Identification and characterization of a novel bacterial ATP-sensitive K<Superscript>+</Superscript> channel

Five bacterial species that are most likely to have putative prokaryotic inward rectifier K⁺ (Kir) channels were selected by in silico sequence homology and membrane topology analyses with respect to the number of transmembrane domains (TMs) and the presence of K⁺ selectivity filter and/or ATP binding sites in reference to rabbit heart inward rectifier K⁺ channel (Kir6.2). A dot blot assay with genomic DNAs when probed with whole rabbit Kir6.2 cDNA further supported the in silico analysis by exhibiting a stronger hybridization in species with putative Kir’s compared to one without a Kir. Among them, Chromobacterium violaceum gave rise to a putative Kir channel gene, which was PCR-cloned into the bacterial expression vector pET30b(+), and its expression was induced in Escherichia coli and confirmed by gel purification and immunoblotting. On the other hand, this putative bacterial Kir channel was functionally expressed in Xenopus oocytes and its channel activity was measured electrophysiologically by using two electrode voltage clamping (TEVC). Results revealed a K⁺ current with characteristics similar to those of the ATP-sensitive K⁺ (K-ATP) channel. Collectively, cloning and functional characterization of bacterial ion channels could be greatly facilitated by combining the in silico analysis and heterologous expression in Xenopus oocytes.     

Characterization of Single Inward Rectifier Potassium Channels from Embryonic Xenopus laevis Myocytes

Single inward rectifier K⁺ channels were studied in Xenopus laevis embryonic myocytes. We have characterized in detail the channel which is most frequently observed (Kir) although we routinely observe three other smaller current levels with the properties of inward rectifier K⁺ channels (Kir_(0.3), Kir_(0.5) and Kir_(0.7)). For Kir, slope conductances of inward currents were 10.3, 20.3, and 27.9 pS, in 60, 120 and 200 mM [K⁺] _o respectively. Extracellular Ba²⁺ blocked the normally high channel activity in a concentration-dependent manner (K _A = 7.8 μm, −90 mV). In whole-cell recordings of inward rectifier K⁺ current, marked voltage dependence of Ba²⁺ block over the physiological range of potentials was observed. We also examined current rectification. Following step depolarizations to voltages positive to E _K , outward currents through Kir channels were not observed even when the cytoplasmic face of excised patches were exposed to Mg²⁺-free solution at pH 9.1. This was probably also true for Kir_(0.3), Kir_(0.5) and Kir_(0.7) channels. We then examined the possibility of modulation of Kir channel activity and found neither ATP nor GTP-γS had any effect on Kir channel activity when added to the solution perfusing the cytoplasmic face of a patch. Kinetic analysis revealed Kir channels with a single open state (mean dwell time 72 msec) and two closed states (time constants 1.4, 79 msec). These results suggest that the native Kir channels of Xenopus myocytes have similar properties to the cloned strong inward rectifier K⁺ channels, in terms of conductance, kinetics and barium block but does show some differences in the effects of modulators of channel activity. Furthermore, skeletal muscle may contain either different inward rectifier channels or a single-channel type which can exist in stable subconductance states. Received: 16 September 1996/Revised: 14 March 1997     

Kir4.1/Kir5.1 channels possess strong intrinsic inward rectification determined by a voltage-dependent K+-flux gating mechanism

Inwardly rectifying potassium (Kir) channels are broadly expressed in both excitable and nonexcitable tissues, where they contribute to a wide variety of cellular functions. Numerous studies have established that rectification of Kir channels is not an inherent property of the channel protein itself, but rather reflects strong voltage dependence of channel block by intracellular cations, such as polyamines and Mg²⁺. Here, we identify a previously unknown mechanism of inward rectification in Kir4.1/Kir5.1 channels in the absence of these endogenous blockers. This novel intrinsic rectification originates from the voltage-dependent behavior of Kir4.1/Kir5.1, which is generated by the flux of potassium ions through the channel pore; the inward K⁺-flux induces the opening of the gate, whereas the outward flux is unable to maintain the gate open. This gating mechanism powered by the K⁺-flux is convergent with the gating of PIP₂ because, at a saturating concentration, PIP₂ greatly reduces the inward rectification. Our findings provide evidence of the coexistence of two rectification mechanisms in Kir4.1/Kir5.1 channels: the classical inward rectification induced by blocking cations and an intrinsic voltage-dependent mechanism generated by the K⁺-flux gating.     

