Zacopride selectively activates the Kir2.1 channel via a PKA signaling pathway in rat cardiomyocytes

We recently reported that zacopride is a selective inward rectifier potassium current (IK1 ) channel agonist, suppressing ventricular arrhythmias without affecting atrial arrhythmias. The present study aimed to investigate the unique pharmacological properties of zacopride. The whole-cell patch-clamp technique was used to study IK1 currents in rat atrial myocytes and Kir2.x currents in human embryonic kidney (HEK)-293 cells transfected with inward rectifier potassium channel (Kir)2.1, Kir2.2, Kir2.3, or mutated Kir2.1 (at phosphorylation site S425L). Western immunoblots were performed to estimate the relative protein expression levels of Kir2.x in rat atria and ventricles. Results showed that zacopride did not affect the IK1 and transmembrane potential of atrial myocytes. In HEK293 cells, zacopride increased Kir2.1 homomeric channels by 40.7%±9.7% at 50 mV, but did not affect Kir2.2 and Kir2.3 homomeric channels, and Kir2.1-Kir2.2, Kir2.1-Kir2.3 and Kir2.2-Kir2.3 heteromeric channels. Western immunoblots showed that similar levels of Kir2.3 protein were expressed in rat atria and ventricles, but atrial Kir2.1 protein level was only 25% of that measured in the ventricle. In addition, 5-hydroxytryptamine (5-HT) 3 receptor was undetectable, whereas 5-HT 4 receptor was weakly expressed in HEK293 cells. The Kir2.1-activating effect of zacopride in these cells was abolished by inhibition of protein kinase A (PKA), but not PKC or PKG. Furthermore, zacopride did not activate the mutant Kir2.1 channel in HEK293 cells but selectively activated the Kir2.1 homomeric channel via a PKA-dependent pathway, independent to that of the 5-HT receptor.     

Differential sensitivity of inward rectifier K+ channels to metabolic inhibitors

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

Modulation of the heteromeric Kir4.1-Kir5.1 channels by P(CO(2)) at physiological levels

Several inward rectifier K(+) (Kir) channels are pH-sensitive, making them potential candidates for CO(2) chemoreception in cells. However, there is no evidence showing that Kir channels change their activity at near physiological level of P(CO(2)), as most previous studies were done using high concentrations of CO(2). It is known that the heteromeric Kir4.1-Kir5.1 channels are highly sensitive to intracellular protons with pKa value right at the physiological pH level. Such a pKa value may allow these channels to regulate membrane potentials with modest changes in P(CO(2)). To test this hypothesis, we studied the Kir4.1-Kir5.1 currents expressed in Xenopus oocytes and membrane potentials in the presence and absence of bicarbonate. Evident inhibition of these currents (by approximately 5%) was seen with P(CO(2)) as low as 8 torr. Higher P(CO(2)) levels (23-60 torr) produced stronger inhibitions (by 30-40%). The inhibitions led to graded depolarizations (5-45 mV with P(CO(2)) 8-60 torr). Similar effects were observed in the presence of 24 mM bicarbonate and 5% CO(2). Indeed, the Kir4.1-Kir5.1 currents were enhanced with 3% CO(2) and suppressed with 8% CO(2) in voltage clamp, resulting in hyper- (-9 mV) and depolarization (16 mV) in current clamp, respectively. With physiological concentration of extracellular K(+), the Kir4.1-Kir5.1 channels conduct substantial outward currents that were similarly inhibited by CO(2) as their inward rectifying currents. These results therefore indicate that the heteromeric Kir4.1-Kir5.1 channels are modulated by a modest change in P(CO(2)) levels. Such a modulation alters cellular excitability, and enables the cell to detect hypercapnia and hypocapnia in the presence of bicarbonate.     

