Four novel splice variants of sulfonylurea receptor 1

ATP-sensitiveK⁺ (K_ATP) channels are composed of pore-formingKir6.x subunits and regulatory sulfonylurea receptor (SUR) subunits. SURs are ATP-binding cassette proteins with two nucleotide-binding folds (NBFs) and binding sites for sulfonylureas, like glibenclamide, and for channel openers. Here we report the identification and functional characterization of four novel splice forms of guinea pigSUR1. Three splice forms originate from alternative splicing of theregion coding for NBF1 and lack exons 17 (SUR117), 19 (SUR119),or both (SUR11719). The fourth (SUR1C) is a COOH-terminal SUR1-fragment formed by exons 31-39 containing the last twotransmembrane segments and the COOH terminus of SUR1. RT-PCR analysisshowed that these splice forms are expressed in several tissues with strong expression of SUR1C in cardiomyocytes. Confocal microscopy usingenhanced green fluorescent protein-tagged SUR or Kir6.x did not provideany evidence for involvement of these splice forms in themitochondrial K_ATP channel. Only SUR1 and SUR117 showed high-affinity binding of glibenclamide (K_d 2 nM in the presence of 1 mM ATP) and formed functional K_ATPchannels upon coexpression with Kir6.2.

     

Potent inhibition of the aortic smooth muscle maxi-K channel by clinical doses of ethanol

We investigated the effects ofclinically relevant ethanol concentrations (5-20 mM) on thesingle-channel kinetics of bovine aortic smooth muscle maxi-K channelsreconstituted in lipid bilayers (1:1palmitoyl-oleoyl-phosphatidylethanolamine:palmitoyl-oleoyl-phosphatidylcholine). Ethanol at 10 and 20 mMdecreased the channel open probability (P_o) by75 ± 20.3% mainly by increasing the mean closed time (+82 to+960%, n = 7). In some instances, ethanol alsodecreased the mean open time (40.8 ± 22.5%). TheP_o-voltage relation in the presence of 20 mMethanol exhibited a rightward shift in the midpoint of voltageactivation (V_1/2  17 mV), a slightlysteeper relationship (change in slope factor, k,  2.5 mV), and a decreased maximum P_o (from~0.82 to ~0.47). Interestingly, channels inhibited by ethanol atlow Ca²⁺ concentrations (2.5 µM) were veryresistant to ethanol in the presence of increased Ca²⁺ ( 20 µM). Alcohol consumption in clinically relevant amounts may alterthe contribution of maxi-K channels to the regulation of arterial tone.

     

Pharmacological modulation of ion transport across wild-type and DeltaF508 CFTR-expressing human bronchial epithelia

