Wanderlust kinetics and variable Ca(2+)-sensitivity of Drosophila, a large conductance Ca(2+)-activated K+ channel, expressed in oocytes

Cloned large conductance Ca(2+)-activated K+ channels (BK or maxi-K+ channels) from Drosophila (dSlo) were expressed in Xenopus oocytes and studied in excised membrane patches with the patch-clamp technique. Both a natural variant and a mutant that eliminated a putative cyclic AMP-dependent protein kinase phosphorylation site exhibited large, slow fluctuations in open probability with time. These fluctuations, termed "wanderlust kinetics," occurred with a time course of tens of seconds to minutes and had kinetic properties inconsistent with simple gating models. Wanderlust kinetics was still observed in the presence of 5 mM caffeine or 50 nM thapsigargin, or when the Ca2+ buffering capacity of the solution was increased by the addition of 5 mM HEDTA, suggesting that the wanderlust kinetics did not arise from Ca2+ release from caffeine and thapsigargin sensitive internal stores in the excised patch. The slow changes in kinetics associated with wanderlust kinetics could be generated with a discrete-state Markov model with transitions among three or more kinetic modes with different levels of open probability. To average out the wanderlust kinetics, large amounts of data were analyzed and demonstrated up to a threefold difference in the [Ca2+]i required for an open probability of 0.5 among channels expressed from the same injected mRNA. These findings indicate that cloned dSlo channels in excised patches from Xenopus oocytes can exhibit large variability in gating properties, both within a single channel and among channels.     

Evidence for mitochondrial Ca(2+)-induced Ca2+ release in permeabilised endothelial cells

Generally most intracellular Ca2+ is stored in the endoplasmic reticulum (ER) and mitochondria. Recently a mitochondrial Ca(2+)-induced Ca2+ release (mCICR) mechanism, unconnected with ryanodine receptors (RyR's), has been shown in tumour cells. The existence of a mitochondrial Ca2+ release mechanism in BAE cells was investigated using saponin-permeabilised BAE cells. When buffered intracellular solution were 'stepped' from 10 nM to 10 microM free Ca2+, the mitochondrial inhibitors CN (2 mM), FCCP (1 microM), and RR (20 microM) significantly reduced total CICR by approximately 25%. The ER Ca(2+)-ATPase inhibitor thapsigargin (100 nM) had no effect. Furthermore, cyclosporin A (200 nM), an inhibitor of the mitochondrial permeability transition pore (PTP), abolished total CICR. Therefore, the novel ryanodine-caffeine insensitive CICR mechanism previously reported in BAE cells involves mitochondrial Ca2 release. It is proposed that in BAE cells, mCICR occurs via the mitochondrial PTP and may be physiologically important in endothelial cell Ca2+ signalling.     

Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel (hslo)

Large conductance calcium- and voltage-sensitive K+ (MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca2+ favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca2+ concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4-5 elementary charges.     

Protein kinase C inhibits the Ca(2+)-activated K+ channel of cultured porcine coronary artery smooth muscle cells

The effect of protein kinase C (C-kinase) on the Ca(2+)-activated K+ channel (KCa-channel) was studied in cultured smooth muscle cells from porcine coronary artery by the patch-clamp technique. In cell-attached patches, bath application of phorbol 12-myristate 13-acetate (PMA, 1 microM), a C-kinase activator, significantly decreased the open probability of the activated KCa-channel in the presence of the calcium ionophore A23187 (20 microM), which increases intracellular Ca2+. This decrease in the open probability was reversed by subsequent application of staurosporine (1 nM), a C-kinase inhibitor. Application of 1-oleoyl-2-acetylglycerol (OAG, 30 microM) or 1,2-dioctanoylglycerol (DG8; 30 microM), activators of C-kinase, also inhibited KCa-channel activation by A23187, and these inhibitions were also reversed by staurosporine. PMA (1 microM) also inhibited KCa-channel activation by dibutylyl cyclic AMP (db-cAMP, 2 mM) or caffeine (30 mM). In inside-out patches, bath application of the C-kinase fraction from rat brain in the presence of ATP (1 mM) and PMA (1 microM) markedly inhibited the KCa-channel. These results indicate that activation of C-kinase inhibits the KCa-channel and may cause membrane depolarization and vascular contraction.     

