, the concentration of GTP at the half-maximal activation (Kd) was 0.2 microM in the Cl- and 10 microM in the SO4(2-) solution. On the other hand, the quasi-steady-state relationship between the concentration of guanosine-5'-o-(3-thiotriphosphate) and the channel activity did not differ significantly between the Cl- and SO4(2-) solutions; i.e., the Hill coefficient was approximately 3-4 and the Kd was approximately 0.06-0.08 microM in both solutions. The decay of channel activity after washout of GTP in the Cl- solution was much slower than that in the SO4(2-) solution. These results suggest that intracellular Cl- does not affect the turn-on reaction but slows the turn-off reaction of GK, resulting in higher sensitivity of the KACh channel for GTP. In the Cl- solution, even in the absence of agonists, GTP (greater than 1 microM) or ATP (greater than 1 mM) alone caused activation of the KACh channel, while neither occurred in the SO4(2-) solution. These observations suggest that the activation of the KACh channel by the basal turn-on reaction of GK or by phosphate transfer to GK by nucleoside diphosphate-kinase may depend at least partly on the intracellular concentration of Cl-.

A functional model for G protein activation of the muscarinic K+ channel in guinea pig atrial myocytes. Spectral analysis of the effect of GTP on single-channel kinetics.

To elucidate the functional interaction between the active G protein subunit (GK*) and the cardiac muscarinic K+ (KACh) channel, the effect of intracellular GTP on the channel current fluctuation in the presence of 0.5 microM extracellular acetylcholine was examined in inside-out patches from guinea pig atrial myocytes using spectral analysis technique. The power density spectra of current fluctuations induced at various concentrations of GTP ([GTP]) were well fitted by the sum of two Lorentzian functions. Because the channel has one open state, the open-close transitions of the channel gate represented by the spectra could be described as C2<-->C1<-->O. As [GTP] was raised, the channel activity increased in a positive cooperative manner. The powers of the two Lorentzian components concomitantly increased, while the corner frequencies and the ratio of the powers at 0 Hz remained almost constant. This indicates that G protein activation did not affect the gating of each channel but mainly increased the number of functionally active channels in the patch to enhance the channel activity. Regulation of the number of functionally active channels could be described by a slow transition of the channel states, U (unavailable)<-->A (available), which is independent of the gating. The equilibrium of this slow transition was shifted by GTP from U to A. Monod-Wyman-Changeux's allosteric model for the channel state transition(U<-->A) could well describe the positive cooperative increase in the channel availability by GTP, assuming that, in the presence of saturating concentrations of ACh, [GK*] linearly increased as [GTP] was raised in our experimental range. The model indicates that the cardiac KACh channel could be described as a multimer composed of four or more functionally identical subunits, to each of which one GK* binds.

On the mechanism of G protein beta gamma subunit activation of the muscarinic K+ channel in guinea pig atrial cell membrane. Comparison with the ATP-sensitive K+ channel.