Biophysical and molecular mechanisms underlying the modulation of heteromeric Kir4.1-Kir5.1 channels by CO2 and pH

CO2 chemoreception may be related to modulation of inward rectifier K+ channels (Kir channels) in brainstem neurons. Kir4.1 is expressed predominantly in the brainstem and inhibited during hypercapnia. Although the homomeric Kir4.1 only responds to severe intracellular acidification, coexpression of Kir4.1 with Kir5.1 greatly enhances channel sensitivities to CO2 and pH. To understand the biophysical and molecular mechanisms underlying the modulation of these currents by CO2 and pH, heteromeric Kir4. 1-Kir5.1 were studied in inside-out patches. These Kir4.1-Kir5.1 currents showed a single channel conductance of 59 pS with open-state probability (P(open)) approximately 0.4 at pH 7.4. Channel activity reached the maximum at pH 8.5 and was completely suppressed at pH 6.5 with pKa 7.45. The effect of low pH on these currents was due to selective suppression of P(open) without evident effects on single channel conductance, leading to a decrease in the channel mean open time and an increase in the mean closed time. At pH 8.5, single-channel currents showed two sublevels of conductance at approximately 1/4 and 3/4 of the maximal openings. None of them was affected by lowering pH. The Kir4.1-Kir5.1 currents were modulated by phosphatidylinositol-4,5-bisphosphate (PIP2) that enhanced baseline P(open) and reduced channel sensitivity to intracellular protons. In the presence of 10 microM PIP2, the Kir4.1-Kir5.1 showed a pKa value of 7.22. The effect of PIP2, however, was not seen in homomeric Kir4.1 currents. The CO2/pH sensitivities were related to a lysine residue in the NH2 terminus of Kir4.1. Mutation of this residue (K67M, K67Q) completely eliminated the CO2 sensitivity of both homomeric Kir4.1 and heteromeric Kir4.1-Kir5.1. In excised patches, interestingly, the Kir4.1-Kir5.1 carrying K67M mutation remained sensitive to low pHi. Such pH sensitivity, however, disappeared in the presence of PIP2. The effect of PIP2 on shifting the titration curve of wild-type and mutant channels was totally abolished when Arg178 in Kir5.1 was mutated. Thus, these studies demonstrate a heteromeric Kir channel that can be modulated by both acidic and alkaline pH, show the modulation of pH sensitivity of Kir channels by PIP2, and provide information of the biophysical and molecular mechanisms underlying the Kir modulation by intracellular protons.     

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

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

Protein kinase C dependent inhibition of the heteromeric Kir4.1-Kir5.1 channel

Heteromultimerization of Kir4.1 and Kir5.1 leads to a channel with distinct functional properties. The heteromeric Kir4.1-Kir5.1 channel is expressed in the eye, kidney and brainstem and has CO(2)/pH sensitivity in the physiological range, suggesting a candidate molecule for the regulation of K(+) homeostasis and central CO(2) chemoreception. It is known that K(+) transport in renal epithelium and brainstem CO(2) chemosensitivity are subject to modulation by hormones and neurotransmitters that activate distinct intracellular signaling pathways. If the Kir4.1-Kir5.1 channel is involved in pH-dependent regulation of cellular functions, it may also be regulated by some of the intracellular signaling systems. Therefore, we undertook studies to determine whether PKC modulates the heteromeric Kir4.1-Kir5.1 channel. The channel expressed using a Kir4.1-Kir5.1 tandem dimer construct was inhibited by the PKC activator PMA in a dose-dependent manner. The channel inhibition was produced via reduction of the P(open). The effect of PMA was abolished by specific PKC inhibitors. In contrast, exposure of oocytes to forskolin (a PKA activator) had no significant effect on Kir4.1-Kir5.1 currents. The channel inhibition appeared to be independent of PIP(2) depletion and PKC-dependent internalization. Several consensus sequences of potential PKC phosphorylation sites were identified in the Kir4.1 and Kir5.1 subunits by sequence scan. Although the C-terminal peptides of both Kir4.1 and Kir5.1 were phosphorylated in vitro, site-directed mutagenesis of individual residues failed to reveal the PKC phosphorylation sites suggesting that the channel may have multiple phosphorylation sites. Taken together, these results suggest that the Kir4.1-Kir5.1 but not the homomeric Kir4.1 channel is strongly inhibited by PKC activation.     

