Molecular characterization of an inwardly rectifying K+ channel from HeLa cells

Retinoid-deficient cultures of airway epithelial cells undergo squamous differentiation. Treatment of such cultures with retinoic acid (RA) leads to restoration of the mucous phenotype. The purpose of our study was to characterize the cellular and molecular changes following RA treatment of retinoid-deficient human tracheobronchial epithelial cell cultures. Of particular interest was to determine when during the conversion of the squamous to the mucous phenotype the mucin genes MUC2, MUC5AC, and MUC5B were expressed. We used cornifin alpha and secreted mucin as markers to monitor the squamous and mucous phenotypes, respectively. Our studies showed that the RA responsiveness of the cultures progressively decreased with protracted retinoid deficiency, requiring higher RA concentrations to restore the mucous phenotype. Within 12 h after the start of RA treatment, cornifin alpha expression decreased, signaling the beginning of a change in cellular phenotype. At 24 h after addition of RA to the cultures, a significant number of mucous cells appeared, and at 72 h mucin was secreted in measurable amounts. Induction of mucin gene expression occurred sequentially: MUC2, MUC5AC, and MUC5B mRNAs were upregulated at 24, 48, and 72 h, respectively. When cultures maintained in 10(-8) M RA were treated with 10(-6) M RA, MUC2 but not MUC5AC and MUC5B mRNA levels were upregulated within 6 h. Our study indicates that MUC2 mRNA is an early marker of mucous differentiation, whereas MUC5AC and MUC5B mRNAs are expressed during more advanced stages of mucous differentiation. Our studies further suggest that each of the mucin genes is regulated by distinct mechanisms.     

Selective block by glyceryl nonivamide of inwardly rectifying K+ current in rat anterior pituitary GH3 cells

The effects of glyceryl nonivamide (GLNVA) on ionic currents were compared and examined in rat pituitary GH3 cells. Hyperpolarization-activated K+ currents in GH3 cells bathed in high-K+ Ca2+-free external solution were studied to assess effects of GLNVA on the an inwardly rectifying K+ current (I(K(IR))). GLNVA is very potent in blocking I(K(IR)) in a concentration-dependent manner, with a half maximal concentrations of 0.1 microM. The complete block of I(K(IR)) achieved with concentrations > or = 1 microM revealed the presence of a non-inactivating current. We also found that GLNVA at a concentration above 30 microM inhibited L-type voltage-dependent Ca2+ current and two components of K+ outward currents, while GLNVA (< or = 3 microM) did not have any effect on them. This study shows that GLNVA, in addition to retaining the capability of eliciting peptidergic neurons, is a selective block of I(K(IR)) in GH3 cells and will provide a useful tool for characterizing I(K(IR)) and understanding its physiological function. In addition, the carefulness should be taken about the interpretation of GLNVA-mediated responses in vivo or in vitro.     

Inhibition by taurine of the inwardly rectifying K+ current in guinea pig ventricular cardiomyocytes

Effects of taurine on the inwardly rectifying K+ current (IK1) in isolated guinea pig ventricular cardiomyocytes were examined using patch voltage-clamp methods. All experiments were performed at 36 degrees C. Taurine (10-20 mM) increased the action potential duration, but failed to affect the resting potential. Holding potential was maintained at -30 mV. The current was activated with an inwardly going rectification, and was completely blocked by Ba2+ (2 mM). Taurine inhibited IK1 at - 120 mV by 28.3+/-1.1% (n=6, P < 0.05) at 10 mM and by 36.0+/-2.1% (n=6, P < 0.01) at 20 mM. The reversal potential was shifted in the hyperpolarizing direction by 3.7+/-0.6 mV (n=6) at 20 mM. In inside-out patch-clamp experiments, the amplitude of unitary channels was -2.7+/-0.3 pA (n=21) at -90 mV. Symmetrical high-K+ (150 mM) solutions in both bath and pipette were used. The channel conductance was 32+/-2 pS (n=9). Taurine did not affect channel conductance, but markedly decreased the open probability at - 120 mV of channel by 21.5+/-2.4% (n=8, P < 0.01) at 10 mM, and by 56.7+/-3.8% (n=8, P < 0.001) at 20 mM. These responses were almost reversible. These results suggest that taurine directly modulates the open probability of the inwardly rectifying K+ current, resulting in regulation of the functions of heart cells.     

