The colonic H+,K+-ATPase functions as a Na+-dependent K+(NH4+)-ATPase in apical membranes from rat distal colon.
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Recent studies have suggested that the colonic H+,K+-ATPase (HKalpha2) can secrete either Na+ or H+ in exchange for K+. If correct, this view would indicate that the transporter could function as either a Na+ or a H+ pump. To investigate this possibility a series of experiments was performed using apical membranes from rat colon which were enriched in colonic H+,K+-ATPase protein. An antibody specific for HKalpha2 was employed to determine whether HKalpha2 functions under physiological conditions as a Na+-dependent or Na+-independent K+-ATPase in this same membrane fraction. K+-ATPase activity was measured as [gamma-32P]ATP hydrolysis. The Na+-dependent K+-ATPase accounted for approximately 80% of overall K+-ATPase activity and was characterized by insensitivity to Sch-28080 but partial sensitivity to ouabain. The Na+-independent K+-ATPase activity was insensitive to both Sch-28080 and ouabain. Both types of K+-ATPase activity substituted NH4+ for K+ in a similar manner. Furthermore, our results demonstrate that when incubated with native distal colon membranes, the blocking antibody inhibited dramatically Na+-dependent K+-ATPase activity. Therefore, these data demonstrate that HKalpha2 can function in native distal colon apical membranes as a Na+-dependent K+-ATPase. Elucidation of the role of the pump as a transporter of Na+ versus H+ or NH4+ versus K+ in vivo will require additional studies. (+info)
Lamellarity of cationic liposomes and mode of preparation of lipoplexes affect transfection efficiency.
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Transfection of NIH-3T3 cells by a human growth hormone expression vector complexed with liposomes composed of N-(1-(2, 3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP) with or without helper lipids was studied. The transfection efficiency was dependent on the lamellarity of the liposomes used to prepare the lipoplexes. Multilamellar vesicles (MLV) were more effective than large unilamellar vesicles (LUV) of approximately 100 nm, irrespective of lipid composition. The optimal DNA/DOTAP mole ratio for transfection was +info)
Membrane phospholipid composition affects function of potassium channels from rabbit colon epithelium.
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We tested the effects of membrane phospholipids on the function of high-conductance, Ca(2+)-activated K(+) channels from the basolateral cell membrane of rabbit distal colon epithelium by reconstituting these channels into planar bilayers consisting of different 1:1 mixtures of phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI). At low ambient K(+) concentrations single-channel conductance is higher in PE/PS and PE/PI bilayers than in PE/PC bilayers. At high K(+) concentrations this difference in channel conductance is abolished. Introducing the negatively charged SDS into PE/PC bilayers increases channel conductance, whereas the positively charged dodecyltrimethylammonium has the opposite effect. All these findings are consistent with modulation of channel current by the charge of the lipid membrane surrounding the channel. But the K(+) that permeates the channel senses only a small fraction of the full membrane surface potential of the charged phospholipid bilayers, equivalent to separation of the conduction pathway from the charged phospholipid head groups by 20 A. This distance appears to insulate the channel entrance from the bilayer surface potential, suggesting large dimensions of the channel-forming protein. In addition, in PE/PC and PE/PI bilayers, but not in PE/PS bilayers, the open-state probability of the channel decreases with time ("channel rundown"), indicating that phospholipid properties other than surface charge are required to maintain channel fluctuations. (+info)
Correlation of miniature synaptic activity and evoked release probability in cultures of cortical neurons.
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Spontaneous miniature synaptic activity is caused by action potential (AP)-independent release of transmitter vesicles and is regulated at the level of single synapses. In cultured cortical neurons we have used this spontaneous vesicle turnover to load the styryl dye FM1-43 into synapses with high rates of miniature synaptic activity. Automated selection procedures restricted analysis to synapses with sufficient levels of miniature activity-mediated FM1-43 uptake. After FM1-43 loading, vesicular FM1-43 release in response to AP stimulation was recorded at single synapses as a measure of release probability. We find that synapses with high rates of miniature activity possess significantly enhanced evoked release rates compared with a control population. Because the difference in release rates between the two populations is [Ca(2+)](o)-dependent, it is most likely caused by a difference in release probability. Within the subpopulation of synapses with high miniature activity, we find that the probabilities for miniature and AP-evoked release are correlated at single synaptic sites. Furthermore, the degree of miniature synaptic activity is correlated with the vesicle pool size. These findings suggest that both evoked and miniature vesicular release are regulated in parallel and that the frequency of miniature synaptic activity can be used as an indicator for evoked release efficacy. (+info)
Voltage-gated K+ currents in freshly isolated myocytes of the pregnant human myometrium.