Ca(2+)-activated K+ channel is present in guinea-pig but lacking in rat hepatocytes

The mechanisms of norepinephrine-induced membrane responses in isolated hepatocytes from guinea-pigs and rats were compared using the suction-pipette, patch-clamp method, and intracellular Ca2+ concentration ([Ca2+]i) was measured using the Ca2+ fluorescent dye, Quin 2. The resting membrane potentials of isolated guinea-pig hepatocytes were -50 +/- 1 mV (mean +/- SD; n = 38), which is similar to that previously reported in rat hepatocytes by Sawanobori et al. (J Cell Physiol 139: 580-585, 1989). In guinea-pig hepatocytes, norepinephrine (6 microM) caused a membrane hyperpolarization, and norepinephrine (6 microM) or Ca(2+)-ionophore (A23187) (0.4 microM) caused a corresponding outward current. The sensitive current produced by norepinephrine and Ca(2+)-ionophore reversed its polarity at -74 +/- 9 mV (n = 7). The single channel recorded by cell-attached patch and inside-out patch had mean conductance of around 20 + 1 pS and was activated by 1 microM [Ca2+]i. On the other hand, neither norepinephrine (6-20 microM) nor Ca(2+)-ionophore (A 23187) (0.4 microM) caused any change in membrane potential and current in rat hepatocytes, whereas norepinephrine increased [Ca2+]i both in rat and guinea-pig hepatocytes to a similar degree. In the single-channel recording, we recorded single channels that had a mean conductance of 109.8 +/- 17.7 pS different from around 20 pS in guinea-pig. In inside-out patches, increased Ca2+ concentration from 10(-6) to 10(-3) M at the intracellular face of the membrane did not modify the single channel of rat hepatocytes. These results indicate that increased [Ca2+]i activates this channel in guinea-pigs, but that the channel activated by increased [Ca2+]i is lacking in rat hepatocytes membrane. Therefore, different mechanism operates in different species of liver cells to keep the constant state.     

Cloned Ca(2+)-dependent K+ channel modulated by a functionally associated protein kinase

Calcium-dependent potassium (KCa) channels carry ionic currents that regulate important cellular functions. Like some other ion channels, KCa channels are modulated by protein phosphorylation. The recent cloning of complementary DNAs encoding Slo KCa channels has enabled KCa channel modulation to be investigated. We report here that protein phosphorylation modulates the activity of Drosophila Slo KCa channels expressed in Xenopus oocytes. Application of ATP-gamma S to detached membrane patches increases Slo channel activity by shifting channel voltage sensitivity. This modulation is blocked by a specific inhibitor of cyclic AMP-dependent protein kinase (PKA). Mutation of a single serine residue in the channel protein also blocks modulation by ATP-gamma S, demonstrating that phosphorylation of the Slo channel protein itself modulates channel activity. The results also indicate that KCa channels in oocyte membrane patches can be modulated by an endogenous PKA-like protein kinase which remains functionally associated with the channels in the detached patch.     

A comparison of the effects of SCA40, NS 004 and NS 1619 on large conductance Ca(2+)-activated K+ channels in bovine tracheal smooth muscle cells in culture