Tissue-specific regulation of G-protein-coupled inwardly rectifying K+ channel expression by muscarinic receptor activation in ovo

We investigated the effects of muscarinic acetylcholine receptor stimulation on the expression levels of the G-protein-coupled inwardly rectifying K+ channel (GIRK) subunits using solution hybridization and immunoblot analyses. We report here that treatment of chick embryos in ovo with muscarinic agonist causes decreases in mRNA levels encoding GIRK1 and GIRK4 in atria but does not alter GIRK1 expression in ventricles. In addition, GIRK1 protein levels also demonstrate a decrease in atria upon muscarinic acetylcholine receptor stimulation. Numerous receptors couple to the activation of the GIRK family of inwardly rectifying K+ channels; thus, these decreases represent a novel mechanism for regulating physiological responses to chronic agonist exposure.     

Soluble or bound laminin elicit in human neuroblastoma cells short- or long-term potentiation of a K+ inwardly rectifying current: relevance to neuritogenesis

Changes in the resting potential (VREST) and in the underlying ionic conductances were measured by the patch-clamp technique in SH-SY5Y human neuroblastoma cells exposed to substrate-bound or soluble Laminin (bLN; sLN), as compared to integrin-independent substrates (polylysine (PL); bovine serum albumin (BSA)). While PL and BSA were ineffective, both forms of LN caused an early (5-15 min) activation of a peculiar type of Inwardly Rectifying K+ current (IIR) characterised by a voltage-dependent inactivation in the range of membrane potentials around -50/0 mV. IIR was blocked by Cs+ ions and by the antiarrhythmic drug E-4031, a specific inhibitor of the HERG-codified channels. In cells adherent to bLN, IIR potentiation (85%) persisted for 90-120 min and was accompanied by a similar, but transient, increase in the leakage conductance (GL). Successively, the persistence of a high IIR conductance and the decrease of GL progressively bring VREST from -12 to approximately -30 mV in about 120 min. On the other hand, in cells adherent to PL, exposure to sLN produced a similar but not persistent activation of IIR, without any increase in GL: this caused a rapid, transient hyperpolarisation of VREST. The effects of bLN and sLN were mimicked by antibodies raised against the integrin beta 1 subunit, suggesting a specific integrin-mediated mechanism. In fact, when bound to the culture dishes, these antibodies simultaneously activated the IIR and GL, whereas in soluble form they only activated IIR. Cells adherent to bLN emitted neurites, a process impaired by the block of IIR by E-4031 and Cs+. On the whole data suggest that the integrin-mediated activation of IIR plays a crucial role in the commitment to neuritogenesis of neuroblastoma cells, independently on the effects of this activation on VREST.     

Neurotransmitter activation of inwardly rectifying potassium current in dissociated hippocampal CA3 neurons: interactions among multiple receptors