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K⁺ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue^1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (G_i and G_o) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K⁺ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders³. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations³.Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC₄(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K⁺ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K⁺ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry⁴ or radiotracer analysis⁵, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.     

Cell proliferation,potassium channels,polyamines and their interactions: a mini review

Polyamines, which are obligatory molecules involved in cell cycling and proliferation, are subject to a change in their free intracellular concentrations during the cell cycle. Potassium (K⁺) channels are also considered, but less well recognized, to be necessary for cell proliferation by either hyperpolarizing or depolarizing cells during the cell cycle. A block of polyamine synthesis as well as block or knockout of K⁺ channels can halt cell proliferation. K⁺ channels like BK (maxi calcium (Ca²⁺)-activated K⁺), Kir (inward rectifier), M-type K⁺-and TASK (two-pore domain K⁺) channels or the delayed rectifier K⁺ channels are modulated in their electrical properties by polyamines. Polyamines are most effective in blocking these channels when applied to the intracellular face of these channels except for TASK channels where they act only from the extracellular side. Quinidine, a general K⁺ channel blocker, was found to reduce putrescine concentrations, to block the ornithine decarboxylase and halt cell proliferation. From these results, the question arises if there is an interaction between polyamines, K⁺ channels and proliferation. It might be speculated that a decrease of intracellular polyamines allows more K⁺ channels to be active, thus inducing hyperpolarization, while an increase of the polyamine concentration may block K⁺ channel activity leading to depolarization of the membrane potential. On the other hand, a block or a deletion of K⁺ channels may cause a decrease of the polyamine concentration in cells. More research is needed to test these hypotheses.     

Dual regulation of renal Kir7.1 potassium channels by protein Kinase A and protein Kinase C

The renal inward rectifier potassium channel Kir7.1 has been proposed to be functionally important for tubular K⁺ recycling and secretion. This study investigated the regulation of Kir7.1 by PKA and PKC.Cloned human Kir7.1 channels were expressed heterologously in Xenopus oocytes. After pharmacological PKC activation, Kir7.1 currents were strongly inhibited. Co-application of PKC inhibitors attenuated this effect. Inactivation of PKC consensus sites also strongly attenuated the effect with a single site (²⁰¹S) being essential for almost the total PKC sensitivity. In contrast, PKA activation induced an increase of Kir7.1 currents. This effect was absent in mutant Kir7.1 channels lacking PKA consensus site ²⁸⁷S.In summary, this study demonstrates the dual regulation of Kir7.1 channel function by PKA and PKC. Structurally, these regulations depend on two key residues in the C-terminal channel domain (^Ser201 for PKC and ^Ser287 for PKA).     

Localization and role of inward rectifier K+ channels in Malpighian tubules of the yellow fever mosquito Aedes aegypti