1. The effects of imidazopyrazine derivative, SCA40, on the activity of single large conductance, Ca(2+)-activated K+ (BKCa) channels in inside-out and outside-out patches from bovine tracheal smooth muscle (BTSM) cells in culture have been compared with those of two established BKCa channel openers, NS 004 and NS 1619. 2. The presence of BKCa channels on inside-out patches of BTSM membranes was confirmed by the single channel conductance (240 pS), selectivity for K+, dependence of channel activity on [Ca2+]i, and sensitivity to the selective BKCa channel blocker, iberiotoxin. 3. NS 004 and ND 1619 (3-30 microM) induced concentration-related increases in open state probability of BKCa channels when applied to either inside-out or outside-out BTSM patches, thus confirming that these compounds are activators of the BKCa channel in this preparation. 4. SCA40 (0.1-10 microM) had no effect on the activity of BKCa channels when applied to either inside-out or outside-out patches which subsequently responded to the application of NS 004 (10-20 microM). 5. It is concluded that SCA40 does not have a direct effect on BKCa channel activity in BTSM patches and that the previously reported relaxant action of SCA40 on tracheal smooth muscle is unlikely to be mediated by this mechanism.     

Isolation and structural characterization of glycosphingolipids of in vitro propagated bovine aortic endothelial cells

Neutral glycosphingolipids and gangliosides were isolated from 3.7 x 10(9) primary bovine aortic endothelial cells and structurally characterized by immunological and chemical methods. Glucosyl- and lactosylceramide were detected as the main neutral glycosphingolipids (28% and 40% of total orcinol stain, respectively); LcOse3Cer and nLcOse4Cer were expressed to somewhat minor amounts (16% and 10% of total orcinol stain, respectively), and nLcOse6Cer occurred only in trace quantities. No neutral glycosphingolipids of the ganglio-series (GgOse3Cer and GgOse4Cer) and the globo-series (GbOse4Cer and the Forssman antigen) have been detected; only traces of GbOse3Cer were identified by TLC immunostaining. Positive CD15 bands obtained by TLC overlay with anti-Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-R antibody indicated the presence of lipid bound Lewisx antigen, whereas the isomeric Lewis(a) structure (Gal beta1-3(Fuc alpha1-4)GlcNAc beta1-R) was not detectable. G(M3) substituted with Neu5Gc and Neu5Ac in a 2:1 ratio was the major ganglioside comprising about 95% within the whole ganglioside fraction. G(M3)-structures were further characterized by FAB-MS and GC-MS of the native compounds and their permethylated derivatives. C18-sphingosine was the only long chain base, whereas variation occurred due to C(24:0,24:1) and C16 fatty acids. Terminally alpha2-3 sialylated neolacto-series gangliosides with nLcOse4- and nLcOse6Cer (<5% of total resorcinol stain) were found in almost equal quantities, whereas no alpha2-6 sialylated counterparts were detected. Fucosylated gangliosides with poly-N-acetyllactosaminyl chains (sialyl Lewis[x], sialyl Lewisa, and VIM-2 antigen) and sulfoglucuronylneolacto series structures with HNK-1 epitope were not detectable in the acidic glycosphingolipid fraction by TLC immunostaining. Gangliotetraose-type gangliosides G(M1) and G(D1a) (<1 % of total resorcinol stain) as well as traces of G(D1b) and G(T1b) have been distinctly identified by combined choleragenoid-TLC-immunostaining and previous neuraminidase treatment. The expression of dominant glycosphingolipids lactosylceramide and G(M3)(Neu5Gc) was proved by indirect immunofluorescence microscopy of cell layers grown in chamber slides, each showing different plasma membrane and subcellular distribution patterns. The results provide the basis for investigation of the role of glycosphingolipids as cell surface antigens of cellular interaction as well as receptors for blood components and macromolecules of the extracellular matrix.     