We characterized potassium current activated by G-protein-coupled receptors in acutely dissociated hippocampal CA3 neurons. Agonists for serotonin, adenosine, and somatostatin receptors reliably activated a potassium-selective conductance that was inwardly rectifying and that was blocked by 1 mM external Ba2+. The conductance had identical properties to that activated by GABAB receptors in the same cells. In one-half of the CA3 neurons that were tested, the metabotropic glutamate agonist 1S,3R-ACPD also activated inwardly rectifying Ba2+-sensitive potassium current. Activation of the current by serotonin and adenosine agonists occurred with a time constant of 200-700 msec after a lag of 50-100 msec; on removal of agonist the current deactivated with a time constant of 1-2 sec after a lag of 200-400 msec. These kinetics are similar to GABAB-activated current and consistent with a direct action of G-protein on the channels. For somatostatin, both activation and deactivation were approximately fourfold slower, probably limited by agonist binding and unbinding. The half-maximally effective agonist concentrations were approximately 75 nM for somatostatin, approximately 100 nM for serotonin, and approximately 400 nM for 2-chloroadenosine. Dose-response relationships had Hill coefficients of 1.2-1.9, suggesting cooperativity in the receptor-to-channel coupling mechanism. At saturating concentrations of agonists, the combined application of baclofen and either somatostatin, serotonin, or 2-chloroadenosine produced effects that were subadditive and often completely occlusive. However, at subsaturating concentrations the effects of baclofen and 2-chloroadenosine were supra-additive. Thus, low levels of different transmitters can act synergistically in activating inwardly rectifying potassium current.     

RGS proteins reconstitute the rapid gating kinetics of gbetagamma-activated inwardly rectifying K+ channels

G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Galpha(i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20-40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of "regulators of G protein signaling" (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor --> GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent "basal" GIRK current was suppressed by approximately 40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of "slow" postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.     

Inwardly rectifying K+ currents in isolated human retinal pigment epithelial cells

PURPOSE: K+ channels in the retinal pigment epithelium (RPE) play a number of important roles, including the establishment of membrane potential, the transport of K+ between the subretinal space and choroid, and the generation of the c-wave of the electroretinogram. Previous studies on amphibian RPE demonstrated that these functions are likely served by an inwardly rectifying K+ channel. The aim of this study was to characterize inwardly rectifying K+ channels in cultured and freshly isolated adult human RPE (hRPE) cells. METHODS: Single cells were dispersed enzymatically from primary cultures of adult hRPE or from fresh adult hRPE-choroid. Ionic currents were recorded using either the perforated-patch or whole-cell configuration of the patch-clamp technique. RESULTS: In 5 mM external K+, roughly 20% of cultured hRPE cells exhibited a strong inwardly rectifying K+ conductance that passed inward but little outward current. This conductance increased when [K+]o was increased and exhibited a voltage-dependent block by external Na+ at negative potentials. In contrast, all freshly isolated hRPE cells exhibited a mild inwardly rectifying K+ conductance that mediated substantial outward current at physiological voltages. This conductance decreased when [K+]o was increased and showed no voltage-dependent block by external Na+. CONCLUSION: The authors conclude that fresh hRPE cells express a mild inwardly rectifying K+ conductance. The operation of this conductance at physiological voltages makes it a likely candidate for the resting K+ conductances of the apical and basolateral membranes. Cultured hRPE cells express a functionally different channel type that may reflect a change in phenotype.     

Pore mutation in a G-protein-gated inwardly rectifying K+ channel subunit causes loss of K+-dependent inhibition in weaver hippocampus

Weaver (wv) mice carry a point mutation in the pore region of a G-protein-gated inwardly rectifying K+ channel subunit (Kir3.2). wvKir3.2 conducts inward currents that may cause the loss of neurons in the cerebellum and substantia nigra. Although Kir3.2 is widely expressed in the CNS, significant morphological or physiological changes have not been reported for other brain areas. We studied the role of wvKir3.2 in hippocampal slices of young [postnatal day (P) 4-18] and adult wv/wv (>/=P24) mice, because protein levels of Kir 3. 1 and Kir3.2 appear to be normal in the first 3 postnatal weeks and only decrease thereafter. In disinhibited slices, the GABAB receptor agonist R-baclofen reduced burst activity in wv/wv mice but was much more potent in wild-type mice. Mean resting membrane potential, slope input resistance, and membrane time constant of CA3 neurons of adult wv/wv and wild-type mice were indistinguishable. However, R-baclofen or chloroadenosine did not induce K+ currents or any other conductance change in wv/wv mice. Moreover, electrical or chemical stimulation of inhibitory neurons did not evoke slow IPSPs in adult wv/wv mice. Only in a few cells of young wv/wv mice did GABAB receptor activation by R-baclofen or presynaptic stimulation induce small inward currents, which were likely caused by a Na+ ion influx through wvKir3.2 channels. The data show that the pore mutation in wvKir3.2 channels results in a hippocampal phenotype resembling Kir3.2-deficient mutants, although it is not associated with the occurrence of seizures.     

