An inwardly rectifying potassium channel in the basolateral membrane of sheep parotid secretory cells

Summary Using whole-cell patch-clamp techniques, we demonstrate that sheep parotid secretory cells have both inwardly and outwardly rectifying currents. The outwardly rectifying current, which is blocked by 10 mmol/liter tetraethylammonium (TEA) applied extracellularly, is probably carried by the 250 pS Ca²⁺-and voltage-activated K⁺ (BK) channel which has been described in previous studies. In contrast, the inwardly rectifying current, which is also carried by K⁺ ions, is not sensitive to TEA. It is similar to the inwardly rectifying currents observed in many excitable tissues in that (i) its conductance is dependent on the square root of the extracellular K⁺, (ii) the voltage range over which it is activated is influenced by the extracellular K⁺ concentration and (iii) it is blocked by the addition of Cs⁺ ions (670 µmol/liter) to the bathing solution. Our previously published cell-attached patch studies have shown that the channel type most commonly observed in the basolateral membrane of unstimulated sheep parotid secretory cells is a K⁺ channel with a conductance of 30 pS and, in this study, we find that its conductance also depends on the square root of the extracellular K⁺ concentration. It thus seems likely that it carries the inwardly rectifying K⁺ current seen in the whole-cell studies.     

Regulation of Drosophila TRPC channels by lipid messengers

The Drosophila TRPC channels TRP and TRPL are the founding members of the TRP superfamily of ion channels, which are important components of calcium influx pathways in virtually all cells. The activation of these channels in the context of fly phototransduction is one of the few in vivo models for TRPC channel activation and has served as a paradigm for understanding TRPC function. TRP and TRPL are activated by G-protein coupled PIP₂ hydrolysis through a mechanism in which IP₃ receptor mediated calcium release seems dispensable. Recent analysis has provided compelling evidence that one or more PIP₂ generated lipid messengers, as well as PIP₂ itself, are essential for regulating TRP and TRPL activity. Evidence on the role of these lipid elements in regulating TRP and TRPL activity is discussed in this review.     

Regulation of an inwardly rectifying K channel in the T84 epithelial cell line by calcium,nucleotides and kinases

Agonists that elevate calcium in T₈₄ cells stimulate chloride secretion by activating K_BIC, an inwardly rectifying K channel in the basolateral membrane. We have studied the regulation of this channel by calcium, nucleotides and phosphorylation using patch clamp and short-circuit current (I _SC) techniques. Open probability (P ₀) was independent of voltage but declined spontaneously with time after excision. Rundown was slower if patches were excised into a bath solution containing ATP (10 m–5 mm), ATP (0.1 mm) + protein kinase A (PKA; 180 nm), or isobutylmethylxanthine (IBMX; 1 mm). Analysis of event durations suggested that the channel has at least two open and two closed states, and that rundown under control conditions is mainly due to prolongation of the long closed time. Channel activity was restimulated after rundown by exposure to ATP, the poorly hydrolyzable ATP analogue AMP-PNP, or ADP. Activity was further enhanced when PKA was added in the presence of MgATP, but only if free calcium concentration was elevated (400 nm). Nucleotide stimulation and inward rectification were both observed in nominally Mg-free solutions. cAMP modulation of basolateral potassium conductance in situ was confirmed by measuring currents generated by a transepithelial K gradient after permeabilization of the apical membrane using -toxin. Finally, protein kinase C (PKC) inhibited single K_BIC channels when it was added directly to excised patches. These results suggest that nonhydrolytic binding of nucleotides and phosphorylation by PKA and PKC modulate the responsiveness of the inwardly rectifying K channel to Ca-mediated secretagogues.This work was supported by the Canadian Cystic Fibrosis Foundation and the Medical Research Council of Canada. J.W.H. is a Chercheur-Boursier of the Fonds de la recherche en santé du Québec.     