Dynamic regulation of intracellular Ca2+ concentration in aortic endothelial cells

In non-excitable cells, a Ca2+ entry pathway is opened after the depletion of intracellular Ca2+ store sites. We have tried to estimate the sensitivity of this pathway to Ca2+ release using bovine aortic endothelial cells. Single application of a high concentration (30 microM) of ATP released almost all stored Ca2+ in Ca(2+)-free extracellular solution, whereas a low concentration of ATP (30 nM) produced a partial (57.3 +/- 3.0%) release of Ca2+. By 10 min of Ca2+ re-perfusion, the Ca2+ store site was reloaded to 97.1% of its initial filling state. When thapsigargin was applied to this cell in Mn2+ solution, Mn(2+)-induced quenching of fura-2 dye started when 19.3 +/- 5.3% of Ca2+ release, produced by 30 nM ATP, had occurred. Therefore, Ca2+ release required for Mn2+ entry was estimated as 11.1 +/- 3.0% of stored Ca2+. These results indicate that intracellular Ca2+ concentration is controlled dynamically by simultaneously occurring Ca2+ release and entry in bovine aortic endothelial cells.     

Ca(2+)-permeable, outwardly-rectifying K+ channels in mesophyll cells of Arabidopsis thaliana

Advances in cannulation techniques and instruments have helped in difficult bile duct cannulation and thus stone extraction. For small common bile duct (CBD) stones, endoscopic papillary balloon dilatation has been proposed as an alternative to endoscopic papillotomy (EPT). The technique must undergo further evaluation before recommending its routine use. For most patients with bile duct stones, EPT remains the method of choice. Out of 8204 patients treated in three surgical endoscopy centers (Chile, Germany, and India), 86% to 91% of all CBD stones could be extracted subsequently after EPT using a Dormia basket; 4% to 7% required mechanical lithotripsy (ML) before removal and 3% to 10% of the patients needed other sophisticated techniques, such as electrohydraulic lithotripsy (EHL), laser-induced shock-wave lithotripsy (LISL), or extracorporeal shock-wave lithotripsy (ESWL). The local expertise and availability of equipment determines the choice of method used. In general, EHL or LISL is used for impacted CBD stones including stones in Mirizzi syndrome refractory to ML. ESWL is best suited for intrahepatic stones. Permanent stenting can be offered to poor risk patients instead of extensive procedures to clear the bile duct. Using currently available nonsurgical techniques, fewer than 1% of all patients with bile duct stones still require surgical intervention.     

Single-channel recording of a store-operated Ca2+ channel in Jurkat T lymphocytes

In T lymphocytes, a store-operated calcium ion (Ca2+) entry mechanism termed the calcium release-activated Ca2+ channel (CRAC channel) underlies the sustained or oscillatory intracellular calcium concentration signal required for interleukin-2 gene expression and cell proliferation. The use of sodium ions as a current carrier enabled single-channel recordings of CRAC channels during activation, inactivation, and blockade of current in the presence of divalent cations. A large conductance of 36 to 40 picosiemens indicates that 100 to 400 CRAC channels are present in T lymphocytes.     

Electrophysiological effects of ketamine on Ca(2+)-activated K+ channel in single rabbit portal vein cell

The effects of ketamine on Ca(2+)-activated K+ channel currents were studied in dispersed single smooth muscle cells from rabbit portal vein using inside-out patch clamp technique. In a near physiological K+ and Ca2+ gradient, three populations of outward rectangular single currents were recorded in isolated cell membrane of rabbit portal vein at +60 mV membrane potential. These currents were judged as Ca(2+)-activated K+ channel currents since application of EGTA or Apamin in the internal solution inhibited these currents. Application of 10(-5)M or 10(-4)M ketamine inhibited the number of occurrences of channel opening and decreased open times, but did not reduce the amplitudes. When the 10(-3)M ketamine was applied, the Ca(2+)-activated K+ channel currents were abolished. We suggest that the depression of Ca(2+)-activated K+ channel currents may explain the continuous contraction observed in rabbit portal vein at a clinical concentration of ketamine from a point of electrophysiological K+ current study.     