Identification of native atrial G-protein-regulated inwardly rectifying K+ (GIRK4) channel homomultimers

G-protein-regulated inwardly rectifying K+ (GIRK) channels play critical inhibitory roles throughout the nervous system, heart, and pancreas. They are believed to be heterotetramers consisting of GIRK1 (Kir3.1) and either GIRK2 (Kir3.2), GIRK3 (Kir3.3), or GIRK4 (Kir3.4) subunits. The GIRK1 subunit is hypothesized to be critical to form GIRK channels with normal channel kinetics based on heterologous expression studies. However, GIRK2 and GIRK3 proteins are present in areas of the brain where no GIRK1 has been detected. Here we demonstrate that GIRK tetramers lacking GIRK1 can be purified from bovine heart atria. We have found that only half of GIRK4 is purified as the GIRK1-GIRK4 heterotetramer, whereas the remaining GIRK4 forms a high molecular weight, SDS-resistant complex that does not contain GIRK1. These GIRK4 complexes, most likely GIRK4 homotetramers, were previously not seen because of their aberrant migration on SDS-polyacrylamide gels. We propose that all of GIRK1 and half of GIRK4 proteins in atria combine to form the heterotetramer IKACh, whereas the remaining GIRK4 forms a novel tetrameric complex. GIRK4 homotetramers form channels with unusual single channel behavior, and their contribution to native currents requires further investigation.     

Distribution of mRNA encoding the inwardly rectifying K+ channel, BIR1 in rat tissues

Chinese hamster P-glycoprotein ("multidrug-resistance protein") was purified and reconstituted in proteoliposomes by the procedure of I. L. Urbatsch, M. K. al-Shawi, and A. E. Senior (1994, Biochemistry 33, 7069-7076). The presence of lipid during the octylglucoside solubilization and Reactive Red 120 chromatography steps was found to be mandatory for retention of ATPase activity. Sheep brain or bovine liver lipid extracts could be substituted for the Escherichia coli lipids used previously. Stimulation of ATPase activity of purified, reconstituted P-glycoprotein by vinblastine, colchicine, and daunomycin was seen with sheep brain and bovine liver lipids, but not with E. coli lipids. Basal (i.e., not drug-stimulated) ATPase activity was different in the three lipids. Azidopine labeling of the drug binding sites in purified, reconstituted P-glycoprotein was carried out; vinblastine, colchicine, and daunomycin competed for labeling in all three lipids. It is therefore evident that the lipid environment can significantly influence the characteristics of purified, reconstituted P-glycoprotein ATPase activity and the apparent coupling between drug-binding and catalytic sites.     

Ceramide inhibits inwardly rectifying K+ currents via a Ras- and Raf-1-dependent pathway in cultured oligodendrocytes

Ceramide is a lipid mediator implicated in apoptosis induced by proinflammatory cytokines in many cell types, including oligodendrocytes (OLGs). To determine whether ceramide modulates transmembrane signaling events in OLGs, we studied its effect on intracellular Ca2+ (Cai), resting membrane potential and inwardly rectifying K+ currents (IKir) in cultured neonatal rat OLGs. We report here that (1) exposure to C2-ceramide (cer) rarely increases OLG Cai, whereas sphingosine elicits sustained increase in Cai; (2) cer causes OLG depolarization, an effect mimicked by sphingosine-1-phosphate but not by sphingosine; and (3) cer, but not its inactive analog dihydroceramide, inhibits OLG IKir. The cer effect is attenuated by Ras antibody Y13-259, by protein kinase C inhibitory peptide (19-36), and by suppression of c-Raf-1 expression with antisense raf-1 oligonucleotides. We conclude that cer-induced OLG depolarization is mediated via inhibition of IKir by a Ras- and raf-1-dependent pathway, which results in the phosphorylation of the inward rectifier K+ channel protein.     

