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.     

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.     

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.     

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.     

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.     

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.     

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.     

Blocking effects of benzyltetrahydropalmatine on delayed rectified K+ currents expressed in Xenopus oocytes and in toad oocytes

The effects of benzyltetrahydropalmatine (BTHP) on delayed rectified K+ currents (Ik) expressed in Xenopus oocytes and Ik of toad (Bufo bufo gargarizans) oocytes were studied. The Ik expressed in Xenopus oocytes was measured after microinjection of mRNA isolated from carp fish (C anratus L.) brains with double -microelectrode voltage clamp technique. The maximum and mean value of Ik expressed in Xenopus oocytes were 600 nA and 360 +/- 104 nA, respectively. BTHP reduced the current amplitude of Ik expressed in Xenopus oocytes in 10-1000 mumol.L-1 dose-dependently, EC50 was 29 mumol.L-1. Also, the reduction of Ik of toad oocytes was 9.1%, 29.1%, 54.7% and 68.6% by BTHP 10, 30, 100 and 1000 mumol.L-1, respectively, EC50 was 33 mumol.L-1. The results showed that BTHP possesses an inhibitory effect on Ik, the main ion mechanism of antiarrhythmic action of BTHP.     

Human D2 and D4 dopamine receptors couple through betagamma G-protein subunits to inwardly rectifying K+ channels (GIRK1) in a Xenopus oocyte expression system: selective antagonism by L-741,626 and L-745,870 respectively

OBJECTIVE: To develop a prognostic model, based on clinical and pathological data, to estimate the probability of micrometastasis in the sentinel lymph node in patients with malignant melanoma. DESIGN: Retrospective analytical study. SETTING: University medical center. PATIENTS: Two hundred fifteen patients with American Joint Committee on Cancer stages I and II cutaneous malignant melanoma underwent sentinel lymph node biopsy. MEASUREMENTS: Presence of microscopic melanoma in the sentinel lymph node(s). Clinical attributes recorded included age, sex, and location of the primary melanoma. Pathological attributes recorded before lymph node evaluation included ulceration, microsatellites, angiolymphatic invasion, mitotic rate, tumor infiltrating lymphocytes, and regression. RESULTS: Forty-six patients (21.4%) overall had a positive sentinel lymph node. Patients with tumor thickness ranging from 3.0 to 3.9 mm had the highest incidence (50%) of nodal involvement, followed by those with tumors 4.0 to 4.9 mm thick (41%). Patients with melanomas measuring greater than 4.9 mm thick and those between 1.0 and 2.9 mm had a similar rate of nodal involvement (16%-17%). Clinical characteristics had minimal correlation with nodal status in multivariate analysis. The total number of histological high-risk features was significantly correlated with sentinel lymph node involvement. Important pathological risk factors included ulceration, high mitotic rate, angiolymphatic invasion, and microsatellites. Patients with tumor thickness greater than 1.0 mm but lacking these features had a 14% risk of occult metastases. CONCLUSION: Among patients with clinically node-negative primary melanoma, the presence of 1 or more high-risk histological features significantly increases the incidence of microscopic nodal involvement and can be used to predict the likelihood of a positive sentinel lymph node biopsy.     

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.     

Purification and G protein subunit regulation of a phospholipase C-beta from Xenopus laevis oocytes