The mechanism of G protein beta gamma subunit (G beta gamma)-induced activation of the muscarinic K+ channel (KACh) in the guinea pig atrial cell membrane was examined using the inside-out patch clamp technique. G beta gamma and GTP-gamma S-bound alpha subunits (G alpha *'s) of pertussis toxin (PT)-sensitive G proteins were purified from bovine brain. Either in the presence or absence of Mg2+, G beta gamma activated the KACh channel in a concentration-dependent fashion. 10 nM G beta gamma almost fully activated the channel in 132 of 134 patches (98.5%). The G beta gamma-induced maximal channel activity was equivalent to or sometimes larger than the GTP-gamma S-induced one. Half-maximal activation occurred at approximately 6 nM G beta gamma. Detergent (CHAPS) and boiled G beta gamma preparation could not activate the KACh channel. G beta gamma suspended by Lubrol PX instead of CHAPS also activated the channel. Even when G beta gamma was pretreated in Mg(2+)-free EDTA internal solution containing GDP analogues (24-48 h) to inactivate possibly contaminating G i alpha *'s, the G beta gamma activated the channel. Furthermore, G beta gamma preincubated with excessive GDP-bound G o alpha did not activate the channel. These results indicate that G beta gamma itself, but neither the detergent CHAPS nor contaminating G i alpha *, activates the KACh channel. Three different kinds of G i alpha * at 10 pM-10 nM could weakly activate the KACh channel. However, they were effective only in 40 of 124 patches (32.2%) and their maximal channel activation was approximately 20% of that induced by GTP-gamma S or G beta gamma. Thus, G i alpha * activation of the KACh channel may not be significant. On the other hand, G i alpha *'s effectively activated the ATP-sensitive K+ channel (KATP) in the ventricular cell membrane when the KATP channel was maintained phosphorylated by the internal solution containing 100 microM Mg.ATP. G beta gamma inhibited adenosine or mACh receptor-mediated, intracellular GTP-induced activation of the KATP channel. G i alpha *'s also activated the phosphorylated KATP channel in the atrial cell membrane, but did not affect the background KACh channel. G beta gamma subsequently applied to the same patch caused prominent KACh channel activation. The above results may indicate two distinct regulatory systems of cardiac K+ channels by PT-sensitive G proteins: G i alpha activation of the KATP channel and G beta gamma activation of the KACh channel.

On the mechanism of basal and agonist-induced activation of the G protein-gated muscarinic K+ channel in atrial myocytes of guinea pig heart.

Using the patch clamp technique, we examined the agonist-free, basal interaction between the muscarinic acetylcholine (m-ACh) receptor and the G protein (GK)-gated muscarinic K+ channel (IK.ACh), and the modification of this interaction by ACh binding to the receptor in single atrial myocytes of guinea pig heart. In the whole cell clamp mode, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma S) gradually increased the IK.ACh current in the absence of agonists (e.g., acetylcholine). This increase was inhibited in cells that were pretreated with islet-activating protein (IAP, pertussis toxin) or N-ethylmaleimide (NEM). In inside-out patches, even in the absence of agonists, intracellular GTP caused openings of IK.ACh in a concentration-dependent manner in approximately 80% of the patches. Channel activation by GTP in the absence of agonist was much less than that caused by GTP-gamma S. The agonist-independent, GTP-induced activation of IK.ACh was inhibited by the A promoter of IAP (with nicotinamide adenine dinucleotide) or NEM. As the ACh concentration was increased, the GTP-induced maximal open probability of IK.ACh was increased and the GTP concentration for the half-maximal activation of IK.ACh was decreased. Intracellular GDP inhibited the GTP-induced openings of IK.ACh in a concentration-dependent fashion. The half-inhibition of IK.ACh openings occurred at a much lower concentration of GDP in the absence of agonists than in the presence of ACh. From these results, we concluded (a) that the interaction between the m-ACh receptor and GK is essential for basal stimulation of IK.ACh, and (b) that ACh binding to the receptor accelerates the turnover of GK and increases GK's affinity to GTP analogues over GDP.

Activation of Ca(2+)-dependent K+ current by acetylcholine and histamine in a human gastric epithelial cell line.