Forskolin,UTP, 1-ethyl-2-benzimidazolinone (1-EBIO), NS004, 8-methoxypsoralen(Methoxsalen; 8-MOP), and genistein were evaluated for theireffects on ion transport across primary cultures of human bronchialepithelium (HBE) expressing wild-type (wt HBE) and F508(F-HBE) cystic fibrosis transmembrane conductance regulator. In wtHBE, the baseline short-circuit current (I_sc)averaged 27.0 ± 0.6 µA/cm² (n = 350). Amiloride reduced this I_sc by 13.5 ± 0.5 µA/cm² (n = 317). In F-HBE,baseline I_sc was 33.8 ± 1.2 µA/cm² (n = 200), and amiloride reducedthis by 29.6 ± 1.5 µA/cm² (n = 116), demonstrating the characteristic hyperabsorption of Na⁺ associated with cystic fibrosis (CF). In wt HBE,subsequent to amiloride, forskolin induced a sustained,bumetanide-sensitive I_sc(I_sc = 8.4 ± 0.8 µA/cm²; n = 119). Addition ofacetazolamide, 5-(N-ethyl-N-isopropyl)-amiloride, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid further reduced I_sc, suggesting forskolin also stimulatesHCO₃ secretion. This was confirmed by ionsubstitution studies. The forskolin-induced I_scwas inhibited by 293B, Ba²⁺, clofilium, and quinine,whereas charybdotoxin was without effect. In F-HBE the forskolinI_sc response was reduced to 1.2 ± 0.3 µA/cm² (n = 30). In wt HBE, mucosal UTPinduced a transient increase in I_sc ( I_sc = 15.5 ± 1.1 µA/cm²;n = 44) followed by a sustained plateau, whereas inF-HBE the increase in I_sc was reduced to5.8 ± 0.7 µA/cm² (n = 13). In wtHBE, 1-EBIO, NS004, 8-MOP, and genistein increased I_sc by 11.6 ± 0.9 (n = 20), 10.8 ± 1.7 (n = 18), 10.0 ± 1.6 (n = 5), and 7.9 ± 0.8 µA/cm²(n = 17), respectively. In F-HBE, 1-EBIO, NS004, and8-MOP failed to stimulate Cl secretion. However, additionof NS004 subsequent to forskolin induced a sustained Clsecretory response (2.1 ± 0.3 µA/cm²,n = 21). In F-HBE, genistein alone stimulatedCl secretion (2.5 ± 0.5 µA/cm²,n = 11). After incubation of F-HBE at 26°C for24 h, the responses to 1-EBIO, NS004, and genistein were allpotentiated. 1-EBIO and genistein increased Na⁺ absorptionacross F-HBE, whereas NS004 and 8-MOP had no effect. Finally,Ca²⁺-, but not cAMP-mediated agonists, stimulatedK⁺ secretion across both wt HBE and F-HBE in aglibenclamide-dependent fashion. Our results demonstrate thatpharmacological agents directed at both basolateral K⁺ andapical Cl conductances directly modulate Clsecretion across HBE, indicating they may be useful in ameliorating theion transport defect associated with CF.

     

PKA and arachidonic acid activation of human recombinant ClC-2 chloride channels

An HEK-293 cell line stably expressing the humanrecombinant ClC-2 Cl channel was used in patch-clampstudies to study its regulation. The relative permeabilityP_x/P_Cl calculated fromreversal potentials was I > Cl = NO₃ = SCNBr. Theabsolute permeability calculated from conductance ratios wasCl = Br = NO₃  SCN > I. The channel was activatedby cAMP-dependent protein kinase (PKA), reduced extracellular pH, oleicacid (C:18 cis9), elaidic acid (C:18trans9), arachidonic acid (AA; C:20cis5,8,11,14), and by inhibitors of AA metabolism,5,8,11,14-eicosatetraynoic acid (ETYA; C:20trans5,8,11,14),-methyl-4-(2-methylpropyl)benzeneacetic acid (ibuprofen), and2-phenyl-1,2-benzisoselenazol-3-[2H]-one (PZ51, ebselen). ClC-2Cl channels were activated by a combination of forskolinplus IBMX and were inhibited by the cell-permeant myristoylated PKAinhibitor (mPKI). Channel activation by reduction of bath pH wasincreased by PKA and prevented by mPKI. AA activation of the ClC-2Cl channel was not inhibited by mPKI or staurosporine andwas therefore independent of PKA or protein kinase C activation.

     

Calsequestrin targeting to sarcoplasmic reticulum of skeletal muscle fibers

Calsequestrin (CS) is the low-affinity, high-capacity calcium binding protein segregated to the lumen of terminal cisternae (TC) of the sarcoplasmic reticulum (SR). The physiological role of CS in controlling calcium release from the SR depends on both its intrinsic properties and its localization. The mechanisms of CS targeting were investigated in skeletal muscle fibers and C₂C₁₂ myotubes, a model of SR differentiation, with four deletion mutants of epitope (hemagglutinin, HA)-tagged CS: CS-HA24_NH2, CS-HA2D, CS-HA3D, and CS-HAHT, a double mutant of the NH₂ terminus and domain III. As judged by immunofluorescence of transfected skeletal muscle fibers, only the double CS-HA mutant showed a homogeneous distribution at the sarcomeric I band, i.e., it did not segregate to TC. As shown by subfractionation of microsomes derived from transfected skeletal muscles, CS-HAHT was largely associated to longitudinal SR whereas CS-HA was concentrated in TC. In C₂C₁₂ myotubes, as judged by immunofluorescence, not only CS-HAHT but also CS-HA3D and CS-HA2D were not sorted to developing SR. Condensation competence, a property referable to CS oligomerization, was monitored for the several CS-HA mutants in C₂C₁₂ myoblasts, and only CS-HA3D was found able to condense. Together, the results indicate that 1) there are at least two targeting sequences at the NH₂ terminus and domain III of CS, 2) SR-specific target and structural information is contained in these sequences, 3) heterologous interactions with junctional SR proteins are relevant for segregation, 4) homologous CS-CS interactions are involved in the overall targeting process, and 5) different targeting mechanisms prevail depending on the stage of SR differentiation. protein-protein interactions; oligomerization; intracellular sorting     

