Identification of an intermolecular disulfide bond in barley hemoglobin

The present study was designed to investigate properties of ion channels in undifferentiated rabbit mesenchymal stem cells (MSCs) from bone marrow using whole-cell patch-clamp and RT-PCR techniques. It was found that three types of outward currents were present in rabbit MSCs, including an inward rectifier K(+) current (I(Kir)), a noise-like Ca(2+)-activated K(+) current (I(KCa)) co-present with delayed rectifier K(+) current (IK(DR)). I(Kir) was inhibited by Ba(2+), while I(KCa) was inhibited by paxilline (a blocker of big conductance I(KCa) channels) and clotrimazole (an inhibitor of intermediate conductance I(KCa) channels). IK(DR) exhibited a slow inactivation, "U-shaped" voltage-dependent inactivation, and slow recovery from inactivation, and the current was inhibited by tetraethylammonium or 4-aminopyridine. RT-PCR revealed the molecular identities for the functional ionic currents, including Kir1.1 (possibly responsible for I(Kir)), KCa1.1 and KCa3.1 (possibly responsible for I(KCa)), and Kv1.2, Kv2.1, and Kv2.2 (possibly responsible for IK(DR)). These results demonstrate for the first time that three types of functional ion channel currents (i.e., I(Kir), I(KCa), and IK(DR)) are present in rabbit MSCs from bone marrow.     

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.     

K+ activation of kir3.1/kir3.4 and kv1.4 K+ channels is regulated by extracellular charges

K+ activates many inward rectifier and voltage-gated K+ channels. In each case, an increase in K+ current through the channel can occur despite a reduced driving force. We have investigated the molecular mechanism of K+ activation of the inward rectifier K+ channel, Kir3.1/Kir3.4, and the voltage-gated K+ channel, Kv1.4. In the Kir3.1/Kir3.4 channel, mutation of an extracellular arginine residue, R155, in the Kir3.4 subunit markedly reduced K+ activation of the channel. The same mutation also abolished Mg2+ block of the channel. Mutation of the equivalent residue in Kv1.4 (K532) abolished K+ activation as well as C-type inactivation of the Kv1.4 channel. Thus, whereas C-type inactivation is a collapse of the selectivity filter, K+ activation could be an opening of the selectivity filter. K+ activation of the Kv1.4 channel was enhanced by acidic pH. Mutation of an extracellular histidine residue, H508, that mediates the inhibitory effect of protons on Kv1.4 current, abolished both K+ activation and the enhancement of K+ activation at acidic pH. These results suggest that the extracellular positive charges in both the Kir3.1/Kir3.4 and the Kv1.4 channels act as "guards" and regulate access of K+ to the selectivity filter and, thus, the open probability of the selectivity filter. Furthermore, these data suggest that, at acidic pH, protonation of H508 inhibits current through the Kv1.4 channel by decreasing K+ access to the selectivity filter, thus favoring the collapse of the selectivity filter.     

Interaction of Ba2+ with the pores of the cloned inward rectifier K+ channels Kir2.1 expressed in Xenopus oocytes.

Interactions of Ba2+ with K+ and molecules contributing to inward rectification were studied in the cloned inward rectifier K+ channels, Kir2.1. Extracellular Ba2+ blocked Kir2.1 channels with first-order kinetics in a Vm-dependent manner. At Vm more negative than -120 mV, the Kd-Vm relationship became less steep and the dissociation rate constants were larger, suggesting Ba2+ dissociation into the extracellular space. Both depolarization and increasing [K+]i accelerated the recovery from extracellular Ba2+ blockade. Intracellular K+ appears to relieve Ba2+ blockade by competitively slowing the Ba2+ entrance rate, instead of increasing its exit rate by knocking off action. Intracellular spermine (100 microM) reduced, whereas 1 mM [Mg2+]i only slightly reduced, the ability of intracellular K+ to repulse Ba2+ from the channel pore. Intracellular Ba2+ also blocked outward IKir2.1 in a voltage-dependent fashion. At Vm >/= +40 mV, where intrinsic inactivation is prominent, intracellular Ba2+ accelerated the inactivation rate of the outward IKir2.1 in a Vm-independent manner, suggesting interaction of Ba2+ with the intrinsic gate of Kir2.1 channels.     