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 EK 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.     

Heterogeneity of IK1 in the mouse heart

Previous studies have shown that cardiac inward rectifier potassium current (I(K1)) channels are heteromers of distinct Kir2 subunits and suggested that species- and tissue-dependent expression of these subunits may underlie variability of I(K1). In this study, we investigated the contribution of the slowly activating Kir2.3 subunit and free intracellular polyamines (PAs) to variability of I(K1) in the mouse heart. The kinetics of activation was measured in Kir2 concatemeric tetramers with known subunit stoichiometry. Inclusion of only one Kir2.3 subunit to a Kir2.1 channel led to an approximate threefold slowing of activation kinetics, with greater slowing on subsequent additions of Kir2.3 subunits. Activation kinetics of I(K1) in both ventricles and both atria was found to correspond to fast-activating Kir2.1/Kir2.2 channels, suggesting no major contribution of Kir2.3 subunits. In contrast, I(K1) displayed significant variation in both the current density and inward rectification, suggesting involvement of intracellular PAs. The total levels of PAs were similar across the mouse heart. Measurements of the free intracellular PAs in isolated myocytes, using transgenically expressed Kir2.1 channels as PA sensors, revealed "microheterogeneity" of I(K1) rectification as well as lower levels of free PAs in atrial myocytes compared with ventricular cells. These findings provide a quantitative explanation for the regional heterogeneity of I(K1).     

Pregnenolone Sulfate Potentiates the Inwardly Rectifying K+ Channel Kir2.3

Background

Neurosteroids have various physiological and neuropsychopharmacological effects. In addition to the genomic effects of steroids, some neurosteroids modulate several neurotransmitter receptors and channels, such as N-methyl-D-aspartate receptors, γ-aminobutyric acid type A (GABA_A) receptors, and σ₁ receptors, and voltage-gated Ca²⁺ and K⁺ channels. However, the molecular mechanisms underlying the various effects of neurosteroids have not yet been sufficiently clarified. In the nervous system, inwardly rectifying K⁺ (Kir) channels also play important roles in the control of resting membrane potential, cellular excitability and K⁺ homeostasis. Among constitutively active Kir2 channels in a major Kir subfamily, Kir2.3 channels are expressed predominantly in the forebrain, a brain area related to cognition, memory, emotion, and neuropsychiatric disorders.

Methodology/Principal Findings

The present study examined the effects of various neurosteroids on Kir2.3 channels using the Xenopus oocyte expression assay. In oocytes injected with Kir2.3 mRNA, only pregnenolone sulfate (PREGS), among nine neurosteroids tested, reversibly potentiated Kir2.3 currents. The potentiation effect was concentration-dependent in the micromolar range, and the current-voltage relationship showed inward rectification. However, the potentiation effect of PREGS was not observed when PREGS was applied intracellularly and was not affected by extracellular pH conditions. Furthermore, although Kir1.1, Kir2.1, Kir2.2, and Kir3 channels were insensitive to PREGS, in oocytes injected with Kir2.1/Kir2.3 or Kir2.2/Kir2.3 mRNA, but not Kir2.1/Kir2.2 mRNA, PREGS potentiated Kir currents. These potentiation properties in the concentration-response relationships were less potent than for Kir2.3 channels, suggesting action of PREGS on Kir2.3-containing Kir2 heteromeric channels.

Conclusions/Significance

The present results suggest that PREGS acts as a positive modulator of Kir2.3 channels. Kir2.3 channel potentiation may provide novel insights into the various effects of PREGS.     

[K+] dependence of open-channel conductance in cloned inward rectifier potassium channels (IRK1, Kir2.1).