Rat liver GTP-binding proteins mediate changes in mitochondrial membrane potential and organelle fusion

The variety of mitochondrial morphology in healthy and diseasedcells can be explained by regulated mitochondrial fusion. Previously, amitochondrial outer membrane fraction containing fusogenic, aluminumfluoride (AlF₄)-sensitiveGTP-binding proteins (mtg) was separated from rat liver (J. D. Cortese,Exp. Cell Res. 240: 122-133,1998). Quantitative confocal microscopy now reveals that mtgtransiently increases mitochondrial membrane potential () when added to permeabilized rat hepatocytes(15%), rat fibroblasts (19%), and rabbit myocytes (10%). This largemtg-induced increment is blocked by fusogenic GTPase-specificmodulators such as guanosine 5'-O-(3-thiotriphosphate),excess GTP (>100 µM), andAlF₄, suggesting a linkage between and mitochondrial fusion. Accordingly, stereometric analysisshows that decreasing or ATP synthesis with respiratory inhibitors limits mtg- andAlF₄-induced mitochondrial fusion. Also, a specific G protein inhibitor(Bordetellapertussis toxin) hyperpolarizesmitochondria and leads to a loss ofAlF₄-dependent mitochondrialfusion. These results place mtg-induced changes upstream ofAlF₄-induced mitochondrial fusion,suggesting that GTPases exert -dependent control of the fusionprocess. Mammalian mitochondrial morphology thus can be modulated bycellular energetics.     

Ca2+ influx inhibits voltage-dependent and augments Ca2+-dependent K+ currents in arterial myocytes

These experiments were performed to determine the effects ofreducing Ca²⁺ influx(Ca_in) onK⁺ currents(I_K) inmyocytes from rat small mesenteric arteries by1) adding externalCd²⁺ or2) lowering externalCa²⁺ to 0.2 mM. When measured froma holding potential (HP) of 20 mV(I_K20),decreasing Ca_in decreasedI_K at voltageswhere it was active (>0 mV). When measured from a HP of 60 mV(I_K60),decreasing Ca_in increasedI_K at voltagesbetween 30 and +20 mV but decreased I_K at voltagesabove +40 mV. Difference currents(I_K) weredetermined by digital subtraction of currents recorded under controlconditions from those obtained whenCa_in was decreased. At testvoltages up to 0 mV,I_K60 exhibitedkinetics similar to controlI_K60, with rapidactivation to a peak followed by slow inactivation. At 0 mV, peakI_K60 averaged75 ± 13 pA (n = 8) withCd²⁺ and 120 ± 20 pA(n = 9) with lowCa²⁺ concentration. At testvoltages from 0 to +60 mV,I_K60 always had an early positive peak phase, but its apparent "inactivation" increased with voltage and its steady value became negative above +20mV. At +60 mV, the initial peakI_K60 averaged115 ± 18 pA with Cd²⁺ and 187 ± 34 pA with low Ca²⁺. With 10 mM pipette BAPTA, Cd²⁺ produced asmall inhibition ofI_K20 but stillincreased I_K60 between 30 and +10 mV. InCa²⁺-free external solution,Cd²⁺ only decreased bothI_K20 andI_K60. In thepresence of iberiotoxin (100 nM) to inhibitCa²⁺-activatedK⁺ channels(K_Ca),Cd²⁺ increasedI_K60 at allvoltages positive to 30 mV while BAY K 8644 (1 µM) decreasedI_K60. Theseresults suggest that Ca_in, through L-type Ca²⁺ channels and perhapsother pathways, increases K_Ca(i.e., I_K20) and decreases voltage-dependent K⁺currents in this tissue. This effect could contribute to membrane depolarization and force maintenance.