Malpighian tubules of adult female yellow fever mosquitoes Aedes aegypti express three inward rectifier K⁺ (Kir) channel subunits: AeKir1, AeKir2B and AeKir3. Here we 1) elucidate the cellular and membrane localization of these three channels in the Malpighian tubules, and 2) characterize the effects of small molecule inhibitors of AeKir1 and AeKir2B channels (VU compounds) on the transepithelial secretion of fluid and electrolytes and the electrophysiology of isolated Malpighian tubules. Using subunit-specific antibodies, we found that AeKir1 and AeKir2B localize exclusively to the basolateral membranes of stellate cells and principal cells, respectively; AeKir3 localizes within intracellular compartments of both principal and stellate cells. In isolated tubules bathed in a Ringer solution containing 34 mM K⁺, the peritubular application of VU590 (10 μM), a selective inhibitor of AeKir1, inhibited transepithelial fluid secretion 120 min later. The inhibition brings rates of transepithelial KCl and fluid secretion to 54% of the control without a change in transepithelial NaCl secretion. VU590 had no effect on the basolateral membrane voltage (V_bl) of principal cells, but it significantly reduced the cell input conductance (g_in) to values 63% of the control within ∼90 min. In contrast, the peritubular application of VU625 (10 μM), an inhibitor of both AeKir1 and AeKir2B, started to inhibit transepithelial fluid secretion as early as 60 min later. At 120 min after treatment, VU625 was more efficacious than VU590, inhibiting transepithelial KCl and fluid secretion to ∼35% of the control without a change in transepithelial NaCl secretion. Moreover, VU625 caused the V_bl and g_in of principal cells to respectively drop to values 62% and 56% of the control values within only ∼30 min. Comparing the effects of VU590 with those of VU625 allowed us to estimate that AeKir1 and AeKir2B respectively contribute to 46% and 20% of the transepithelial K⁺ secretion when the tubules are bathed in a Ringer solution containing 34 mM K⁺. Thus, we uncover an important role of AeKir1 and stellate cells in transepithelial K⁺ transport under conditions of peritubular K⁺ challenge. The physiological role of AeKir3 in intracellular membranes of both stellate and principal cells remains to be determined.     

Cloning and functional characterization of inward-rectifying potassium (Kir) channels from Malpighian tubules of the mosquito Aedes aegypti

Inward-rectifying K⁺ (Kir) channels play critical physiological roles in a variety of vertebrate cells/tissues, including the regulation of membrane potential in nerve and muscle, and the transepithelial transport of ions in osmoregulatory epithelia, such as kidneys and gills. It remains to be determined whether Kir channels play similar physiological roles in insects. In the present study, we sought to 1) clone the cDNAs of Kir channel subunits expressed in the renal (Malpighian) tubules of the mosquito Aedes aegypti, and 2) characterize the electrophysiological properties of the cloned Kir subunits when expressed heterologously in oocytes of Xenopus laevis. Here, we reveal that three Kir subunits are expressed abundantly in Aedes Malpighian tubules (AeKir1, AeKir2B, and AeKir3); each of their full-length cDNAs was cloned. Heterologous expression of the AeKir1 or the AeKir2B subunits in Xenopus oocytes elicits inward-rectifying K⁺ currents that are blocked by barium. Relative to the AeKir2B-expressing oocytes, the AeKir1-expressing oocytes 1) produce larger macroscopic currents, and 2) exhibit a modulation of their conductive properties by extracellular Na⁺. Attempts to functionally characterize the AeKir3 subunit in Xenopus oocytes were unsuccessful. Lastly, we show that in isolated Aedes Malpighian tubules, the cation permeability sequence of the basolateral membrane of principal cells (Tl⁺ > K⁺ > Rb⁺ > NH₄⁺) is consistent with the presence of functional Kir channels. We conclude that in Aedes Malpighian tubules, Kir channels contribute to the majority of the barium-sensitive transepithelial transport of K⁺.     

Mechanism of high K+ and Tl+ uptake in cultured human glioma cells

Summary 1. The aim of this study was to elucidate if the K⁺ uptake was higher in cultured human glioma cells than in cells from other malignant tumors and to analyze the importance of membrane potential and K⁺ channels for the uptake.2. K⁺ transport properties were studied with the isotopes⁴²K and the K-analogue²⁰¹Tl.3. Comparison with cultured cells from other malignant tumors showed that the specific steady-state accumulation of Tl⁺ was significantly higher in glioma cells (U-251MG and Tp-378MG).4. In Ringer's solution at 37°C the rates of K⁺ and Tl⁺ uptake were both inhibited by about 55% in ouabain and 60% in furosemide, bumetanide, or Na⁺-or Cl^–-free medium. This indicated that the routes for K⁺ and Tl⁺ uptake were similar and due to Na,K-ATPase-dependent transport and to Na-K-Cl cotransport.5. About 10% of the uptake was neither ouabain nor bumetanide sensitive. Ba²⁺, which is known to block inward-rectifying K⁺ channels and to depolarize glial cells, and other K⁺ channel blockers (Cs⁺ and bupivacaine), had no effect on Tl⁺ uptake.6. Metabolic inhibition with dinitrophenol reduced the uptake rate to 17%.7. The washout of Tl⁺ was unaffected by bumetanide and K⁺ channel blockers, but dinitrophenol caused a transient increase of 75%, an effect which persisted in the presence of K⁺ channel blockers.8. It was concluded that the high specific K⁺ and Tl⁺ accumulation in cultured human glioma cells was due not to the presence of inwardly rectifying K⁺ channels or other identified K⁺ channels, but to Na,K-ATPase dependent transport and Na-K-Cl cotransport.     