Expression of an inwardly rectifying K channel from rat basophilic leukemia cell mRNA in Xenopus oocytes

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K⁺ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K⁺ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K⁺ equilibrium potential (E_K), (2)decreased in slope conductance near E_K, (3) was dependent on [K⁺]_o and (4) was blocked by external Ba²⁺ and Cs⁺. These properties were similar to those of the inwardly rectifying K⁺ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K⁺ channels.     

Structural and functional determinants of conserved lipid interaction domains of inward rectifying Kir6.2 channels

All members of the inward rectifiier K(+) (Kir) channel family are activated by phosphoinositides and other amphiphilic lipids. To further elucidate the mechanistic basis, we examined the membrane association of Kir6.2 fragments of K(ATP) channels, and the effects of site-directed mutations of these fragments and full-length Kir6.2 on membrane association and K(ATP) channel activity, respectively. GFP-tagged Kir6.2 COOH terminus and GFP-tagged pleckstrin homology domain from phospholipase C delta1 both associate with isolated membranes, and association of each is specifically reduced by muscarinic m1 receptor-mediated phospholipid depletion. Kir COOH termini are predicted to contain multiple beta-strands and a conserved alpha-helix (residues approximately 306-311 in Kir6.2). Systematic mutagenesis of D307-F315 reveals a critical role of E308, I309, W311 and F315, consistent with residues lying on one side of a alpha-helix. Together with systematic mutation of conserved charges, the results define critical determinants of a conserved domain that underlies phospholipid interaction in Kir channels.     

Regulation of cloned ATP-sensitive K channels by adenine nucleotides and sulfonylureas: interactions between SUR1 and positively charged domains on Kir6.2

K(ATP) channels, comprised of the pore-forming protein Kir6.x and the sulfonylurea receptor SURx, are regulated in an interdependent manner by adenine nucleotides, PIP2, and sulfonylureas. To gain insight into these interactions, we investigated the effects of mutating positively charged residues in Kir6.2, previously implicated in the response to PIP2, on channel regulation by adenine nucleotides and the sulfonylurea glyburide. Our data show that the Kir6.2 "PIP2-insensitive" mutants R176C and R177C are not reactivated by MgADP after ATP-induced inhibition and are also insensitive to glyburide. These results suggest that R176 and R177 are required for functional coupling to SUR1, which confers MgADP and sulfonylurea sensitivity to the K(ATP) channel. In contrast, the R301C and R314C mutants, which are also "PIP2-insensitive," remained sensitive to stimulation by MgADP in the absence of ATP and were inhibited by glyburide. Based on these findings, as well as previous data, we propose a model of the K(ATP) channel whereby in the presence of ATP, the R176 and R177 residues on Kir6.2 form a specific site that interacts with NBF1 bound to ATP on SUR1, promoting channel opening by counteracting the inhibition by ATP. This interaction is facilitated by binding of MgADP to NBF2 and blocked by binding of sulfonylureas to SUR1. In the absence of ATP, since K(ATP) channels are not blocked by ATP, they do not require the counteracting effect of NBF1 interacting with R176 and R177 to open. Nevertheless, channels in this state remain activated by MgADP. This effect may be explained by a direct stimulatory interaction of NBF2/MgADP moiety with another region of Kir6.2 (perhaps the NH2 terminus), or by NBF2/MgADP still promoting a weak interaction between NBF1 and Kir6.2 in the absence of ATP. The region delimited by R301 and R314 is not involved in the interaction with NBF1 or NBF2, but confers additional PIP2 sensitivity.     