     

Activation of Delta F508 CFTR in an epithelial monolayer

The F508 mutation leads to retention of cystic fibrosistransmembrane conductance regulator (CFTR) in the endoplasmic reticulum and rapid degradation by the proteasome and other proteolytic systems.In stably transfected LLC-PK₁(porcine kidney) epithelial cells, F508 CFTR conforms to thisparadigm and is not present at the plasma membrane. WhenLLC-PK₁ cells or human nasal polyp cells derived from a F508 homozygous patient are grown on plastic dishes and treated with an epithelial differentiating agent (DMSO, 2%for 4 days) or when LLC-PK₁ cellsare grown as polarized monolayers on permeable supports, plasmamembrane F508 CFTR is significantly increased. Moreover, whenconfluent LLC-PK₁ cells expressingF508 CFTR were treated with DMSO and mounted in an Ussing chamber, afurther increase in cAMP-activated short-circuit current (i.e., ~7µA/cm²;P < 0.00025 compared with untreatedcontrols) was observed. No plasma membrane CFTR was detected after DMSOtreatment in nonepithelial cells (mouse L cells) expressing F508CFTR. The experiments describe a way to augment F508 CFTR maturationin epithelial cells that appears to act through a novel mechanism andallows insertion of functional F508 CFTR in the plasma membranes oftransporting cell monolayers. The results raise the possibility thatincreased epithelial differentiation might increase the delivery ofF508 CFTR from the endoplasmic reticulum to the Golgi, where theF508 protein is shielded from degradative pathways such as theproteasome and allowed to mature.

     

Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein

The patch-clamp technique was used to investigate the effects ofthe isoflavone genistein on disease-causing mutations (G551D andF508) of the cystic fibrosis transmembrane conductance regulator (CFTR). In HeLa cells recombinantly expressing thetrafficking-competent G551D-CFTR, the forskolin-stimulated Cl currentswere small, and average open probability of G551D-CFTR wasP_o = 0.047 ± 0.019. Addition of genistein activated Cl currents~10-fold, and the P_o of G551D-CFTRincreased to 0.49 ± 0.12, which is aP_o similar towild-type CFTR. In cystic fibrosis (CF) epithelial cells homozygous forthe trafficking-impaired F508 mutation, forskolin and genistein activated Cl currents only after 4-phenylbutyrate treatment. These datasuggested that genistein activated CFTR mutants that were present inthe cell membrane. Therefore, we tested the effects of genistein in CFpatients with the G551D mutation in nasal potential difference (PD)measurements in vivo. The perfusion of the nasal mucosa of G551D CFpatients with isoproterenol had no effect; however, genisteinstimulated Cl-dependent nasal PD by, on average, 2.4 ± 0.6 mV, which corresponds to 16.9% of the responses (to -adrenergicstimulation) found in healthy subjects.

     

Effect of reactive oxygen species on NH4+ permeation in Xenopus laevis oocytes

To investigate theeffects of reactive oxygen species (ROS) on NHpermeation in Xenopus laevis oocytes, we used intracellulardouble-barreled microelectrodes to monitor the changes in membranepotential (V_m) and intracellular pH(pH_i) induced by a 20 mM NH₄Cl-containingsolution. Under control conditions, NH₄Cl exposure induceda large membrane depolarization (to V_m = 4.0 ± 1.5 mV; n = 21) and intracellularacidification [reaching a change in pH_i(pH_i) of 0.59 ± 0.06 pH units in 12 min]; theinitial rate of cell acidification (dpH_i/dt) was0.06 ± 0.01 pH units/min. Incubation of the oocytes in thepresence of H₂O₂ or -amyloid protein had nomarked effect on the NH₄Cl-induced pH_i. Bycontrast, in the presence of photoactivated rose bengal (RB),tert-butyl-hydroxyperoxide (t-BHP), orxanthine/xanthine oxidase (X/XO), the same experimental maneuverinduced significantly greater pH_i anddpH_i/dt. These increases in pH_iand dpH_i/dt were prevented by the ROS scavengershistidine and desferrioxamine, suggesting involvement of the reactivespecies ¹gO₂ and ·OH. Using thevoltage-clamp technique to identify the mechanism underlying theROS-measured effects, we found that RB induced a large increase in theoocyte membrane conductance (G_m). ThisRB-induced G_m increase was prevented by 1 mMdiphenylamine-2-carboxylate (DPC) and by a low Na⁺concentration in the bath. We conclude that RB, t-BHP, andX/XO enhance NH influx into the oocyte via activationof a DPC-sensitive nonselective cation conductance pathway.