Cell surface expression of the ROMK (Kir 1.1) channel is regulated by the aldosterone-induced kinase,SGK-1, and protein kinase A

The Kir1.1 (ROMK) subtypes of inward rectifier K+ channels mediate potassium secretion and regulate sodium chloride reabsorption in the kidney. The density of ROMK channels on the cortical collecting duct apical membrane is exquisitely regulated in concert with physiological demands. Although protein kinase A-dependent phosphorylation of one of the three phospho-acceptors in Kir1.1, Ser-44, also a canonical serum-glucocorticoid-regulated kinase (SGK-1) phosphorylation site, controls the number of active channels, it is unknown whether this involves activating dormant channels already residing on the plasma membrane or recruiting new channels to the cell surface. Here we explore the mechanism and test whether SGK-1 phosphorylation of ROMK regulates cell surface expression. Removal of the phosphorylation site by point mutation (Kir1.1, S44A) dramatically attenuated the macroscopic current density in Xenopus oocytes. As measured by antibody binding of external epitope-tagged forms of Kir1.1, surface expression of Kir1.1 S44A was inhibited, paralleling the reduction in macroscopic current. In contrast, surface expression and macroscopic current density was augmented by a phosphorylation mimic mutation, Kir1.1 S44D. In vitro phosphorylation assays revealed that Ser-44 is a substrate of SGK-1 phosphorylation, and expression of SGK-1 with the wild type channel increased channel density to the same level as the phosphorylation mimic mutation. Moreover, the stimulatory effect of SGK-1 was completely abrogated by mutation of the phosphorylation site. In conclusion, SGK-1 phosphorylation of Kir1.1 drives expression on the plasmalemma. Because SGK-1 is an early aldosterone-induced gene, our results suggest a possible molecular mechanism for aldosterone-dependent regulation of the secretory potassium channel in the kidney.     

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

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

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.     

Sex differences in and hormonal regulation of Kv1 potassium channel gene expression in the electric organ: molecular control of a social signal

Electric fish communicate with electric organ (EO) discharges (EODs) that are sexually dimorphic, hormone-sensitive, and often individually distinct. The cells of the EO (electrocytes) of the weakly electric fish Sternopygus possess delayed rectifying K+ currents that systematically vary in their activation and deactivation kinetics, and this precise variation in K+ current kinetics helps shape sex and individual differences in the EOD. Because members of the Kv1 subfamily produce delayed rectifier currents, we cloned a number of genes in the Kv1 subfamily from the EO of Sternopygus. Using our sequences and those from genome databases, we found that in teleost fish Kv1.1 and Kv1.2 exist as duplicate pairs (Kv1.1a&b, Kv1.2a&b) whereas Kv1.3 does not. Using real-time quantitative RT-PCR, we found that Kv1.1a and Kv1.2a, but not Kv1.2b, expression in the EO is higher in high EOD frequency females (which have fast EO K+ currents) than in low EOD frequency males (which have slow EO K+ currents). Systemic treatment with dihydrotestosterone decreased Kv1.1a and Kv1.2a, but not Kv1.2b, expression in the EO, whereas treatment with human chorionic gonadotropin (hCG) increased Kv1.2a but not Kv1.1a or Kv1.2b expression in the EO. Thus, systematic variation in the ratios of Kv1 channels expressed in the EO is correlated with individual differences in and sexual dimorphism of a communication signal.     

Shared requirement for dynein function and intact microtubule cytoskeleton for normal surface expression of cardiac potassium channels