Expression of an inwardly rectifying K+ channel from rat basophilic leukemia cell mRNA in Xenopus oocytes

Rat basophilic leukemia cells (RBL-2H3) have previously been shown to contain a single type of voltage-activated channel, namely an inwardly rectifying K⁺ channel, under normal recording conditions. Thus, RBL-2H3 cells seemed like a logical source of mRNA for the expression cloning of inwardly rectifying K⁺ channels. Injection of mRNA isolated from RBL-2H3 cells into Xenopus oocytes resulted in the expression of an inward current which (1) activated at potentials negative to the K⁺ equilibrium potential (E_K), (2)decreased in slope conductance near E_K, (3) was dependent on [K⁺]_o and (4) was blocked by external Ba²⁺ and Cs⁺. These properties were similar to those of the inwardly rectifying K⁺ current recorded from RBL-2H3 cells using whole-cell voltage clamp. Injection of size-fractionated mRNA into Xenopus oocytes revealed that the current was most strongly expressed from the fraction containing mRNA of approximately 4–5 kb. Expression of this channel represents a starting point for the expression cloning of a novel class of K⁺ channels.     

Synergistic activation of G protein-gated inwardly rectifying potassium channels by the betagamma subunits of G proteins and Na(+) and Mg(2+) ions.

Native and recombinant G protein-gated inwardly rectifying potassium (GIRK) channels are directly activated by the betagamma subunits of GTP-binding (G) proteins. The presence of phosphatidylinositol-bis-phosphate (PIP(2)) is required for G protein activation. Formation (via hydrolysis of ATP) of endogenous PIP(2) or application of exogenous PIP(2) increases the mean open time of GIRK channels and sensitizes them to gating by internal Na(+) ions. In the present study, we show that the activity of ATP- or PIP(2)-modified channels could also be stimulated by intracellular Mg(2+) ions. In addition, Mg(2+) ions reduced the single-channel conductance of GIRK channels, independently of their gating ability. Both Na(+) and Mg(2+) ions exert their gating effects independently of each other or of the activation by the G(betagamma) subunits. At high levels of PIP(2), synergistic interactions among Na(+), Mg(2+), and G(betagamma) subunits resulted in severalfold stimulated levels of channel activity. Changes in ionic concentrations and/or G protein subunits in the local environment of these K(+) channels could provide a rapid amplification mechanism for generation of graded activity, thereby adjusting the level of excitability of the cells.     

新型钾通道开放剂对心血管ATP-敏感性钾通道基因表达的调节作用

目的：研究脂肪胺类的新型钾通道开放剂（KCO）埃他卡林（Ipt）和氰胍类的KCO吡那地尔（Pin）对大鼠心血管ATP-敏感性钾通道（KATP）的亚基SUR1、SUR2、Kir6．1和Kir6．2等在mRNA水平的调节作用。方法：SD大鼠给药1周后处死并取组织,提取总RNA,利用反转录-聚合酶链式反应（RT-PCR）研究以上基因在mRNA水平的改变。结果：与正常对照相比,心脏组织中,Ipt和Pin对KATP的4个亚基在mRNA水平均无显著影响;主动脉平滑肌上,Ipt对4个亚基的mRNA表达无显著影响,但Pin可显著上调SUR2的mRNA表达;尾动脉平滑肌上,Ipt对Kit6．1／Kit6．2、Pin对SUR2／Kir6．1均有显著下调的作用。结论：心肌、大动脉平滑肌和小动脉平滑肌KATP基因表达的调控不同,Ipt选择性调节小动脉平滑肌Kit6．1／Kit6．2;Ipt对心血管KATP基因表达的调节作用不同于Pin。     

Protein kinase A-dependent activation of inward rectifier potassium channels by adenosine in rabbit coronary smooth muscle cells