     

Cryptdin-3 induces novel apical conductance(s) in Cl- secretory, including cystic fibrosis, epithelia

Opening ofanion-conductive pathways in apical membranes of secretory cells liningmucosal surfaces is a critical step in salt and water secretion and,thus, hydration of sites including airway and intestine. In intestine,Paneth cells are positioned at the base of the secretory gland (crypt)and release defensin peptide, in mice termed cryptdins, into the cryptlumen. Because at least some defensins have been shown to formanion-conductive channels in phospholipid bilayers, we tested whetherthese endogenous antimicrobial peptides could act as soluble inducersof channel-like activity when applied to apical membranes. To directlyevaluate the possibility of cryptdin-3-mediated apical anionconductance (G_ap), we have utilized amphotericinB to selectively permeabilize basolateral membranes of electricallytight monolayers of polarized human intestinal secretory epithelia (T84cells), thus isolating the apical membrane for study. Cryptdin-3induces G_ap that is voltage independent(G_ap = 1.90 ± 0.60 mS/cm²) and exhibits ion selectivity contrasting to thatelicited by forskolin or thapsigargin (for cryptdin-3,Cl = gluconate; for forskolin and thapsigargin,Cl gluconate). We cannot exclude the possibility thatthe macroscopic current induced by cryptdin could be the sum of cationand Cl currents. Cryptdin-3 induces a current inbasolaterally permeabilized epithelial monolayers derived from airwaycells harboring the F508 mutation of cystic fibrosis (CF;G_ap = 0.80 ± 0.06 mS/cm²), demonstrating that cryptdin-3 restores anionsecretion in CF cells; this occurs independently of the CFtransmembrane conductance regulator channel. These results support theidea that cryptdin-3 may associate with apical membranes ofCl-secreting epithelia and self-assemble into conductingchannels capable of mediating a physiological response.

     

Role of beta 1- and beta 3-adrenoceptors in the regulation of lipolysis and thermogenesis in rat brown adipocytes

To evaluate the physiological functions of₁-,₂-, and₃-adrenoceptors (ARs) in brownadipose tissue, the lipolytic and respiratory effects of variousadrenergic agonists and antagonists were studied in rat brownadipocytes. The -agonists stimulated both lipolysis and respiration(8-10 times above basal levels), with the following order ofpotency (concentration eliciting 50% of maximum response):CL-316243 (₃) > BRL-37344(₃) > isoproterenol (mainly₁/₂) > norepinephrine (NE; mainly₁/₂) > epinephrine (mainly₁/₂) dobutamine (₁)  procaterol (₂). Schild plot coefficients of competitive inhibition experiments using ICI-89406 (₁ antagonist) revealed thatmore than one type of receptor mediates NE action. It is concluded fromour results that 1) NE, at low plasma levels (1-25 nM), stimulates lipolysis and respiration mainly through ₁-ARs,2) NE, at higher levels, stimulateslipolysis and respiration via both₁- and₃-ARs,3)₂-ARs play only a minor role,and 4)₃-ARs may represent thephysiological receptors for the high NE concentrations in the synapticcleft, where the high-affinity₁-ARs are presumablydesensitized. It is also suggested that lipolysis represents theflux-generating step regulating mitochondrial respiration.