The effects of acetylcholine (ACh) and histamine (His) on the membrane potential and current were examined in JR-1 cells, a mucin-producing epithelial cell line derived from human gastric signet ring cell carcinoma. The tight-seal, whole cell clamp technique was used. The resting membrane potential, the input resistance, and the capacitance of the cells were approximately -12 mV, 1.4 G ohms, and 50 pF, respectively. Under the voltage-clamp condition, no voltage-dependent currents were evoked. ACh or His added to the bathing solution hyperpolarized the membrane by activating a time- and voltage-independent K+ current. The ACh-induced hyperpolarization and K+ current persisted, while the His response desensitized quickly (< 1 min). These effects of ACh and His were mediated predominantly by m3-muscarinic and H1-His receptors, respectively. The K+ current induced by ACh and His was inhibited by charybdotoxin, suggesting that it is a Ca(2+)-activated K+ channel current (IK.Ca). The measurement of intracellular Ca2+ ([Ca2+]i) using Indo-1 revealed that both agents increased [Ca2+]i with similar time courses as they increased IK.Ca. When EGTA in the pipette solution was increased from 0.15 to 10 mM, the induction of IK.Ca by ACh and His was abolished. Thus, both ACh and His activate IK.Ca by increasing [Ca2+]i in JR-1 cells. In the Ca(2+)-free bathing solution (0.15 mM EGTA in the pipette), ACh evoked IK.Ca transiently. Addition of Ca2+ (1.8 mM) to the bath immediately restored the sustained IK.Ca. These results suggest that the ACh response is due to at least two different mechanisms; i.e., the Ca2+ release-related initial transient activation and the Ca2+ influx-related sustained activation of IK.Ca. Probably because of desensitization, the Ca2+ influx-related component of the His response could not be identified. Intracellularly applied inositol 1,4,5-trisphosphate (IP3), with and without inositol 1,3,4,5-tetrakisphosphate (IP4), mimicked the ACh response. IP4 alone did not affect the membrane current. Under the steady effect of IP3 or IP3 plus IP4, neither ACh nor His further evoked IK.Ca. Intracellular application of heparin or of the monoclonal antibody against the IP3 receptor, mAb18A10, inhibited the ACh and His responses in a concentration-dependent fashion. Neomycin, a phospholipase C (PLC) inhibitor, also inhibited the agonist-induced response in a concentration-dependent fashion. Although neither pertussis toxin (PTX) nor N-ethylmaleimide affected the ACh or His activation of IK,Ca, GDP beta S attenuated and GTP gamma S enhanced the agonist response.(ABSTRACT TRUNCATED AT 400 WORDS)

Molecular aspects of ATP-sensitive K+ channels in the cardiovascular system and K+ channel openers.

ATP-sensitive K+ (K(ATP)) channels are inhibited by intracellular ATP (ATPi) and activated by intracellular nucleoside diphosphates and thus, provide a link between cellular metabolism and excitability. K(ATP) channels are widely distributed in various tissues and may be associated with diverse cellular functions. In the heart, the K(ATP) channel appears to be activated during ischemic or hypoxic conditions, and may be responsible for the increase of K+ efflux and shortening of the action potential duration. Therefore, opening of this channel may result in cardioprotective, as well as proarrhythmic, effects. These channels are clearly heterogeneous. The cardiac K(ATP) channel is the prototype of K(ATP) channels possessing approximately 80 pS of single-channel conductance in the presence of approximately 150 mM extracellular K+ and opens spontaneously in the absence of ATPi. A vascular K(ATP) channel called a nucleoside diphosphate-dependent K+ (K(NDP)) channel exhibits properties significantly different from those of the cardiac K(ATP) channel. The K(NDP) channel has the single-channel conductance of approximately 30-40 pS in the presence of approximately 150 mM extracellular K+, is closed in the absence of ATPi, and requires intracellular nucleoside di- or triphosphates, including ATPi to open. Nevertheless, K(ATP) and K(NDP) channels are both activated by K+ channel openers, including pinacidil and nicorandil, and inhibited by sulfonylurea derivatives such as glibenclamide. It recently was found that the cardiac K(ATP) channel is composed of a sulfonylurea receptor (SUR)2A and a two-transmembrane-type K+ channel subunit Kir6.2, while the vascular K(NDP) channel may be the complex of SUR2B and Kir6.1. By precisely comparing the functional properties of the SUR2A/Kir6.2 and the SUR2B/Kir6.1 channels, we shall show that the single-channel characteristics and pharmacological properties of SUR/Kir6.0 channels are determined by Kir and SUR subunits, respectively, while responses to intracellular nucleotides are determined by both SUR and Kir subunits.

G-protein control of cardiac potassium channels.

Two cardiac potassium (K(+)) channels are activated by pertussis toxin (PTX)-sensitive G proteins either directly or in a "membrane-delimited" manner. They are muscarinic K(+)(K(ACH)) and ATP-sensitive K(+)(K(ATP)) channels. K(ACH) channels are responsible for acetylcholine (ACh)- or adenosine-induced deceleration of the heartbeat and atrioventricular conduction, while K(ATP) channels are responsible for the ischemia-induced shortening of the cardiac action potential and possibly for the adenosine-mediated protection from ischemic damage. Distinct molecular mechanisms underlie G-protein activation of these cardiac K(+) channels; the α subunit of PTX-sensitive G proteins activates the K(ATP) channels, while ßγ subunits activate the K(ACh) channel. The physiologic significance of this heterogeneous mechanism remains to be determined.