We studied the effect of adenosine on the Ba(2+)-sensitive K(IR) channels in the smooth muscle cells isolated from the small-diameter (<100microm) coronary arteries of rabbit. Adenosine increased K(IR) currents in concentration-dependent manner (EC(50)=9.4+/-1.4microM, maximum increase of 153%). The adenosine-induced stimulation of K(IR) current was blocked by adenylyl cyclase inhibitor, SQ22536 and was mimicked by adenylyl cyclase activator, forskolin. The adenosine-induced increase of current was blocked by cyclic AMP-dependent protein kinase (PKA) inhibitors, KT 5720 and Rp-8-CPT-cAMPs. The adenosine-induced increase of K(IR) currents was blocked by an A(3)-selective antagonist MRS1334, while the antagonists of other subtypes (DPCPX for A(1), ZM241385 for A(2A), and alloxazine for A(2B)) were all ineffective. Furthermore, an A(3)-selective agonist, 2-Cl-IB-MECA induced increase of K(IR) currents. We also examined the effect of adenosine on coronary blood flow (CBF) rate by using the Langendorff-perfused heart. In the presence of glibenclamide to exclude the effects of ATP-sensitive K(+) (K(ATP)) channels, CBF was increased by adenosine (10microM), which was blocked by the addition of Ba(2+) (50microM). Above results suggest that adenosine increases K(IR) current via A(3) subtype through the activation of PKA in rabbit small-diameter coronary arterial smooth muscle cells.     

Agonist-induced localization of Gq-coupled receptors and G protein-gated inwardly rectifying K+ (GIRK) channels to caveolae determines receptor specificity of phosphatidylinositol 4,5-bisphosphate signaling

G protein-gated inwardly rectifying K(+) (GIRK) channels are parasympathetic effectors in cardiac myocytes that act as points of integration of signals from diverse pathways. Neurotransmitters and hormones acting on the Gq protein regulate GIRK channels by phosphatidylinositol 4,5-bisphosphate (PIP(2)) depletion. In previous studies, we found that endothelin-1, but not bradykinin, inhibited GIRK channels, even though both of them hydrolyze PIP(2) in cardiac myocytes, showing receptor specificity. The present study assessed whether the spatial organization of the PIP(2) signal into caveolar microdomains underlies the specificity of PIP(2)-mediated signaling. Using biochemical analysis, we examined the localization of GIRK and Gq protein-coupled receptors (GqPCRs) in mouse atrial myocytes. Agonist stimulation induced a transient co-localization of GIRK channels with endothelin receptors in the caveolae, excluding bradykinin receptors. Such redistribution was eliminated by caveolar disruption with methyl-β-cyclodextrin (MβCD). Patch clamp studies showed that the specific response of GIRK channels to GqPCR agonists was abolished by MβCD, indicating the functional significance of the caveolae-dependent spatial organization. To assess whether low PIP(2) mobility is essential for PIP(2)-mediated signaling, we blocked the cytoskeletal restriction of PIP(2) diffusion by latrunculin B. This abolished the GIRK channel regulation by GqPCRs without affecting their targeting to caveolae. These data suggest that without the hindered diffusion of PIP(2) from microdomains, PIP(2) loses its signaling efficacy. Taken together, these data suggest that specific targeting combined with restricted diffusion of PIP(2) allows the PIP(2) signal to be compartmentalized to the targets localized closely to the GqPCRs, enabling cells to discriminate between identical PIP(2) signaling that is triggered by different receptors.     

Pharmacology of mitochondrial potassium channels: dark side of the field

Mitochondrial potassium channels play an important role in cytoprotection. Potassium channels in the inner mitochondrial membrane are modulated by inhibitors and activators (potassium channel openers) previously described for plasma membrane potassium channels. The majority of mitochondrial potassium channel modulators exhibit a broad spectrum of off-target effects. These include uncoupling properties, inhibition of the respiratory chain and effects on cellular calcium homeostasis. Therefore, the rational application of channel inhibitors or activators is crucial to understanding the cellular consequences of mitochondrial channel inhibition or activation. Moreover, understanding their side-effects should facilitate the design of a specific mitochondrial channel opener with cytoprotective properties. In this review, we discuss the complex interactions of potassium channel inhibitors and activators with cellular structures.     