Potassium conduction through unblocked inwardly rectifying (IRK1, Kir2.1) potassium channels was measured in inside-out-patches from Xenopus oocytes, after removal of polyamine-induced strong inward rectification. Unblocked IRK1 channel current-voltage (I-V) relations show very mild inward rectification in symmetrical solutions, are linearized in nonsymmetrical solutions that bring the K+ reversal potential to extreme negative values, and follow Goldman-Hodgkin-Katz constant field equation at extreme positive E alpha. When intracellular K+ concentration (KIN) was varied, at constant extracellular K+ concentration (KOUT) the conductance at the reversal potential (GREV) followed closely the predictions of the Goldman-Hodgkin-Katz constant field equation at low concentrations and saturated sharply at concentrations of > 150 mM. Similarly, when KOUT was varied, at constant KIN, GREV saturated at concentrations of > 150 mM. A square-root dependence of conductance on KOUT is a well-known property of inward rectifier potassium channels and is a property of the open channel. A nonsymmetrical two-site three-barrier model can qualitatively explain both the I-V relations and the [K+] dependence of conductance of open IRK1 (Kir2.1) channels.     

Inhibition of electrical activity by retroviral infection with Kir2.1 transgenes disrupts electrical differentiation of motoneurons

Network-driven spontaneous electrical activity in the chicken spinal cord regulates a variety of developmental processes including neuronal differentiation and formation of neuromuscular structures. In this study we have examined the effect of chronic inhibition of spinal cord activity on motoneuron survival and differentiation. Early spinal cord activity in chick embryos was blocked using an avian replication-competent retroviral vector RCASBP (B) carrying the inward rectifier potassium channel Kir2.1. Chicken embryos were infected with one of the following constructs: RCASBP(B), RCASBP(B)-Kir2.1, or RCASBP(B)-GFP. Infection of chicken embryos at E2 resulted in widespread expression of the viral protein marker p27 gag throughout the spinal cord. Electrophysiological recordings revealed the presence of functional Kir2.1 channels in RCASBP(B)-Kir2.1 but not in RCASBP(B)-infected embryos. Kir2.1 expression significantly reduced the generation of spontaneous motor movements in chicken embryos developing in ovo. Suppression of spontaneous electrical activity was not due to a reduction in the number of surviving motoneurons or the number of synapses in hindlimb muscle tissue. Disruption of the normal pattern of activity in chicken embryos resulted in a significant downregulation in the functional expression of large-conductance Ca(2+)-dependent K(+) channels. Reduction of spinal cord activity also generates a significant acceleration in the inactivation rate of A-type K(+) currents without any significant change in current density. Kir2.1 expression did not affect the expression of voltage-gated Na(+) channels or cell capacitance. These experiments demonstrate that chronic inhibition of chicken spinal cord activity causes a significant change in the electrical properties of developing motoneurons.     

Moving the pH Gate of the Kir1.1 Inward Rectifier Channel

Both structural and functional studies suggest that pH gating of the inward rectifier potassium (K) channel, Kir1.1 (ROMK), is mediated by the convergence of 4 hydrophobic leucines (one from each subunit) near the cytoplasmic bundle-crossing of the inner transmembrane helices. We tested this hypothesis by moving the putative leucine gate from the L160-Kir1.1b to other positions along the inner transmembrane helix, and measuring inward current and conductance as functions of internal pH, using the Xenopus oocyte heterologous expression system. Results of these studies indicated that it was possible to replace the putative inward rectifier pH gate at L160-Kir1.1b by either a leucine or methionine at 157-Kir1.1b (G157L-L160G or G157M-L160G). Although both leucine and methionine gated the channel at 157-Kir1.1b, residues of similar hydrophobicity (tyrosine and valine) did not. Hence, hydrophobicity was a necessary but not a sufficient condition for steric gating at 157. This was in contrast to the 160-Kir1.1b locus, where side-chain hydrophobicity was both a necessary and sufficient property for steric gating. Homology models were constructed for all mutants that expressed significant whole-cell currents, using the closed-state coordinates of the prokaryotic inward rectifier, KirBac1.1. Models of mutants that retained pH gating were too narrow at the bundle crossing to permit hydrated K ion permeation in the closed-state. On the other hand, mutants that lost pH gating had ample space at the bundle crossing for hydrated K permeation in the closed-state. These results support our hypothesis that hydrophobic leucines at the cytoplasmic end of the inner transmembrane helices comprise the principal pH gate of Kir1.1, a gate that can be relocated from 160-Kir1.1b to 157-Kir1.1b.     