Xenopus oocytes exhibit both pertussis toxin-sensitive and -insensitive inositol lipid signaling responses to G protein-coupled receptor activation. The G protein subunits Galphai, Galphao, Galphaq, Galphas, and Gbetagamma all have been proposed to function as activators of phospholipase C in oocytes. Ma et al. (Ma, H.-W., Blitzer, R. D., Healy, E. C., Premont, R. T., Landau, E. M., and Iyengar, R. J. Biol. Chem. 268, 19915-19918) cloned a Xenopus phospholipase C (PLC-betaX) that exhibits homology to the PLC-beta class of isoenzymes. Although this enzyme was proposed to function as a signaling protein in the pertussis toxin-sensitive inositol lipid signaling pathway of oocytes, its regulation by G protein subunits has not been directly assessed. As such we have utilized baculovirus-promoted overexpression of PLC-betaX in Sf9 insect cells and have purified a recombinant 150-kDa isoenzyme. PLC-betaX catalyzes hydrolysis of phosphatidylinositol(4,5)bisphosphate and phosphatidylinositol(4)monophosphate, and reaction velocity is dependent on Ca2+. Recombinant PLC-betaX was activated by both Galphaq and Gbetagamma. PLC-betaX exhibited a higher apparent affinity for Galphaq than Gbetagamma, and Galphaq was more efficacious than Gbetagamma at lower concentrations of PLC-betaX. Relative to other PLC-beta isoenzymes, PLC-betaX was less sensitive to stimulation by Galphaq than PLC-beta1 but similar to PLC-beta2 and PLC-betaT. PLC-betaX was more sensitive to stimulation by Gbetagamma than PLC-beta1 but less sensitive than PLC-beta2 and PLC-betaT. In contrast PLC-betaX was not activated by the pertussis toxin substrate G proteins Galphai1, Galphai2, Galphai3, or Galphao. These results are consistent with the idea that PLC-betaX is regulated by alpha-subunits of the Gq family and by Gbetagamma and do not support the idea that alpha-subunits of pertussis toxin-sensitive G proteins are directly involved in regulation of this protein.     

Shaker and ether-à-go-go K+ channel subunits fail to coassemble in Xenopus oocytes

Members of different voltage-gated K+ channel subfamilies usually do not form heteromultimers. However, coassembly between Shaker and ether-à-go-go (eag) subunits, members of two distinct K+ channel subfamilies, was suggested by genetic and functional studies (Zhong and Wu. 1991. Science. 252: 1562-1564; Chen, M.-L., T. Hoshi, and C.-F. Wu. 1996. Neuron. 17:535-542). We investigated whether Shaker and eag form heteromultimers in Xenopus laevis oocytes using electrophysiological and biochemical approaches. Coexpression of Shaker and eag subunits produced K+ currents that were virtually identical to the sum of separate Shaker and eag currents, with no change in the kinetics of Shaker inactivation. According to the results of dominant negative and reciprocal coimmunoprecipitation experiments, the Shaker and eag proteins do not interact. We conclude that Shaker and eag do not coassemble to form heteromultimers in Xenopus oocytes.     

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.     

Number and stoichiometry of subunits in the native atrial G-protein-gated K+ channel, IKACh

The G-protein-regulated, inwardly rectifying K+ (GIRK) channels are critical for functions as diverse as heart rate modulation and neuronal post-synaptic inhibition. GIRK channels are distributed predominantly throughout the heart, brain, and pancreas. In recent years, GIRK channels have received a great deal of attention for their direct G-protein betagamma (Gbetagamma) regulation. Native cardiac IKACh is composed of GIRK1 and GIRK4 subunits (Krapivinsky, G., Gordon, E. A., Wickman, K. A., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). Here, we examine the quaternary structure of IKACh using a variety of complementary approaches. Complete cross-linking of purified atrial IKACh protein formed a single adduct with a total molecular weight that was most consistent with a tetramer. In addition, partial cross-linking of purified IKACh produced subsets of molecular weights consistent with monomers, dimers, trimers, and tetramers. Within the presumed protein dimers, GIRK1-GIRK1 and GIRK4-GIRK4 adducts were formed, indicating that the tetramer was composed of two GIRK1 and two GIRK4 subunits. This 1:1 GIRK1 to GIRK4 stoichiometry was confirmed by two independent means, including densitometry of both silver-stained and Western-blotted native atrial IKACh. Similar experimental results could potentially be obtained if GIRK1 and GIRK4 subunits assembled randomly as 2:2 and equally sized populations of 3:1 and 1:3 tetramers. We also show that GIRK subunits may form homotetramers in expression systems, although the evidence to date suggests that GIRK1 homotetramers are not functional. We conclude that the inwardly rectifying atrial K+ channel, IKACh, a prototypical GIRK channel, is a heterotetramer and is most likely composed of two GIRK1 subunits and two GIRK4 subunits.     