Intracellular acidification and ADP enhance nicorandil induction of ATP sensitive potassium channel current in cardiomyocytes.

OBJECTIVE: Potassium channel openers target ATP sensitive potassium (KATP) channels, and might be beneficial in the treatment of ischaemic cardiac conditions. Ischaemia is associated with an increase in the concentrations of intracellular nucleoside diphosphates and protons, two regulators of KATP channels. Since it is largely unknown whether these factors modulate the action of potassium channel openers, the aim of this study was to define the effects of ADP and intracellular acidification on KATP channel activation by nicorandil, a potassium channel opener currently in clinical use. METHODS: To measure ionic currents, the whole cell patch clamp technique was employed in guinea pig single ventricular myocytes. Induction of KATP channel current by pinacidil, a potassium channel opener chemically unrelated to nicorandil, was used for comparison. RESULTS: Nicorandil (300 microM) and pinacidil (100 microM) induced a time independent K+ current, which was inhibited by glibenclamide (6 microM), a selective blocker of KATP channels. ADP in the pipette solution was required for the nicorandil induced activation of KATP channels when the pH of the pipette solution was 7.2. In the absence of ADP, lowering the pH of the pipette solution to 6.5 was necessary for nicorandil to induce KATP channel current. For pinacidil to induce KATP channel current neither the addition of intracellular ADP nor acidification was required. CONCLUSIONS: Nicorandil may activate KATP channel current more effectively in conditions associated with a change in intracellular proton and ADP concentrations such as cardiac ischaemia.

Platelet-activating factor activates cardiac GK via arachidonic acid metabolites.

Platelet-activating factor (PAF), added to the bathing solution, stimulated the cardiac muscarinic K+ channel (KACh) in the cell-attached patch (no agonist in the pipette). The PAF-induced KACh channel activation was blocked by WEB2086, a PAF-receptor inhibitor, indicating that the PAF-receptor mediated the response. PAF-induced activation was prevented by nordihydroguaieretic acid, a lipoxygenase inhibitor, and AA-861, a 5-lipoxygenase inhibitor, but was not affected by indomethacin, a cyclo-oxygenase inhibitor. The PAF-induced KACh channel activity disappeared upon formation of inside-out patch. In this inside-out patch, intracellular GTP alone induced maximal channel reactivation, which was inhibited by GDP-beta S. These results suggest that 5-lipoxygenase metabolites of PAF-released arachidonic acid cause a persistent stimulation of GK but not the KACh channel itself, resulting in a receptor-independent activation of the KACh channel by GTP.

Phosphorylation-independent inhibition by intracellular cyclic nucleotides of brain inwardly rectifying K+ current expressed in Xenopus oocytes.

An inwardly rectifying K+ current, which was heterologously expressed in Xenopus oocytes, was inhibited by isoproterenol, a fadrenergic agonist. Poly(A)+ mRNA isolated from guinea-pig brain was injected into oocytes 2-3 days before experiments. Isoproterenol inhibition of the K+ current was time-and voltage-dependent: the inhibition became faster and more pronounced as the command voltage steps were applied to more negative potentials. This inhibition was prevented by propranolol. Dibutylyl cyclic (dB-c) AMP could mimic the effect of isoproterenol, while injection of the catalytic subunit of cAMP-dependent protein kinase into the oocytes did not affect the K+ current. Inhibitors of the protein kinases, WIPTIDE and H-8, did not prevent the inhibition by dB-cAMP. Furthermore, dB-cGMP also inhibited the K+ current in a similar time- and voltage-dependent manner. We propose that the phosphorylation-independent action of cyclic nucleotides mediates beta-adrenergic inhibition of brain inwardly rectifying K+ channels expressed in Xenopus oocytes.