An inwardly rectifying whole cell current induced by Gq-coupled receptors

Ca(2+) influx across the plasma membrane after stimulation of G protein-coupled receptors is important for many physiological functions. Here we studied the regulation of an inwardly rectifying whole cell current and its putative role in Ca(2+) entry in Xenopus oocytes. Expression of P2Y(1) or M1 receptors in Xenopus oocytes elicited a characteristic inwardly rectifying current without receptor stimulation. This current displayed distinct activation and inactivation kinetics and was highly Ca(2+)-dependent. After stimulation of endogenous G(q)-coupled receptors in water-injected cells similar currents were observed. We therefore speculated that the current could be activated via Ca(2+) store depletion induced by constitutive stimulation of the IP(3) cascade in cells overexpressing G(q)-coupled receptors. Receptor-independent Ca(2+) store depletion also induced the current. In conclusion, this current is activated after store depletion suggesting a role in Ca(2+) entry after stimulation of G(q)-coupled receptors. Finally, our data do not support the proposed ionotropic properties of the P2Y(1) receptor.     

Stabilization of the activity of ATP-sensitive potassium channels by ion pairs formed between adjacent Kir6.2 subunits

ATP-sensitive potassium (KATP) channels are formed by the coassembly of four Kir6.2 subunits and four sulfonylurea receptor subunits (SUR). The cytoplasmic domains of Kir6.2 mediate channel gating by ATP, which closes the channel, and membrane phosphoinositides, which stabilize the open channel. Little is known, however, about the tertiary or quaternary structures of the domains that are responsible for these interactions. Here, we report that an ion pair between glutamate 229 and arginine 314 in the intracellular COOH terminus of Kir6.2 is critical for maintaining channel activity. Mutation of either residue to alanine induces inactivation, whereas charge reversal at positions 229 and 314 (E229R/R314E) abolishes inactivation and restores the wild-type channel phenotype. The close proximity of these two residues is demonstrated by disulfide bond formation between cysteine residues introduced at the two positions (E229C/R314C); disulfide bond formation abolishes inactivation and stabilizes the current. Using Kir6.2 tandem dimer constructs, we provide evidence that the ion pair likely forms by residues from two adjacent Kir6.2 subunits. We propose that the E229/R314 intersubunit ion pairs may contribute to a structural framework that facilitates the ability of other positively charged residues to interact with membrane phosphoinositides. Glutamate and arginine residues are found at homologous positions in many inward rectifier subunits, including the G-protein-activated inwardly rectifying potassium channel (GIRK), whose cytoplasmic domain structure has recently been solved. In the GIRK structure, the E229- and R314-corresponding residues are oriented in opposite directions in a single subunit such that in the tetramer model, the E229 equivalent residue from one subunit is in close proximity of the R314 equivalent residue from the adjacent subunit. The structure lends support to our findings in Kir6.2, and raises the possibility that a homologous ion pair may be involved in the gating of GIRKs.     

Activation of inwardly rectifying potassium channels by muscarinic receptor-linked G protein in isolated human ventricular myocytes

Muscarinic receptor-linked G protein, G _i , can directely activate the specific K⁺ channel (I _K(ACh)) in the atrium and in pacemaker tissues in the heart. Coupling of G _i to the K⁺ channel in the ventricle has not been well defined. G protein regulation of K⁺ channels in isolated human ventricular myocytes was examined using the patch-clamp technique. Bath application of 1 μm acetylcholine (ACh) reversibly shortened the action potential duration to 74.4 ± 12.1% of control (at 90% repolarization, mean ±sd, n= 8) and increased the whole-cell membrane current conductance without prior β-adrenergic stimulation in human ventricular myocytes. The ACh effect was reversed by atropine (1 μm). In excised inside-out patch configurations, application of GTPγS (100 μm) to the bath solution (internal surface) caused activation of I _K(ACh) and/or the background inwardly-rectifying K⁺ channel (I _K1) in ventricular cell membranes. I _K(ACh) exhibited rapid gating behavior with a slope conductance of 44 ± 2 pS (n= 25) and a mean open lifetime of 1.8 ± 0.3 msec (n= 21). Single channel activity of GTPγS-activated I _K1 demonstrated long-lasting bursts with a slope conductance of 30 ± 2 pS (n= 16) and a mean open lifetime of 36.4 ± 4.1 msec (n= 12). Unlike I _K(ACh), G protein-activated I _K1 did not require GTP to maintain channel activity, suggesting that these two channels may be controlled by G proteins with different underlying mechanisms. The concentration of GTP at half-maximal channel activation was 0.22 μm in I _K(ACh) and 1.2 μm in I _K1. Myocytes pretreated with pertussis toxin (PTX) prevented GTP from activating these channels, indicating that muscarinic receptor-linked PTX-sensitive G protein, G _i , is essential for activation of both channels. G protein-activated channel characteristics from patients with terminal heart failure did not differ from those without heart failure or guinea pig. These results suggest that ACh can shorten the action potential by activating I _K(ACh) and I _K1 via muscarinic receptor-linked G _i proteins in human ventricular myocytes. Received: 23 September 1996/Revised: 18 December 1996     