Inhibition by mibefradil, a novel calcium channel antagonist, of Ca(2+)- and volume-activated Cl- channels in macrovascular endothelial cells

1. We have studied the effects of mibefradil, a novel calcium antagonist, on the resting potential and ion channel activity of macrovascular endothelial cells (calf pulmonary artery endothelial cells, CPAE). The patch clamp technique was used to measure ionic currents and the Fura-II microfluorescence technique to monitor changes in the intracellular Ca2+ concentration, [Ca2+]i. 2. Mibefradil (10 microM) hyperpolarized the membrane potential of CPAE cells from its mean control value of -26.6 +/- 0.6 mV (n = 7) to -59.8 +/- 1.7 mV (n = 6). A depolarizing effect was observed at higher concentrations (-13.7 +/- 0.6 mV, n = 4, 30 microM mibefradil). 3. Mibefradil inhibited Ca(2+)-activated Cl- currents, ICl,Ca, activated by loading CPAE cells via the patch pipette with 500 nM free Ca2+ (Ki = 4.7 +/- 0.18 microM, n = 8). 4. Mibefradil also inhibited volume-sensitive Cl- currents, ICl,vol, activated by challenging CPAE cells with a 27% hypotonic solution (Ki = 5.4 +/- 0.22 microM, n = 6). 5. The inwardly rectifying K+ channel, IRK, was not affected by mibefradil at concentrations up to 30 microM. 6. Ca2+ entry activated by store depletion, as assessed by the rate of [Ca2+]i-increase upon reapplication of 10 mM extracellular Ca2+ to store-depleted cells, was inhibited by 17.6 +/- 6.5% (n = 8) in the presence of 10 microM mibefradil. 7. Mibefradil inhibited proliferation of CPAE cells. Half-maximal inhibition was found at 1.7 +/- 0.12 microM (n = 3), which is similar to the concentration for half-maximal block of Cl- channels. 8. These actions of mibefradil on Cl- channels and the concomitant changes in resting potential might, in addition to its effect on T-type Ca2+ channels, be an important target for modulation of cardiovascular function under normal and pathological conditions.     

A Ba(2+)-insensitive K+ conductance in the basolateral membrane of rabbit cortical thick ascending limb cells

The nature of the K+ exit across the basolateral membrane of microperfused rabbit cortical thick ascending limbs (cTALs) was investigated using the transepithelial and transmembrane potential difference (PDte, PDbl) and conductance measurements. An increase in bath K+ concentration from 4 to 10, 25, 50 mmol/l depolarized the basolateral membrane in a concentration-dependent manner, accompanied by a decrease in the fractional resistance of the basolateral membrane (FRbl). The Cl- channel blocker, 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB), did not prevent these effects. The effect of Ba2+ on PDbl was bimodally distributed: paradoxically, in the tubules in which Ba2+ largely depolarized, the effects on PDbl of the bath K+ concentration increases were not inhibited by extracellular Ba2+, in tubules in which Ba2+ moderately depolarized, Ba2+ partially inhibited the K+ concentration increase-induced depolarization of the basolateral membrane. However, the parallel decrease in FRbl was Ba2+ insensitive, indicating that the K+ channel of the basolateral membrane was not modified by extracellular Ba2+. The Ba(2+)-induced depolarizations were prevented by furosemide suggesting that Ba2+ acts by inhibiting basolateral KCl extrusion. Finally, the K+ concentration increase-induced depolarizations were insensitive to tetraethylammonium, charybdotoxin, apamin and verapamil. In conclusion, the present study provides evidence that, in addition to a Ba(2+)-sensitive KCl cotransport system, the basolateral membrane of rabbit cTAL cells possesses a K+ conductance which is insensitive to extracellular Ba2+.     