Differential distribution of classical inwardly rectifying potassium channel mRNAs in the brain: comparison of IRK2 with IRK1 and IRK3.

Distribution of IRK2 inwardly rectifying potassium channel mRNA in the mouse brain was studied using in situ hybridization histochemistry and compared with those of other classical inwardly rectifying potassium channel (IRK1 and IRK3) mRNAs. All these IRK channel mRNAs were detected in neurons, but not in glial cells. Their distribution patterns in the brain were, however, quite divergent: IRK2 mRNA was detected extremely high in granule cells of cerebellum, relatively high in motor trigeminal nucleus and moderate in olfactory bulb, piriform cortex, cerebral cortex, CA1 through CA3 regions of hippocampus, dentate gyrus and pontine nucleus. On the other hand, IRK1 mRNA was expressed throughout whole brain but in particular subsets of neurons, and IRK3 mRNA was in forebrain. Expression of these three IRK mRNAs overlapped in hippocampus, olfactory bulb, and cerebral cortex. This differential distribution of IRK mRNAs suggests that each of these channels has its specific function in regulation of the excitability of brain neurons.

Molecular cloning, functional expression and localization of a novel inward rectifier potassium channel in the rat brain.

We have cloned a novel inward rectifier potassium channel from a rat brain cDNA library and designated it RB-IRK2. The rat brain cDNA library was screened using a fragment of the mouse macrophage IRK1 cDNA as a probe. The amino acid sequence of RB-IRK2 shares 70%, 40% and 45% identity to mouse IRK1, rat ROMK1 and rat GIRK1, respectively. Xenopus oocytes injected with cRNA derived from RB-IRK2 expressed a potassium current which showed inward-rectifying channel characteristics similar to the IRK1 current, but distinct from the ROMK1 or the GIRK1 currents. However, the localization of RB-IRK2 mRNA in rat tissues, assessed by the Northern blot analysis, differed from that of mouse IRK1. These results indicate that the IRK family is composed of multiple genes, which express in different tissues and therefore may play heterogenous functional roles in various organs, including rat central nervous system.

Molecular cloning and functional expression of a novel brain-specific inward rectifier potassium channel.

We have cloned a novel brain-specific inward rectifier K+ channel from a mouse brain cDNA library and designated it MB-IRK3. The mouse brain cDNA library was screened using a fragment of the mouse macrophage inward rectifier K+ channel (IRK1) cDNA as a probe. The amino acid sequence of MB-IRK3 shares 61% and 64% identity to MB-IRK1 and RB-IRK2, respectively. Xenopus oocytes injected with cRNA derived from this clone expressed a potassium current which showed inward-rectifying channel characteristics similar to MB-IRK1 and RB-IRK2 currents, but distinct from ROMK1 or GIRK1 current. However, the single channel conductance of MB-IRK3 was approximately 10 pS with 140 mM extracellular K+, which was distinct from that of MB-IRK1 (20 pS). MB-IRK3 mRNA expressed specifically in the forebrain, which clearly differed from MB-IRK1 and RB-IRK2 mRNAs. These results indicate that members of the IRK family with distinct electrophysiological properties express differentially and may play heterogenous functional roles in brain functions.

High-resolution immunogold cytochemistry indicates that AQP4 is concentrated along the basal membrane of parietal cell in rat stomach.

Gastric parietal cells secrete hydrochloric acid in stomach. Because the secreted HCl solution is isotonic with the plasma fluid, it should accompany the water transport across the membranes of parietal cells. Aquaporins (AQPs) are water channel proteins that play the central role in the cellular handling of water in various mammalian tissues. Using immunocytochemistry, we found that AQP4 was expressed only in parietal cells of rat gastric mucosa. Immunogold electron microscopy study further demonstrated that AQP4 was mostly localized at the basal membrane of parietal cells. In the basal membrane, AQP4 was prominently enriched on the portion contacting with the basement membrane surrounding gastric glands. These results suggest that the contact between basement membrane and basal membrane may generate the signal involved in the targeting of AQP4 in gastric parietal cells.