     

Regulation of a cloned epithelial Na+ channel by its beta - and gamma -subunits

Using the Xenopus oocyteexpression system, we examined the mechanisms by which the - and-subunits of an epithelial Na⁺channel (ENaC) regulate -subunit channel activity and the mechanisms by which -subunit truncations cause ENaC activation. Expression of-ENaC alone produced small amiloride-sensitive currents (43 ± 10 nA, n = 7). These currentsincreased >30-fold with the coexpression of - and -ENaC to1,476 ± 254 nA (n = 20).This increase was accompanied by a 3.1- and 2.7-fold increase ofmembrane fluorescence intensity in the animal and vegetal poles of theoocyte, respectively, with use of an antibody directed against the-subunit of ENaC. Truncation of the last 75 amino acids of the-subunit COOH terminus, as found in the original pedigree ofindividuals with Liddle's syndrome, caused a 4.4-fold(n = 17) increase of theamiloride-sensitive currents compared with wild-type -ENaC.This was accompanied by a 35% increase of animal pole membranefluorescence intensity. Injection of a 30-amino acid peptide withsequence identity to the COOH terminus of the human -ENaCsignificantly reduced the amiloride-sensitive currents by 40-50%.These observations suggest a tonic inhibitory role on the channel'sopen probability (P_o) by the COOH terminus of -ENaC. We conclude that the changes of current observed with coexpression of the - and -subunits or those observed with -subunit truncation are likely the result ofchanges of channel density in combination with large changes ofP_o.

     

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells

HumanNa⁺-K⁺-ATPase₁₁,₂₁, and ₃₁heterodimers were expressed individually in yeast, and ouabainbinding and ATP hydrolysis were measured in membrane fractions. Theouabain equilibrium dissociation constant was 13-17 nM for₁₁ and ₃₁at 37°C and 32 nM for ₂₁, indicatingthat the human -subunit isoforms have a similar high affinity forcardiac glycosides. K_0.5 values for antagonism of ouabain binding by K⁺ were ranked in order as follows:₂ (6.3 ± 2.4 mM) > ₃(1.6 ± 0.5 mM)  ₁ (0.9 ± 0.6 mM),and K_0.5 values for Na⁺ antagonismof ouabain binding to all heterodimers were 9.5-13.8 mM. Themolecular turnover for ATP hydrolysis by₁₁ (6,652 min¹) was abouttwice as high as that by ₃₁ (3,145 min¹). These properties of the human heterodimersexpressed in yeast are in good agreement with properties of the humanNa⁺-K⁺-ATPase expressed in Xenopusoocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-DHorisberger, L Lelievie, and K Geering. J Biol Chem275: 1976-1986, 2000). In contrast to Na⁺ pumpsexpressed in Xenopus oocytes, the₂₁ complex in yeast membranes wassignificantly less stable than ₁₁ or₃₁, resulting in a lower functionalexpression level. The ₂₁ complex was also more easily denatured by SDS than was the₁₁ or the₃₁ complex.

     

An immunologically distinct beta -adaptin on tubulovesicles of gastric oxyntic cells

Clathrin and the -adaptin subunit of the AP-1 clathrinadaptor have been previously identified on H-K-ATPase-richtubulovesicles from gastric acid secretory (oxyntic) cells [C. T. Okamoto, S. M. Karam, Y. Y. Jeng, J. G. Forte, and J. Goldenring.Am. J. Physiol. 274 (Cell Physiol. 43):C1017-C1029]. We further characterized this AP-1 adaptorfrom rabbit and hog tubulovesicles biochemically and immunologically.Clathrin coat proteins were stripped from purified tubulovesicularmembranes and fractionated by hydroxyapatite chromatography. The AP-1adaptor appears to elute at 200 mM sodium phosphate, based on thepresence of proteins in this fraction that are immunoreactive withantibodies against three of the four subunits of this heterotetramericcomplex: the -, µ₁-, and₁-adaptin subunits. Althoughthe putative -adaptin subunit in this fraction is not immunoreactivewith the anti--adaptin monoclonal antibody (MAb), this -adaptinis immunoreactive with polyclonal antibodies (PAbs) directed againstthe peptide sequenceGly⁶²⁵-Asp-Leu-Leu-Gly-Asp-Leu-Leu-Asn-Leu-Asp-Leu-Gly-Pro-Pro-Val⁶⁴⁰,a region conserved between ₁-and ₂-adaptins that is thought to be involved in the binding of clathrin heavy chain.Immunoprecipitation of the AP-1 adaptor complex from this fraction withanti--adaptin MAb 100/3 resulted in the coimmunoprecipitation of the-adaptin that did not react with the anti--adaptin MAb but didreact with the anti--adaptin PAbs. In contrast, immunoprecipitationof the AP-1 adaptor complex from crude clathrin-coated vesicles from brain resulted in the coimmunoprecipitation of a -adaptin that wasrecognized by both the anti--adaptin MAb and PAbs. These resultssuggest that the tubulovesicular AP-1 adaptor complex may be distinctfrom that found in the trans-Golgi network and may contain animmunologically distinct -adaptin. This immunologically distinct-adaptin may be diagnostic of apical tubulovesicular endosomes ofepithelial cells.