Modulation of Kir4.1 and Kir4.1-Kir5.1 channels by extracellular cations

This work demonstrates that extracellular Na⁺ modulates the cloned inwardly rectifying K⁺ channels Kir4.1 and Kir4.1-Kir5.1. Whole-cell patch clamp studies on astrocytes have previously indicated that inward potassium currents are regulated by external Na⁺. We expressed Kir4.1 and Kir4.1-Kir5.1 in Xenopus oocytes to disclose if Kir4.1 and/or Kir4.1-Kir5.1 at the molecular level are responsible for the observed effect of [Na⁺]_o and to investigate the regulatory mechanism of external cations further. Our results showed that Na⁺ has a biphasic modulatory effect on both Kir4.1 and Kir4.1-Kir5.1 currents. Depending on the Na⁺-concentration and applied voltage, the inward Kir4.1/Kir4.1-Kir5.1 currents are either enhanced or reduced by extracellular Na⁺. The Na⁺ activation was voltage-independent, whereas the Na⁺-induced reduction of the Kir4.1 and Kir4.1-Kir5.1 currents was both concentration-, time- and voltage-dependent. Our data indicate that the biphasic effect of extracellular Na⁺on the Kir4.1 and Kir4.1-Kir5.1 channels is caused by two separate mechanisms.     

Nicotine depresses the functions of multiple cardiac potassium channels.

Nicotine is the main constituent of tobacco smoke responsible for the elevated risk of the cardiovascular disease and sudden coronary death associated with smoking, presumably by provoking cardiac arrhythmias. The cellular mechanisms may be related to the ability of nicotine to prolong action potentials and to depolarize membrane potential. However, the underlying ionic mechanisms remained unknown. We showed here that nicotine blocked multiple types of K+ currents, including the native currents in canine ventricular myocytes and the cloned channels expressed in Xenopus oocytes: A-type K+ currents (I(to)/Kv4.3), delayed rectifier K+ currents (I(Kr)/HERG) and inward rectifier K+ currents (I(K1)/Kir2.1). Most noticeably, nicotine at a concentration as low as of 10 nM significantly suppressed I(to) and Kv4.3 by approximately 20%. The effects of nicotine were independent of nicotinic receptor simulation or catecholamine release. Our results indicate that nicotine is a non-specific blocker of K+ channels and the inhibitory effects are the consequence of direct interactions between nicotine molecules and the channel proteins. Our study provided for the first time the evidence for the direct inhibition of cardiac K+ channels by nicotine and established a novel aspect of nicotine pharmacology.     

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.     

Subunit positional effects revealed by novel heteromeric inwardly rectifying K+ channels.

Kir 4.1 is an inward rectifier potassium channel subunit isolated from rat brain which forms homomeric channels when expressed in Xenopus oocytes; Kir 5.1 is a structurally related subunit which does not. Co-injection of mRNAs encoding Kir 4.1 and Kir 5.1 resulted in potassium currents that (i) were much larger than those seen from expression of Kir 4.1 alone, (ii) increased rather than decreased during several seconds at strongly negative potentials and (iii) had an underlying unitary conductance of 43 pS rather than the 12 pS seen with Kir 4.1 alone. In contrast, the properties of Kir 1.1, 2.1, 2.3, 3.1, 3.2 or 3.4 were not altered by coexpression with Kir 5.1. Expression of a concatenated cDNA encoding two or four linked subunits produced currents with the properties of co-expressed Kir 4.1 and Kir 5.1 when the subunits were connected 4-5 or 4-5-4-5, but not when they were connected 4-4-5-5. The results indicate that Kir 5.1 associates specifically with Kir 4.1 to form heteromeric channels, and suggest that they do so normally in the subunit order 4-5-4-5. Further, the relative order of subunits within the channel contributes to their functional properties.     

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.