Loss of inwardly rectifying potassium currents by human retinal glial cells in diseases of the eye

We compared the inward K+ currents of Müller glial cells from healthy and pathologically changed human retinas. To this purpose, the whole-cell voltage-clamp technique was performed on noncultured Müller cells acutely isolated from human retinas. Cells originated from retinas of four healthy organ donors and of 24 patients suffering from different vitreoretinal and chorioretinal diseases. Müller cells from organ donors displayed inward K+ currents in the whole-cell mode similar to those found in other species. In contrast, this pattern was clearly changed in the Müller cells from patient retinas. In whole-cell recordings many Müller cells had strongly decreased inward K+ current amplitudes or lost these currents completely. Thus, the mean input resistance of Müller cells from patients was significantly increased to 1,129 +/- 812 M omega, compared to 279 +/- 174 M omega in Müller cells from healthy organ donor retinas. Accordingly, since the membrane potential is mainly determined by the K+ inward conductance in healthy Müller cells, a large amount of Müller cells from patient retinas had a membrane potential which was significantly lower than that of Müller cells from control eyes. The mean membrane potentials were -37 +/- 24 mV and -63 +/- 25 mV for patient and donor Müller cells, respectively. The newly described membrane characteristic changes of Müller cells from patient eyes are assumed to interfere severely with normal retinal function: (1) the retinal K+ homeostasis, which is partly regulated by the Müller cell-mediated spatial buffering, should be disturbed, and (2) the diminished membrane potential should influence voltage-dependent transporter systems of the Müller cells, e.g., the Na(+)-dependent glutamate uptake.     

Influence of flupirtine on a G-protein coupled inwardly rectifying potassium current in hippocampal neurones

Zinc deficiency has been associated with growth deficits, reduced dietary intake and appetite, and has been hypothesized to result in reduced activity. This randomized, double-blind, placebo-controlled study examined whether 10 mg of oral zinc as zinc sulfate, given daily for up to 7 mo, affected activity patterns of 85 Guatemalan infants recruited at 6-9 mo of age. Infant activity was assessed by time sampling-observation method at 10-min intervals during a 12-h data collection period, at base line, 3 and 7 mo follow-up. Motor development and the percentage of time infants were observed in various positions (being carried, lying down, sitting, crawling, standing or walking) and engaged in various activities (eating, sleeping, resting, crying/whining or playing) were compared by treatment group. No differences in motor development were observed by treatment group. However, at follow-up 2 (after 7 mo of supplementation), zinc-supplemented infants were significantly more frequently observed sitting up compared with lying down, and were playing during 4.18 +/- 1.95% (P < 0.05) more observations than unsupplemented infants. They were also somewhat less likely to be observed crying or whining (P < 0.10) compared with those receiving the placebo. These effects are independent of other factors including infant age, motor development, sex, maternal education, family socioeconomic status and nutritional status at base line. Further research must be conducted to determine the long-term developmental importance of these differences in activity patterns associated with zinc supplementation in this setting.     

Adenovirus-mediated expression of 5-HT1B receptors in cardiac ventricle myocytes; coupling to inwardly rectifying K+ channels

The 5-HT1B receptor is expressed on nerve terminals where it inhibits neurotransmitter release. When expressed ectopically in fibroblasts, the 5-HT1B receptor inhibits adenylyl cyclase. However, in the central nervous system, the effect of this receptor on neurotransmitter release appears to be cAMP-independent. We therefore investigated alternative effector systems that might be activated by the 5-HT1B receptor. We constructed a recombinant adenovirus that allows expression of high levels of the 5-HT1B receptor in a variety of cells. We chose cardiac ventricle myocytes because they express a muscarinic-gated, inwardly rectifying K+ channel (i[KACh]). In infected ventricle cells, both 5-HT and the muscarinic receptor agonist, carbachol, elicited a similar inwardly rectifying K+ current. The currents elicited by these agonists were pertussis-toxin sensitive and were not additive. These results suggest a common signal transduction pathway for 5-HT1B and muscarinic receptors in ventricle cells.     