Potassium channels at the cardiomyocyte surface must eventually be internalized and degraded, and changes in cardiac potassium channel expression are known to occur during myocardial disease. It is not known which trafficking pathways are involved in the control of cardiac potassium channel surface expression, and it is not clear whether all cardiac potassium channels follow a common pathway or many pathways. In the present study we have surveyed the role of retrograde microtubule-dependent transport in modulating the surface expression of several cardiac potassium channels in ventricular myocytes and heterologous cells. The disruption of microtubule transport in rat ventricular myocytes with nocodazole resulted in significant changes in potassium currents. A-type currents were enhanced 1.6-fold at +90 mV, rising from control densities of 20.9 +/- 2.8 to 34.0 +/- 5.4 pA/pF in the nocodazole-treated cells, whereas inward rectifier currents were reduced by one-third, perhaps due to a higher nocodazole sensitivity of Kir channel forward trafficking. These changes in potassium currents were associated with a significant decrease in action potential duration. When expressed in heterologous human embryonic kidney (HEK-293) cells, surface expression of Kv4.2, known to substantially underlie A-type currents in rat myocytes, was increased by nocodazole, by the dynein inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride, and by p50 overexpression, which specifically interferes with dynein motor function. Peak current density was 360 +/- 61.0 pA/pF in control cells and 658 +/- 94.5 pA/pF in cells overexpressing p50. The expression levels of Kv2.1, Kv3.1, human ether-a-go-go-related gene, and Kir2.1 were similarly increased by p50 overexpression in this system. Thus the regulation of potassium channel expression involves a common dynein-dependent process operating similarly on the various channels.     

Evidence against functional heteromultimerization of the KATP channel subunits Kir6.1 and Kir6.2

K(ATP) channels consist of pore-forming potassium inward rectifier (Kir6.x) subunits and sulfonylurea receptors (SURs). Although Kir6.1 or Kir6.2 coassemble with different SUR isoforms to form heteromultimeric functional K(ATP) channels, it is not known whether Kir6.1 and Kir6.2 coassemble with each other. To define the molecular identity of K(ATP) channels, we used adenoviral gene transfer to express wild-type and dominant-negative constructs of Kir6.1 and Kir6.2 in a heterologous expression system (A549 cells) and in native cells (rabbit ventricular myocytes). Dominant-negative (DN) Kir6.2 gene transfer suppressed current through heterologously expressed SUR2A + Kir6.2 channels. Conversely, DN Kir6.1 suppressed SUR2B + Kir6.1 current but had no effect on coexpressed SUR2A + Kir6. 2. We next probed the ability of Kir6.1 and Kir6.2 to affect endogenous K(ATP) channels in adult rabbit ventricular myocytes, using adenoviral vectors to achieve efficient gene transfer. Infection with the DN Kir6.2 virus for 72 h suppressed pinacidil-inducible K(ATP) current density measured by whole-cell patch clamp. However, there was no effect of infection with the DN Kir6.1 on the K(ATP) current. Based on these functional assays, we conclude that Kir6.1 and Kir6.2 do not heteromultimerize with each other and that Kir6.2 is the sole K(ATP) pore-forming subunit in the surface membrane of heart cells.     

Kir2.3 knock-down decreases IK1 current in neonatal rat cardiomyocytes

Inward rectifier potassium Kir2.x channels mediate cardiac inward rectifier potassium currents (I(K1)). As a subunit of Kir2.x, the physiological role of Kir2.3 in native cardiomyocytes has not been reported. This study shows that Kir2.3 knock-down remarkably down-regulates Kir2.3 expression (Kir2.3 protein was reduced to 19.91+/-3.24% on the 2nd or 3rd day) and I(K1) current densities (at -120 mV, control vs. knock-down: -5.03+/-0.24 pA/pF, n=5 vs. -1.16+/-0.19 pA/pF, n=7, P<0.001) in neonatal rat cardiomyocytes. The data suggest that Kir2.3 plays a potentially important role in I(K1) currents in neonatal rat cardiomyocytes.     

Molecular correlates of altered expression of potassium currents in failing rabbit myocardium

Action potential (AP) prolongation is a hallmark of failing myocardium. Functional downregulation of K currents is a prominent feature of cells isolated from failing ventricles. The detailed changes in K current expression differ depending on the species, the region of the heart, and the mechanism of induction of heart failure. We used complementary approaches to study K current downregulation in pacing tachycardia-induced heart failure in the rabbit. The AP duration (APD) at 90% repolarization was significantly longer in cells isolated from failing hearts compared with controls (539 +/- 162 failing vs. 394 +/- 114 control, P < 0.05). The major K currents in the rabbit heart, inward rectifier potassium current (I(K1)), transient outward (I(to)), and delayed rectifier current (I(K)) were functionally downregulated in cells isolated from failing ventricles. The mRNA levels of Kv4.2, Kv1.4, KChIP2, and Kir2.1 were significantly downregulated, whereas the Kv4.3, Erg, KvLQT1, and minK were unaltered in the failing ventricles compared with the control left ventricles. Significant downregulation in the long splice variant of Kv4.3, but not in the total Kv4.3, Kv4.2, and KChIP2 immunoreactive protein, was observed in cells isolated from the failing ventricle with no change in Kv1.4, KvLQT1, and in Kir2.1 immunoreactive protein levels. Multiple cellular and molecular mechanisms underlie the downregulation of K currents in the failing rabbit ventricle.     