KCR1, a membrane protein that facilitates functional expression of non-inactivating K+ currents associates with rat EAG voltage-dependent K+ channels

Cerebellar granule neurons possess a non-inactivating K+ current, which controls resting membrane potentials and modulates the firing rate by means of muscarinic agonists. kcr1 was cloned from the cerebellar cDNA library by suppression cloning. KCR1 is a novel protein with 12 putative transmembrane domains and enhances the functional expression of the cerebellar non-inactivating K+ current in Xenopus oocytes. KCR1 also accelerates the activation of rat EAG K+ channels expressed in Xenopus oocytes or in COS-7 cells. Far-Western blotting revealed that KCR1 and EAG proteins interacted with each other by means of their C-terminal regions. These results suggest that KCR1 is the regulatory component of non-inactivating K+ channels.     

Cloning and functional expression of a voltage-gated calcium channel alpha1 subunit from jellyfish

Voltage-gated Ca2+ channels in vertebrates comprise at least seven molecular subtypes, each of which produces a current with distinct kinetics and pharmacology. Although several invertebrate Ca2+ channel alpha1 subunits have also been cloned, their functional characteristics remain unclear, as heterologous expression of a full-length invertebrate channel has not previously been reported. We have cloned a cDNA encoding the alpha1 subunit of a voltage-gated Ca2+ channel from the scyphozoan jellyfish Cyanea capillata, one of the earliest existing organisms to possess neural and muscle tissue. The deduced amino acid sequence of this subunit, named CyCaalpha1, is more similar to vertebrate L-type channels (alpha1S, alpha1C, and alpha1D) than to non-L-type channels (alpha1A, alpha1B, and alpha1E) or low voltage-activated channels (alpha1G). Expression of CyCaalpha1 in Xenopus oocytes produces a high voltage-activated Ca2+ current that, unlike vertebrate L-type currents, is only weakly sensitive to 1,4-dihydropyridine or phenylalkylamine Ca2+ channel blockers and is not potentiated by the agonist S(-)-BayK 8644. In addition, the channel is less permeable to Ba2+ than to Ca2+ and is more permeable to Sr2+. CyCaalpha1 thus represents an ancestral L-type alpha1 subunit with significant functional differences from mammalian L-type channels.     

Progesterone-induced DNA polymerase activity in full-grown oocytes of Xenopus laevis (1)

DNA polymerase activity increases in full-grown oocytes of Xenopus laevis during in vitro progesterone-induced maturation. This increase is inhibited by cycloheximide. The presence of the oocyte's nucleus (germinal vesicle) seems essential for the induction of this increase: in previously enucleated oocytes, the level of DNA polymerase activity does not change during progesterone treatment. Furthermore, a new form of DNA polymerase is detectable by DEAE chromatography in in vitro matured oocytes.     

Expression of a human renal sodium nucleoside cotransporter in Xenopus laevis oocytes

In this study, Xenopus laevis oocytes injected with poly(A)+ RNA (mRNA) isolated from human kidney were used to express a Na(+)-nucleoside cotransporter. Na(+)-stimulated [3H]thymidine uptake was enhanced 2-3-fold in oocytes injected with 50 ng poly(A)+ RNA and 4-5-fold in oocytes injected with 20 ng of a size-fractionated human renal cortex mRNA fragment (2-3 kb) in comparison with water-injected oocytes. Na(+)-dependent thymidine uptake in oocytes injected with the 2-3 kb mRNA fragment was inhibited significantly by thymidine and guanosine but not by formycin B, consistent with the N4 Na(+)-nucleoside cotransporter. The Km (28 microM) of Na(+)-dependent thymidine uptake in the oocytes injected with the 2-3 kb mRNA fragment was similar to the Km (27 microM) of Na(+)-dependent thymidine uptake obtained in human renal brush border membrane vesicles. These data suggest for the first time that a Na(+)-nucleoside cotransporter from human kidney can be expressed in X. laevis oocytes.