Gating Properties of Inward-Rectifier Potassium Channels: Effects of Permeant Ions

Two inward-rectifier K⁺ channels, ROMK2 (Kir1.1b) and IRK1 (Kir2.1), were expressed in Xenopus oocytes and their gating properties were studied in cell-attached membrane patches. The gating properties depended strongly on the ion being conducted (K⁺, NH₄ ⁺, Rb⁺, or Tl⁺), suggesting tight coupling between permeation and gating. Mean open times were strongly dependent on the nature of the conducted ion. For ROMK2 the order from the longest to the shortest times was K⁺ > Rb⁺ > Tl⁺ > NH₄ ⁺. For IRK1 the sequence was K⁺ > NH₄ ⁺ > Tl⁺. In both cases the open times decreased monotonically as the membrane voltage was hyperpolarized. Both the absolute values and the voltage dependence of closed times were dependent on the conducted species. ROMK2 showed a single closed state whose mean lifetimes were biphasic functions of voltage. The maxima were at various voltages for different ions. IRK1 had at least two closed states whose lifetimes decreased monotonically with K⁺, increased monotonically with Tl⁺, and were relatively constant with NH₄ ⁺ as the conducted ion. We explain the ion-dependence of gating by assuming that the ions bind to a site within the permeation pathway, resulting in a stable, ion-dependent, closed state of the channel. The patterns of voltage-dependence of closed-state lifetimes, which are specific for different ions, can be explained by variations in the rate at which the bound ions leave the pore toward the inside or the outside of the cell. Received: 18 April 2001/Revised: 28 June 2001     

Mislocalization of K channels causes the renal salt wasting in EAST/SeSAME syndrome

The Kir4.1/Kir5.1 channel mediates basolateral K⁺ recycling in renal distal tubules; this process is critical for Na⁺ reabsorption at the tubules. Mutations in Kir4.1 are associated with EAST/SeSAME syndrome, a genetic disorder characterized by renal salt wasting. In this study, we found that MAGI-1 anchors Kir4.1 channels (Kir4.1 homomer and Kir4.1/Kir5.1 heteromer) and contributes to basolateral K⁺ recycling. The Kir4.1 A167V mutation associated with EAST/SeSAME syndrome caused mistrafficking of the mutant channels and inhibited their expression on the basolateral surface of tubular cells. These findings suggest mislocalization of the Kir4.1 channels contributes to renal salt wasting.     

Burst Kinetics of Co-expressed Kir6.2/SUR1 Clones: Comparison of Recombinant with Native ATP-sensitive K+ Channel Behavior