Dominant negative effects of a Gbeta mutant on G-protein coupled inward rectifier K+ channel

HEK293 cells were transfected with cDNAs for Gbeta1(W332A) [a mutant Gbeta1], Ggamma2, and inward rectifier K+ channels (Kir3.1/Kir3.2). Application of Gbeta1gamma2 protein to these cells activated the K+ channels only slightly. When mu-opioid receptors and Kir3.1/Kir3.2 were transfected, application of a mu-opioid agonist induced a Kir3 current. However, co-expression of Gbeta1(W332A) suppressed this current. Most likely, Gbeta1(W332A) inhibited the action of the endogenous Gbeta. Such a dominant negative effect of Gbeta1(W332A) was also observed in neuronal Kir3 channels in locus coeruleus. The mutant, Gbeta1(W332A) protein, although inactive, retains its ability to bind Kir3 and prevents the wild type Gbeta from activating the channel.     

Electrophysiological properties of human adipose tissue-derived stem cells

Human adipose tissue-derived stem cells (hASCs) represent a potentially valuable cell source for clinical therapeutic applications. The present study was designed to investigate properties of ionic channel currents present in undifferentiated hASCs and their impact on hASCs proliferation. The functional ion channels in hASCs were analyzed by whole-cell patch-clamp recording and their mRNA expression levels detected by RT-PCR. Four types of ion channels were found to be present in hASCs: most of the hASCs (73%) showed a delayed rectifier-like K(+) current (I(KDR)); Ca(2+)-activated K(+) current (I(KCa)) was detected in examined cells; a transient outward K(+) current (I(to)) was recorded in 19% of the cells; a small percentage of cells (8%) displayed a TTX-sensitive transient inward sodium current (I(Na.TTX)). RT-PCR results confirmed the presence of ion channels at the mRNA level: Kv1.1, Kv2.1, Kv1.5, Kv7.3, Kv11.1, and hEAG1, possibly encoding I(KDR); MaxiK, KCNN3, and KCNN4 for I(KCa); Kv1.4, Kv4.1, Kv4.2, and Kv4.3 for I(to) and hNE-Na for I(Na.TTX). The I(KDR) was inhibited by tetraethyl ammonium (TEA) and 4-aminopyridine (4-AP), which significantly reduced the proliferation of hASCs in a dose-dependent manner (P < 0.05), as suggested by bromodeoxyurindine (BrdU) incorporation. Other selective potassium channel blockers, including linopiridine, iberiotoxin, clotrimazole, and apamin also significantly inhibited I(KDR). TTX completely abolished I(Na.TTX). This study demonstrates for the first time that multiple functional ion channel currents such as I(KDR), I(KCa), I(to), and I(Na.TTX) are present in undifferentiated hASCs and their potential physiological function in these cells as a basic understanding for future in vitro experiments and in vivo clinical investigations.     

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium

Inward-rectifier K channel: using macroscopic voltage clamp and single- channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current- voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell- attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. Conclusions: under physiological ionic gradients, a 15-pS inward- rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.     

Development of recombinant cell line co-expressing mutated Nav1.5, Kir2.1, and hERG for the safety assay of drug candidates