Action potential-like responses due to the inward rectifying potassium channel

Summary This paper describes experiments carried out in the absence of sodium and calcium in the external solution. Frog atrial trabeculae were stimulated in current clamp with the double sucrose gap technique. The voltage responses looked like slow action potentials with a clear threshold. These responses were not suppressed in the presence of EGTA, in the presence of sodium or calcium channel blockers, or when sulfate ions replaced chloride. Guinea pig isolated ventricular myocytes were studied in whole cell clamp mode with a pathch pipette. Under current clamp, they displayed also voltage responses with a threshold. These responses were resistant to cadmium (5mm), and were suppressed by barium (0.5mm). A negative slope conductance is required to take into account these results. The membrane current in current clamp can be estimated by plotting the response in the phase plane. This analysis shows that on both types of preparations, the current responsible for the negative slope is not time dependent. This current is suppressed by barium. It can be concluded that it is the outward current flowing through the inward rectifying potassium channels. To confirm this hypothesis, data obtained in voltage clamp on the same preparations were introduced into a computer model to predict the response in current clamp. The results were in agreement with the experiments. Similar responses could be recorded and analyzed on skeletal muscle in isotonic potassium solution. These results show that the inward rectifier can induce by itself properties looking like excitability on different preparations. The physiological significance of this effect in normal conditions is discussed. The voltage responses described in this paper look similar to the slow action potentials on heart, which are sensitive to modifications of the calcium channels, but also of the potassium channels. Some implications in cardiac pharmacology are discussed.     

The inwardly rectifying potassium current of embryonic chick hepatocytes

The single channel and whole-cell properties of an inward, rectifying potassium current in cultured embryonic chick hepatocytes were studied at 20°C. In cell-attached patches, channels open upon membrane hyperpolarization and are present in about 90% of cellattached patches. With 145 mm potassium in the pipette, inward current has a slope conductance of 80 pS. The conductance is not a linear function of the external potassium concentration. Current saturates at high external potassium and has a Michaelis-Menten affinity constant of 275 mm potassium. Substitution of gluconate for chloride in the external solution has no significant effect on conductance, and the reversal potential shifts approximately 18 mV with a change in external potassium from 72.5 to 145 mm indicating potassium selectivity. Channel openings are characterized by multiple brief closures during a burst. The channel is inhibited by external cesium in a concentration-dependent manner. Block is characterized by an increased frequency of transient closures. Whole-cell dialysis with 145 mm CsCl of cells bathed in 145 mm KCl reveals time-independent inward currents that reverse at 0 mV in response to 200 msecvoltage steps. Although voltage ramps evoke currents that are 75% potassium dependent and cesium sensitive, the mean chord conductance (425 pS) indicates that less than five channels are open at any instant. We suggest that the inwardly rectifying potassium channel is partially inactivated in the dialysed hepatocyte.We thank K. Paula S. Hettiaratchi and Eunice Y. Wang for expert cell isolation and culture technique, and the Natural Sciences and Engineering Research Council of Canada for supporting this work.     