Effect of the general anesthetic halothane on the activity of the transverse tubule Ca(2+)-activated K+ channel

The effect of cycloheximide on increased splanchnic prostacyclin release following acute hemorrhage was studied in the rat. Male Sprague-Dawley rats were anesthetized and subjected to acute hemorrhage to 30 mm Hg for 30 min (shock) or sham shock. The superior mesenteric artery was cannulated and removed with its end organ intestine (SV + SI preparation) and perfused in vitro with oxygenated Krebs-Henseleit buffer. Cycloheximide was infused in half of the sham and acute hemorrhage SV + SI preparations at 50 micrograms/ml. Venous effluent from all groups were analyzed for prostanoid release by radioimmunoassay. The SV + SI released 10-fold more 6-keto-prostaglandin (PG) F1 alpha than PGE2 and thromboxane. Acute hemorrhage increased SV + SI release of 6-keto-PGF1 alpha 3-fold compared to sham. Cycloheximide abolished the increased 6-keto-PGF1 alpha following acute hemorrhage but not the basal release in the sham group. Indomethacin decreased PG synthesis in all groups by 90%. Sham PG release was dependent on a stable pool of cyclooxygenase with a long half-life and was not affected by cycloheximide treatment. Acute hemorrhage stimulated a rapid induction of enzymes (cyclooxygenase, prostacyclin synthase) responsible for prostacyclin synthesis and release which were dependent on de novo protein synthesis.     

Ca2+ mobilization in bovine corneal endothelial cells by P2 purinergic receptors

PURPOSE: To characterize Ca2+ mobilization by P2 receptors in the bovine corneal endothelial cells (BCEC). METHODS: Changes in intracellular Ca2+ ([Ca2+]i) were measured by fluorescence imaging of cultured and fresh BCEC cells loaded with the Ca2+-sensitive dye Fura-PE3. Relative rates of Ca2+ influx were measured employing Mn2+ as a surrogate for Ca2+. RESULTS: Exposure of cultured cells to uridine 5'-triphosphate (UTP), 2-methyl-thio ATP (msATP) and ATP caused biphasic changes in [Ca2+]i consisting of a peak followed by a plateau phase. Based on the peak responses to 100 microM agonist, the magnitude of UTP responses were similar to that of ATP but greater than that of msATP or ADP. UTP and msATP stimulated Mn2+ influx following [Ca2+]i peak similar to that observed in response to cyclopiazonic acid (CPA), an inhibitor of ER Ca2+-ATPase. Under Ca2+-free conditions, peak responses were similar to those in the presence of external Ca2+, but reduced when the cells were pre-exposed to CPA. Reactive Blue-2 (RB2), inhibited msATP responses by 60.4 +/- 18.8% but UTP responses by only 10.6 +/- 9.5%. Repeated exposures to UTP or msATP reduced [Ca2+]i mobilization indicating homologous desensitization. Response to UTP was not affected by a prior exposure to msATP. However, response to msATP was reduced by a prior exposure to UTP indicating mixed heterologous desensitization. Fresh cells responded to UTP (50 microM) with temporal characteristics of [Ca2+]i mobilization similar to that of cultured cells. CONCLUSION: BCEC express P2 receptors belonging to the P2Y subfamily. The emptying of the IP3-sensitive stores, leading to the initial peak in [Ca2+]i response, subsequently caused capacitative Ca2+ influx leading to the onset of the plateau phase. A significant homologous desensitization to UTP and msATP, selective heterologous desensitization between UTP and msATP, and selective inhibition by RB2 indicate the coexistence of multiple P2Y receptors.     

Characterization of acidic Ca(2+)-independent phospholipase A2 of bovine lung

The Gram-positive bacterium Staphylococcus aureus infects diverse tissues and causes a wide spectrum of diseases, suggesting that it possesses a repertoire of distinct molecular mechanisms promoting bacterial survival in disparate in vivo environments. Signature-tag transposon mutagenesis screening of a 1520-member library identified numerous S. aureus genetic loci affecting growth and survival in four complementary animal infection models including mouse abscess, bacteraemia and wound and rabbit endocarditis. Of a total of 237 in vivo attenuated mutants identified by the murine models, less than 10% showed attenuation in all three models, emphasizing the advantage of screening in diverse disease environments. The largest gene class identified by these analyses encoded peptide and amino acid transporters, some of which were important for S. aureus survival in all animal infection models tested. The identification of staphylococcal loci affecting growth, persistence and virulence in multiple tissue environments provides insight into the complexities of human infection and on the molecular mechanisms that could be targeted by new antibacterial therapies.     