Co-expression of clones encoding Kir6.2, a K⁺ inward rectifier, and SUR1, a sulfonylurea receptor, reconstitutes elementary features of ATP-sensitive K⁺ (K_ATP) channels. However, the precise kinetic properties of Kir6.2/SUR1 clones remain unknown. Herein, intraburst kinetics of Kir6.2/SUR1 channel activity, heterologously co-expressed in COS cells, displayed mean closed times from 0.7 ± 0.1 to 0.4 ± 0.03 msec, and from 0.4 ± 0.1 to 2.0 ± 0.2 msec, and mean open times from 1.9 ± 0.4 to 4.5 ± 0.8 msec, and from 12.1 ± 2.4 to 5.0 ± 0.2 msec between −100 and −20 mV, and +20 to +80 mV, respectively. Burst duration for Kir6.2/SUR1 activity was 17.9 ± 1.8 msec with 5.6 ± 1.5 closings per burst. Burst kinetics of the Kir6.2/SUR1 activity could be fitted by a four-state kinetic model defining transitions between one open and three closed states with forward and backward rate constants of 1905 ± 77 and 322 ± 27 sec⁻¹ for intraburst, 61.8 ± 6.6 and 23.9 ± 5.8 sec⁻¹ for interburst, 12.4 ± 6.0 and 13.6 ± 2.9 sec⁻¹ for intercluster events, respectively. Intraburst kinetic properties of Kir6.2/SUR1 clones were essentially indistinguishable from pancreatic or cardiac K_ATP channel phenotypes, indicating that intraburst kinetics per se were insufficient to classify recombinant Kir6.2/SUR1 amongst native K_ATP channels. Yet, burst kinetic behavior of Kir6.2/SUR1 although similar to pancreatic, was different from that of cardiac K_ATP channels. Thus, expression of Kir6.2/SUR1 proteins away from the pancreatic micro-environment, confers the burst kinetic identity of pancreatic, but not cardiac K_ATP channels. This study reports the kinetic properties of Kir6.2/SUR1 clones which could serve in the further characterization of novel K_ATP channel clones. Received: 12 March 1997/Revised: 5 May 1997     

Differential modulation of cardiac potassium channels by Grb adaptor proteins

Scaffolding growth factor receptor-bound (Grb) adaptor proteins are components of macromolecular signaling complexes at the plasma membrane and thus are putative regulators of ion channel activity. The present study aimed to define the impact of Grb adaptor proteins on the function of cardiac K⁺ channels. To this end channel proteins were coinjected with the adaptor proteins in Xenopus oocytes and channel activity analyzed with two-electrode voltage-clamp. It is shown that coexpression of Grb adaptor proteins can reduce current amplitudes of coexpressed channels. Grb7 and 10 significantly inhibited functional currents generated by hERG, Kv1.5 and Kv4.3 channels. Only Grb10 significantly inhibited KCNQ1/KCNE1 K⁺ channels, and only Grb7 reduced Kir2.3 activity, whereas neither Grb protein significantly affected the closely related Kir2.1 and Kir2.2 channels. The present observations for the first time provide evidence for a selective and modulatory role of Grb adaptor proteins in the functional expression of cardiac K⁺ channels.     

V-ATPase B1-subunit promoter drives expression of EGFP in intercalated cells of kidney, clear cells of epididymis and airway cells of lung in transgenic mice

Inward rectifier K⁺ channels (Kir) are a significant determinant of endothelial cell (EC) membrane potential, which plays an important role in endothelium-dependent vasodilatation. In the present study, several complementary strategies were applied to determine the Kir2 subunit composition of human aortic endothelial cells (HAECs). Expression levels of Kir2.1, Kir2.2, and Kir2.4 mRNA were similar, whereas Kir2.3 mRNA expression was significantly weaker. Western blot analysis showed clear Kir2.1 and Kir2.2 protein expression, but Kir2.3 protein was undetectable. Functional analysis of endothelial inward rectifier K⁺ current (I_K) demonstrated that 1) I_K current sensitivity to Ba²⁺ and pH were consistent with currents determined using Kir2.1 and Kir2.2 but not Kir2.3 and Kir2.4, and 2) unitary conductance distributions showed two prominent peaks corresponding to known unitary conductances of Kir2.1 and Kir2.2 channels with a ratio of 4:6. When HAECs were transfected with dominant-negative (dn)Kir2.x mutants, endogenous current was reduced 50% by dnKir2.1 and 85% by dnKir2.2, whereas no significant effect was observed with dnKir2.3 or dnKir2.4. These studies suggest that Kir2.2 and Kir2.1 are primary determinants of endogenous K⁺ conductance in HAECs under resting conditions and that Kir2.2 provides the dominant conductance in these cells. potassium channels; inward rectifier potassium channel     