Cloning of a Xenopus laevis inwardly rectifying K+ channel subunit that permits GIRK1 expression of IKACh currents in oocytes

Xenopus oocytes injected with GIRK1 mRNA express inwardly rectifying K+ channels resembling IKACh. Yet IKACh, the atrial G protein-regulated ion channel, is a heteromultimer of GIRK1 and CIR. Reasoning that an oocyte protein might be substituting for CIR, we cloned XIR, a CIR homolog endogenously expressed by Xenopus oocytes. Coinjecting XIR and GIRK1 mRNAs produced large, inwardly rectifying K+ currents responsive to m2-muscarinic receptor stimulation. The m2-stimulated currents of oocytes expressing GIRK1 alone decreased 80% after injecting antisense oligonucleotides specific to the 5' untranslated region of XIR, but GIRK1/CIR currents were unaffected. Thus, GIRK1 without XIR or CIR only ineffectively produces currents in oocytes. This result suggests that GIRK1 does not form native homomultimeric channels.     

Control of channel activity through a unique amino acid residue of a G protein-gated inwardly rectifying K+ channel subunit

G protein-gated inwardly rectifying K+ (GIRK) channels, which are important regulators of membrane excitability both in heart and brain, appear to function as heteromultimers. GIRK1 is unique in the GIRK channel family in that although it is by itself inactive, it can associate with the other family members (GIRK2-GIRK5) to enhance their activity and alter their single-channel characteristics. By generating a series of chimeras, we identified a phenylalanine residue, F137, in the pore region of GIRK1 that critically controls channel activity. F137 is found only in GIRK1, while the remaining GIRK channels possess a conserved serine residue in the analogous position. The single-point mutant GIRK4(S143F) behaved as a GIRK1 analog, forming multimers with GIRK2, GIRK4, or GIRK5 channels that exhibited prolonged single-channel open-time duration and enhanced activity compared with that of homomultimers. Expression of the corresponding GIRK1 (F137S) mutant alone resulted in appreciable channel activity with novel characteristics that was further enhanced upon coexpression with other GIRK subunits. Thus, although the F137 residue renders the GIRK1 subunit inactive, when combined with other GIRK heteromeric partners it alters their gating and contributes to their enhanced activity.     

Characterization of an inwardly rectifying potassium channel in the rabbit superior lacrimal gland

We have investigated the underlying assumptions in estimating cross-correlation rates between chemical shift anisotropy (CSA) and dipolar coupling mechanisms in a scalar-coupled two-spin IS system, from laboratory frame relaxation experiments. It has ben shown that for an arbitrary relaxation delay, the difference in relaxation rates of the individual components of an in-phase (or antiphase) doublet is not related to the CSA-dipolar coupling cross-correlation rate in a simple way. This is especially true in the case where the difference in the decay rates of the in-phase and antiphase terms of the density matrix becomes comparable to the magnitude of the scalar coupling between the two spins. Improved means of extracting cross-correlation rates in these cases are presented.     

Partial gene structure and assignment to chromosome 2q37 of the human inwardly rectifying K+ channel (Kir7.1) gene (KCNJ13)

The novel weakly inward rectifying potassium channel Kir7.1 is a low-conductance channel that is predominantly expressed in epithelial cells. Here we describe a partial genomic characterization and the chromosomal assignment of the human Kir7.1 gene (KCNJ13). Analysis of the genomic structure using a PCR-based approach revealed a single 2088-bp intron in the coding region of KCNJ13. PCR analysis of monochromosomal and radiation hybrid panels assigns KCNJ13 to band 2q37 between markers D2S331 and D2S345. In addition, a single nucleotide polymorphism (C524-->T), leading to an exchange of a Thr with an Ile residue at amino acid position 175, was found.