     

Evidence for functional role of epsilonPKC isozyme in the regulation of cardiac Na(+) channels

Investigation of the role ofindividual protein kinase C (PKC) isozymes in the regulation ofNa⁺ channels has been largely limited by the lack ofisozyme-selective modulators. Here we used a novel peptide-specificactivator (V1-7) of PKC and other peptide isozyme-specificinhibitors in addition to the general PKC activator phorbol12-myristate 13-acetate (PMA) to dissect the role of individual PKCs inthe regulation of the human cardiac Na⁺ channel hH1,heterologously expressed in Xenopus oocytes. Peptides wereinjected individually or in combination into the oocyte. Whole cellNa⁺ current (I_Na) was recorded usingtwo-electrode voltage clamp. V1-7 (100 nM) and PMA (100 nM)inhibited I_Na by 31 ± 5% and 44 ± 8% (at 20 mV), respectively. These effects were not seen with thescrambled peptide for V1-7 (100 nM) or the PMA analog4-phorbol 12,13-didecanoate (100 nM). However, V1-7-and PMA-induced I_Na inhibition was abolished byV1-2, a peptide-specific antagonist of PKC. Furthermore,PMA-induced I_Na inhibition was not altered by100 nM peptide-specific inhibitors for -, -, -, or PKC. PMAand V1-7 induced translocation of PKC from soluble toparticulate fraction in Xenopus oocytes. This translocationwas antagonized by V1-2. In native rat ventricular myocytes,PMA and V1-7 also inhibited I_Na; thisinhibition was antagonized by V1-2. In conclusion, the resultsprovide evidence for selective regulation of cardiac Na⁺channels by PKC isozyme.

     

Modulation of human neuronal alpha 1E-type calcium channel by alpha 2delta -subunit

Calcium channels are composed of a pore-forming subunit,₁, and at least two auxiliarysubunits, - and₂-subunits. It is well knownthat -subunits regulate most of the properties of the channel. Thefunction of ₂-subunit isless understood. In this study, the effects of the calcium channel₂-subunit on the neuronal_1E voltage-gated calciumchannel expressed in Xenopus oocyteswas investigated without and with simultaneous coexpression of eitherthe _1b- or the_2a-subunit. Most aspects of_1E function were affected by₂. Thus₂ caused a shift in thecurrent-voltage and conductance-voltage curves toward more positivepotentials and accelerated activation, deactivation, and theinstallation of the inactivation process. In addition, the efficiencywith which charge movement is coupled to pore opening assessed bydetermining ratios of limiting conductance to limiting charge movementwas decreased by ₂ byfactors that ranged from 1.6 (P < 0.01) for _1E-channels to 3.0 (P < 0.005) for_1E1b-channels. These results indicate that₂ facilitates the expressionand the maturation of_1E-channels and converts thesechannels into molecules responding more rapidly to voltage.