Phosphoinositide regulation of non-canonical transient receptor potential channels

Transient receptor potential (TRP) channels are involved in a wide range of physiological processes, and characterized by diverse activation mechanisms. Phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate [PIP₂, or PtdIns(4,5)P₂] recently emerged as regulators of many TRP channels. Several TRP channels require PIP₂ for activity, and depletion of the lipid inhibits them. For some TRP channels, however, phosphoinositide regulation seems more complex, both activating and inhibitory effects have been reported. This review will discuss phosphoinositide regulation of members of the TRPM (Melastatin), TRPV (Vanilloid), TRPA (Ankyrin) and TRPP (Polycystin) families. Lipid regulation of TRPC (Canonical) channels is discussed elsewhere in this volume.     

Inhibition of the cardiac inward rectifier potassium currents by KB-R7943

KB-R7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea) was developed as a specific inhibitor of the sarcolemmal sodium–calcium exchanger (NCX) with potential experimental and therapeutic use. However, KB-R7943 is shown to be a potent blocker of several ion currents including inward and delayed rectifier K⁺ currents of cardiomyocytes. To further characterize KB-R7943 as a blocker of the cardiac inward rectifiers we compared KB-R7943 sensitivity of the background inward rectifier (I_K1) and the carbacholine-induced inward rectifier (I_KACh) currents in mammalian (Rattus norvegicus; rat) and fish (Carassius carassius; crucian carp) cardiac myocytes. The basal I_K1 of ventricular myocytes was blocked with apparent IC50-values of 4.6 × 10^− 6 M and 3.5 × 10^− 6 M for rat and fish, respectively. I_KACh was almost an order of magnitude more sensitive to KB-R7943 than I_K1 with IC₅₀-values of 6.2 × 10^− 7 M for rat and 2.5 × 10^− 7 M for fish. The fish cardiac NCX current was half-maximally blocked at the concentration of 1.9–3 × 10^− 6 M in both forward and reversed mode of operation. Thus, the sensitivity of three cardiac currents to KB-R7943 block increases in the order I_K1 ~ I_NCX < I_KACh. Therefore, the ability of KB-R7943 to block inward rectifier potassium currents, in particular I_KACh, should be taken into account when interpreting the data with this inhibitor from in vivo and in vitro experiments in both mammalian and fish models.     

ATP-sensitive potassium channels and vascular function

Fluorescence-based approaches provide powerful techniques to directly report structural dynamics underlying gating processes in Shaker K_V channels. Here, following on from work carried out in Shaker channels, we have used voltage clamp fluorimetry for the first time to study voltage sensor motions in mammalian K_V1.5 channels, by attaching TMRM fluorescent probes to substituted cysteine residues in the S3-S4 linker of K_V1.5 (A397C). Compared with the Shaker channel, there are significant differences in the fluorescence signals that occur on activation of the channel. In addition to a well-understood fluorescence quenching signal associated with S4 movement, we have recorded a unique partial recovery of fluorescence after the quenching that is attributable to gating events at the outer pore mouth,¹ that is not seen in Shaker despite significant homology between it and Kv1.5 channels in the S5-P loop-S6 region. Extracellular potassium is known to modulate C-type inactivation in Shaker and K_V channels at sites in the outer pore mouth, and so here we have measured the concentration-dependence of potassium effects on the fluorescence recovery signals from A397C. Elevation of extracellular K⁺ inhibits the rapid fluorescence recovery, with complete abolition at 99 mM K⁺, and an IC₅₀ of 29 mM K⁺_o. These experiments suggest that the rapid fluorescence recovery reflects early gating movements associated with inactivation, modulated by extracellular K⁺, and further support the idea that outer pore motions occur rapidly after K_V1.5 channel opening and can be observed by fluorophores attached to the S3-S4 linker.