Regulation and possible physiological role of the Ca(2+)-dependent K+ channel of cortical collecting ducts of the rat

In the luminal membrane of rat cortical collecting duct (CCD) a big Ca(2+)-dependent and a small Ca(2+)-independent K+ channel have been described. Whereas the latter most likely is responsible for the K+ secretion in this nephron segment, the function of the large-conductance K+ channel is unknown. The regulation of this channel and its possible physiological role were examined with the conventional cell-free and the cell-attached nystatin patch-clamp techniques. Patch-clamp recordings were obtained from the luminal membrane of isolated perfused CCD segments and from freshly isolated CCD cells. Intracellular calcium was measured using the calcium-sensitive dye fura-2. The large-conductance K+ channel was strongly voltage- and calcium-dependent. At 3 mumol/l cytosolic Ca2+ activity it was half-maximally activated. At 1 mmol/l it was neither regulated by cytosolic pH nor by ATP. At 1 mumol/l Ca2+ activity the open probability (Po) of this channel was pH-dependent. At pH 7.0 Po was decreased to 4 +/- 2% (n = 9) and at pH 8.5 it was increased to 425 +/- 52% (n = 9) of the control. At this low Ca2+ activity the Po of the channel was reduced by 1 mmol/l ATP to 8 +/- 4% (n = 6). Cell swelling activated the large-conductance K+ channel (n = 14) and hyperpolarized the membrane potential of the cells by 9 +/- 1 mV (n = 23). Intracellular Ca2+ activity increased after hypotonic stress. This increase depended on the extracellular Ca2+ activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Developmental- and eye-specific transcriptional control elements in an intronic region of a Ca(2+)-activated K+ channel gene

The range of electrical properties that a neuron or muscle cell can manifest is determined by which ion channel genes it expresses and in what amounts. The Drosophila slowpoke Ca(2+)-activated K+ channel gene has four distinct promoters. Here we assess the role that a downstream intronic region, called the C2/C3 region, plays in modulating Promoter C1 and Promoter C2 activity. Promoter C1 and Promoter C2 appear to be responsible for all neuronal and muscle expression, respectively. Transgenic flies were used to determine the expression pattern from each promoter in the presence and absence of the C2/C3 region. Deletion of this region silences Promoter C1 in adult but not larval CNS and causes a substantial reduction in Promoter C2 activity in adult but not larval muscle. The C2/C3 region also activates Promoter C1 in the animal's eye. By placing the C2/C3 region adjacent to a basal HSP70 promoter we have demonstrated that it contains elements that can specifically activate a heterologous promoter in the eye and in adult but not larval muscle. These results demonstrate that the C2/C3 region has a important role in regulating slowpoke developmental expression in the CNS and musculature and in regulating eye expression.     

Endoplasmic reticulum Ca(2+)-ATPase in microsomal vesicles isolated from bovine retinae

Active Ca2+ transport was measured in microsomal vesicles prepared from bovine retinae and was compared with that in disk membranes of the photoreceptor cells of the same retina. The 45Ca uptake was dependent on the presence of Mg(2+)-ATP and was inhibited by vanadate or when GTP substituted for ATP. The dependence of calcium uptake on the external free Ca2+ concentration gave a KM = 13 microM or a KM = 0.1 microM for disks and microsomal vesicles, respectively. A phosphorylated intermediate (E-P) of Ca(2+)-ATPase of about 100 kDa was isolated in microsomal vesicles. The E-P formation was strongly inhibited by thapsigargin and partially by 2,5-di-(-butyl)benzohydroquinone. Digestion of disks or microsomes with calpain had no effect on the phosphorylated intermediate, while digestion with trypsin produced two fragments of approximately 55 kDa and 35 kDa. These results suggest that bovine retinal microsomes contain a calcium pump belonging to the SERCA family.