To provide a high-throughput screening method for human ether-a-go-go-gene-related gene (hERG) K(+) channel inhibition, a new recombinant cell line, in which single action potential (AP)-induced cell death was produced by gene transfection. Mutated human cardiac Na(+) channel Nav1.5 (IFM/Q3), which shows extremely slow inactivation, and wild-type inward rectifier K(+) channel, Kir2.1, were stably co-expressed in HEK293 cells (IFM/Q3+Kir2.1). In IFM/Q3+Kir2.1, application of single electrical stimulation (ES) elicited a long AP lasting more than 30 s and led cells to die by more than 70%, whereas HEK293 co-transfected with wild-type Nav1.5 and Kir2.1 fully survived. The additional expression of hERG K(+) channels in IFM/Q3+Kir2.1 shortened the duration of evoked AP and thereby markedly reduced the cell death. The treatment of the cells with hERG channel inhibitors such as nifekalant, E-4031, cisapride, terfenadine, and verapamil, recovered the prolonged AP and dose-dependently facilitated cell death upon ES. The EC(50) values to induce the cell death were 3 μM, 19 nM, 17 nM, 74 nM, and 3 μM, respectively, whereas 10 μM nifedipine did not induce cell death. Results indicate the high utility of this cell system for hERG K(+) channel safety assay.     

Thyroid hormone regulates mRNA expression and currents of ion channels in rat atrium

Atrial fibrillation is one of the common arrhythmias associated with hyperthyroidism. This study examined the effects of thyroid hormone (T3) on mRNA expression and currents of major ionic channels determining the action potential duration (APD) in the rat atrium using the RNase protection assay and the whole-cell patch-clamp technique, respectively. T3 increased the Kv1.5 mRNA expression and decreased the L-type calcium channel mRNA expression, while the Kv4.2 mRNA expression did not change. APD was shorter in hyperthyroid than in euthyroid myocytes. The ultrarapid delayed rectifier potassium currents were remarkably increased in hyperthyroid than in euthyroid myocytes, whereas the transient outward potassium currents were unchanged. L-type calcium currents were decreased in hyperthyroid than in euthyroid myocytes. T3 shifted the current-voltage relationship for calcium currents negatively. In conclusion, T3 increased the outward currents and decreased the inward currents. The resultant changes of ionic currents shortened APD, providing a substrate for atrial fibrillation.     

Exaggerated Mg2+ Inhibition of KCNJ2 as a Consequence of Reduced PIP2 Sensitivity in Andersen Syndrome

Andersen syndrome is an autosomal dominant disorder characterized by cardiac arrhythmias, periodic paralysis and dysmorphic features. Many Andersen syndrome cases have been associated with loss-of-function mutations in the inward rectifier K+ channel Kir2.1 encoded by KCNJ2. Using engineered concatenated tetrameric channels we determined the mechanism for dominant loss-of-function associated with a trafficking-competent missense mutation, Kir2.1-T74A. This mutation alters a conserved threonine residue in an N-terminal domain analogous to the slide helix identified in the structure of a bacterial inward rectifier. Incorporation of a single mutant subunit in channel tetramers was sufficient to cause a selective impairment of whole-cell outward current, but no difference in the level of inward current compared with wild-type (WT) tetramers. The presence of two mutant subunits resulted in greatly reduced outward and impaired inward currents. Experiments using excised inside-out membrane patches revealed that tetramers with one mutant subunit exhibited increased Mg2+ inhibition. Additional experiments demonstrated that concatenated tetramers containing one T74A subunit had reduced PIP2 sensitivity, and that outward current carried by mutant tetramers could be restored by addition of PIP2 in the absence of Mg2+. Our results are consistent with the involvement of the Kir2.1 N-terminus in PIP2 modulation of channel activity and support the existence of an inverse relationship between PIP2 sensitivity and Mg2+ inhibition of Kir2.1 channels. Our data also indicate that a single mutant subunit is sufficient to explain dominant-negative behavior of Kir2.1-T74A in Andersen syndrome.     

Do voltage-gated Kv1.1 and inward rectifier Kir2.1 potassium channels form heteromultimers?

Possible heteromultimer formation between Kv- and Kir-type K⁺ channels was investigated, in connection with the known functional diversity of K⁺ channels in vivo. Voltage-clamp experiments were performed on Xenopus oocytes, either injected with concatenated Kir2.1-Kv1.1 mRNA, or co-injected with Kv1.1 and Kir2.1 mRNA. K⁺ currents could be approximated by the algebraic sum of the 2 K⁺ current types alone. The tandem construct did not show functional expression, although it could be detected by Western blotting. We conclude that Kv1.1 and Kir2.1 α-subunit proteins fail to assemble and do not contribute functional diversity to K⁺ channels.