Modulation of Kir4.2 rectification properties and pHi-sensitive run-down by association with Kir5.1

Inwardly rectifying K⁺ channels (Kir) comprise seven subfamilies that can be subdivided further on the basis of cytosolic pH (pHi) sensitivity, rectification strength and kinetics, and resistance to run-down. Although distinct residues within each channel subunit define these properties, heteromeric association with other Kir subunits can modulate them. We identified such an effect in the wild-type forms of Kir4.2 and Kir5.1 and used this to further understand how the functional properties of Kir channels relate to their structures. Kir4.2 and a Kir4.2-Kir5.1 fusion protein were expressed in HEK293 cells. Inward currents from Kir4.2 were stable over 10 min and pHi-insensitive (pH 6 to 8). Conversely, currents from Kir4.2-Kir5.1 exhibited a pHi-sensitive run-down at slightly acidic pHi. At pHi 7.2, currents in response to voltage steps positive to E_K were essentially time independent for Kir4.2 indicating rapid block by Mg²⁺. Coexpression with Kir5.1 significantly increased the blocking time constant, and increased steady-state outward current characteristic of weak rectifiers. Recovery from blockade at negative potentials was voltage dependent and 2 to 10 times slower in the homomeric channel. These results show that Kir5.1 converts Kir4.2 from a strong to a weak rectifier, rendering it sensitive to pHi, and suggesting that Kir5.1 plays a role in fine-tuning Kir4.2 activity.     

Identification of a Novel Bacterial K<Superscript>+</Superscript> Channel

In an attempt to explore unknown K⁺ channels in mammalian cells, especially ATP-sensitive K⁺ (K_ATP) channels, we compared the sequence homology of Kir6.1 and Kir6.2, two pore-forming subunits of mammalian K_ATP channel genes, with bacterial genes that code for selective proteins with confirmed or putative ion transport properties. BLAST analysis revealed that a prokaryotic gene (ydfJ) expressed in Escherichia coli K12 strain shared 8.6% homology with Kir6.1 and 8.3% with Kir6.2 genes. Subsequently, we cloned and sequenced ydfJ gene from E. coli K12 and heterologously expressed it in mammalian HEK-293 cells. The whole-cell patch-clamp technique was used to record ion channel currents generated by ydfJ-encoded protein. Heterologous expression of ydfJ gene in HEK-293 cells yielded a novel K⁺ channel current that was inwardly rectified and had a reversal potential close to K⁺ equilibrium potential. The expressed ydfJ channel was blocked reversibly by low concentration of barium in a dose-dependent fashion. Specific K_ATP channel openers or blockers did not alter the K⁺ current generated by ydfJ expression alone or ydfJ coexpressed with rvSUR1 or rvSUR2B subunits of K_ATP channel complex. Furthermore, this coexpressed ydfJ/rvSUR1 channels were not inhibited by ATP dialysis. On the other hand, ydfJ K⁺ currents were inhibited by protopine (a nonspecific K⁺ channel blocker) but not by dofetilide (a HERG channel blocker). In summary, heterologously expressed prokaryotic ydfJ gene formed a novel functional K⁺ channel in mammalian cells.     

Inward Rectifier K+ Currents Are Regulated by CaMKII in Endothelial Cells of Primarily Cultured Bovine Pulmonary Arteries