     

Genistein activates CFTR-mediated Cl(-) secretion in the murine trachea and colon

The action of the isoflavonegenistein on the cystic fibrosis transmembrane conductance regulator(CFTR) has been studied in many cell systems but not in intact murinetissues. We have investigated the action of genistein on murine tissuesfrom normal and cystic fibrosis (CF) mice. Genistein increased theshort-circuit current (I_sc) in tracheal(16.4 ± 2.8 µA/cm²) and colonic (40.0 ± 4.4 µA/cm²) epithelia of wild-type mice. This increase wasinhibited by furosemide, diphenylamine-2-carboxylate, andglibenclamide, but not by DIDS. In contrast, genistein produced nosignificant change in the I_sc of the trachealepithelium (0.9 ± 1.1 µA/cm²) and decreased theI_sc of colons from CF null (13.1 ± 2.3 µA/cm²) and F508 mice (10.3 ± 1.3 µA/cm²). Delivery of a human CFTRcDNA-liposome complex to the airways of CF null mice restored thegenistein response in the tracheas to wild-type levels. Tracheas fromF508 mice were also studied: 46% of trachea showed no response togenistein, whereas 54% gave an increase in I_scsimilar to that in wild type. We conclude that genistein activatesCFTR-mediated Cl secretion in the murine trachea anddistal colon.

     

Astrocytes from Na(+)-K(+)-Cl(-) cotransporter-null mice exhibit absence of swelling and decrease in EAA release

We reported previously that inhibition ofNa⁺-K⁺-Cl cotransporter isoform 1 (NKCC1) by bumetanide abolishes high extracellular K⁺concentration ([K⁺]_o)-induced swelling andintracellular Cl accumulation in rat cortical astrocytes.In this report, we extended our study by using cortical astrocytes fromNKCC1-deficient (NKCC1^/) mice. NKCC1 protein andactivity were absent in NKCC1^/ astrocytes.[K⁺]_o of 75 mM increased NKCC1 activityapproximately fourfold in NKCC1^+/+ cells (P < 0.05) but had no effect in NKCC1^/ astrocytes.Intracellular Cl was increased by 70% inNKCC1^+/+ astrocytes under 75 mM[K⁺]_o (P < 0.05) butremained unchanged in NKCC1^/ astrocytes. Baselineintracellular Na⁺ concentration([Na⁺]_i) in NKCC1^+/+ astrocyteswas 19.0 ± 0.5 mM, compared with 16.9 ± 0.3 mM[Na⁺]_i in NKCC1^/ astrocytes(P < 0.05). Relative cell volume ofNKCC1^+/+ astrocytes increased by 13 ± 2% in 75 mM[K⁺]_o, compared with a value of 1.0 ± 0.5% in NKCC1^/ astrocytes (P < 0.05).Regulatory volume increase after hypertonic shrinkage was completelyimpaired in NKCC1^/ astrocytes.High-[K⁺]_o-induced ¹⁴C-labeledD-aspartate release was reduced by ~30% inNKCC1^/ astrocytes. Our study suggests that stimulationof NKCC1 is required for high-[K⁺]_o-inducedswelling, which contributes to glutamate release from astrocytes underhigh [K⁺]_o.

     

Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells

We investigated theeffects of epidermal growth factor (EGF) on activeNa⁺ absorption by alveolarepithelium. Rat alveolar epithelial cells (AEC) were isolated andcultivated in serum-free medium on tissue culture-treated polycarbonatefilters. mRNA for rat epithelial Na⁺ channel (rENaC) -, -,and -subunits and Na⁺ pump₁- and₁-subunits were detected inday 4 monolayers by Northern analysisand were unchanged in abundance in day5 monolayers in the absence of EGF. Monolayerscultivated in the presence of EGF (20 ng/ml) for 24 h fromday 4 to day5 showed an increase in both₁ and₁Na⁺ pump subunit mRNA but noincrease in rENaC subunit mRNA. EGF-treated monolayers showed parallelincreases in Na⁺ pump₁- and₁-subunit protein by immunoblotrelative to untreated monolayers. Fixed AEC monolayers demonstratedpredominantly membrane-associated immunofluorescent labeling withanti-Na⁺ pump₁- and₁-subunit antibodies, withincreased intensity of cell labeling for both subunits seen at 24 hfollowing exposure to EGF. These changes inNa⁺ pump mRNA and protein precededa delayed (>12 h) increase in short-current circuit (measure ofactive transepithelial Na⁺transport) across monolayers treated with EGF compared with untreated monolayers. We conclude that EGF increases activeNa⁺ resorption across AECmonolayers primarily via direct effects onNa⁺ pump subunit mRNA expressionand protein synthesis, leading to increased numbers of functionalNa⁺ pumps in the basolateralmembranes.