Endothelium lines the interior surface of vascular walls and regulates vascular tones. The endothelial cells sense and respond to chemical and mechanical stimuli in the circulation, and couple the stimulus signals to vascular smooth muscles, in which inward rectifier K⁺ currents (Kir) play an important role. Here we applied several complementary strategies to determine the Kir subunit in primarily cultured pulmonary arterial endothelial cells (PAECs) that was regulated by the Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII). In whole-cell voltage clamp, the Kir currents were sensitive to micromolar concentrations of extracellular Ba²⁺. In excised inside-out patches, an inward rectifier K⁺ current was observed with single-channel conductance 32.43 ± 0.45 pS and P_open 0.27 ± 0.04, which were consistent with known unitary conductance of Kir 2.1. RT-PCR and western blot results showed that expression of Kir 2.1 was significantly stronger than that of other subtypes in PAECs. Pharmacological analysis of the Kir currents demonstrated that insensitivity to intracellular ATP, pinacidil, glibenclamide, pH, GDP-β-S and choleratoxin suggested that currents weren’t determined by K_ATP, Kir2.3, Kir2.4 and Kir3.x. The currents were strongly suppressed by exposure to CaMKII inhibitor W-7 and KN-62. The expression of Kir2.1 was inhibited by knocking down CaMKII. Consistently, vasodilation was suppressed by Ba²⁺, W-7 and KN-62 in isolated and perfused pulmonary arterial rings. These results suggest that the PAECs express an inward rectifier K⁺ current that is carried dominantly by Kir2.1, and this K⁺ channel appears to be targeted by CaMKII-dependent intracellular signaling systems.     

The mechano-gated K<Subscript>2P</Subscript> channel TREK-1

The versatility of neuronal electrical activity is largely conditioned by the expression of different structural and functional classes of K⁺ channels. More than 80 genes encoding the main K⁺ channel alpha subunits have been identified in the human genome. Alternative splicing, heteromultimeric assembly, post-translational modification and interaction with auxiliary regulatory subunits further increase the molecular and functional diversity of K⁺ channels. Mammalian two-pore domain K⁺ channels (K_2P) make up one class of K⁺ channels along with the inward rectifiers and the voltage- and/or calcium-dependent K⁺ channels. Each K_2P channel subunit is made up of four transmembrane segments and two pore-forming (P) domains, which are arranged in tandem and function as either homo- or heterodimeric channels. This novel structural arrangement is associated with unusual gating properties including “background” or “leak” K⁺ channel activity, in which the channels show constitutive activity at rest. In this review article, we will focus on the lipid-sensitive mechano-gated K_2P channel TREK-1 and will emphasize on the polymodal function of this “unconventional” K⁺ channel. EBSA Satellite meeting: Ion channels, Leeds, July 2007.     

Multi-ion distributions in the cytoplasmic domain of inward rectifier potassium channels

Inward rectifier potassium (Kir) channels act as cellular diodes, allowing unrestricted flow of potassium (K⁺) into the cell while preventing currents of large magnitude in the outward direction. The rectification mechanism by which this occurs involves a coupling between K⁺ and intracellular blockers—magnesium (Mg²⁺) or polyamines—that simultaneously occupy the permeation pathway. In addition to the transmembrane pore, Kirs possess a large cytoplasmic domain (CD) that provides a favorable electronegative environment for cations. Electrophysiological experiments have shown that the CD is a key regulator of both conductance and rectification. In this study, we calculate and compare averaged equilibrium probability densities of K⁺ and Cl⁻ in open-pore models of the CDs of a weak (Kir1.1-ROMK) and a strong (Kir2.1-IRK) rectifier through explicit-solvent molecular-dynamics simulations in ∼1 M KCl. The CD of both channels concentrates K⁺ ions greater than threefold inside the cytoplasmic pore while IRK shows an additional K⁺ accumulation region near the cytoplasmic entrance. Simulations carried out with Mg²⁺ or spermine (SPM⁴⁺) show that these ions interact with pore-lining residues, shielding the surface charge and reducing K⁺ in both channels. The results also show that SPM⁴⁺ behaves differently inside these two channels. Although SPM⁴⁺ remains inside the CD of ROMK, it diffuses around the entire volume of the pore. In contrast, this polyatomic cation finds long-lived conformational states inside the IRK pore, interacting with residues E224, D259, and E299. The strong rectifier CD is also capable of sequestering an additional SPM⁴⁺ at the cytoplasmic entrance near a cluster of negative residues D249, D274, E275, and D276. Although understanding the actual mechanism of rectification blockade will require high-resolution structural information of the blocked state, these simulations provide insight into how sequence variation in the CD can affect the multi-ion distributions that underlie the mechanisms of conduction, rectification affinity, and kinetics.