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1. Voltage-gated K+ currents in human myometrium are not well characterized, and were therefore investigated, using the whole-cell patch clamp technique, in freshly isolated myometrial smooth muscle cells from pregnant women at term. 2. Three types of voltage-gated K+ currents were identified. IK1 was a 4-aminopyridine-insensitive current with a negative half-inactivation (V0.5 = -61 to -67 mV) and negative activation characteristics (threshold between -60 and -40 mV) and slow kinetics. IK2 was a 4-aminopyridine-sensitive current (half-maximal block at approximately 1 mM) with relatively positive half-inactivation (V0.5 = -30 mV) and activation characteristics (threshold between -40 and -30 mV) and faster kinetics. IK,A was a 4-aminopyridine-sensitive current with a negative inactivation and very fast inactivation kinetics. 3. Both IK1 and IK2 were sensitive to high concentrations of tetraethylammonium (half-maximal block at approximately 3 mM) and low concentrations of clofilium (half-maximal block by 3-10 microM). 4. IK1 and IK2 were unevenly distributed between myometrial cells, most cells possessing either IK1 (30 cells) or IK2 (24 cells) as the predominant current. 5. The characteristics of these currents suggest a possible function in the control of membrane potentials and smooth muscle quiescence in the pregnant human myometrium. (+info)
Canalicular membrane transport is primarily responsible for the difference in hepatobiliary excretion of triethylmethylammonium and tributylmethylammonium in rats.
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Two structurally similar quaternary ammonium compounds, triethylmethylammonium (TEMA, M(r) 116) and tributylmethylammonium (TBuMA, M(r) 200) were used as model compounds to identify the unit process of hepatobiliary excretion that is responsible for markedly different biliary excretion of organic cations (OCs). Cumulative biliary excretion (in percentage of dose; i.v., 12 micromol/kg) was 0.17 for TEMA and 34.5 for TBuMA. In vivo uptake clearance into the liver was 0.686 +/- 0.020 ml/min for TEMA and 0.421 +/- 0.028 ml/min for TBuMA. When the uptake clearance was examined in an isolated hepatocyte system, comparable clearance between TEMA and TBuMA was obtained, consistent with the in vivo result. These observations suggest that uptake into the liver is not the major determinant for the difference in biliary excretion of the OCs. Coadministration of colchicine, an inhibitor of microtubule formation, had no effect on biliary excretion of the model compounds, and the primary site of subcellular distribution of the OCs appears to be the cytosol, suggesting that intracellular movement does not play a major role in the markedly different biliary excretion of the OCs. In contrast, in vivo excretion clearance across the canalicular membrane for TBuMA was 180-fold greater than that for TEMA, and in vitro efflux clearance of TBuMA was smaller than that of TEMA (p <.01), indicative of involvement of these processes in the markedly different biliary excretion of the OCs. Therefore, these data indicate that canalicular transport is primarily responsible for the markedly different biliary excretion of TEMA and TBuMA. (+info)
A method for determining transmembrane helix association and orientation in detergent micelles using small angle x-ray scattering.
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Solution small angle x-ray scattering can be used to study the association of transmembrane proteins solubilized in detergent micelles. We have used the alpha-helical transmembrane domain of the human erythrocyte glycophorin A (GpA) fused to the carboxyl terminus of monomeric staphylococcal nuclease (SN/GpA) as a model system for study. By matching the average electron density of the detergent micelles to that of the buffer solution, the micelle contribution to the small angle scattering vanishes, and the molecular weight and the radius of gyration of the proteins can be determined. SN/GpA has been found to dimerize in a zwitterionic detergent micelle, N-dodecyl-N,N-(dimethylammonio)butyrate (DDMAB), whose average electron density naturally matches the electron density of an aqueous buffer. The dimerization occurs through the transmembrane domains of GpA. With the aid of the nuclease domain scattering, the orientation of the helices within a dimer can be determined to be parallel by radius of gyration analysis. The association constant of a mutant (G83I) that weakens the GpA dimerization has been determined to be 24 microM in the DDMAB environment. The experimental methods established here could be used to apply solution small angle x-ray scattering to studying the association and interactions of other membrane proteins. (+info)
Use of a mathematical model of rodent in vitro benzene metabolism to predict human in vitro metabolism data.
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Benzene, a ubiquitous environmental pollutant, is known to cause leukemia and aplastic anemia in humans and hematotoxicity and myelotoxicity in rodents. Toxicity is thought to be exerted through oxidative metabolites formed in the liver, primarily via pathways mediated by cytochrome P450 2E1 (CYP2E1). Phenol, hydroquinone and trans-trans-muconaldehyde have all been hypothesized to be involved in benzene-induced toxicity. Recent reports indicate that benzene oxide is produced in vitro and in vivo and may be sufficiently stable to reach the bone marrow. Our goal was to improve existing mathematical models of microsomal benzene metabolism by including time course data for benzene oxide, by obtaining better parameter estimates and by determining if enzymes other than CYP2E1 are involved. Microsomes from male B6C3F1 mice and F344 rats were incubated with [(14)C]benzene (14 microM), [(14)C]phenol (303 microM) and [(14)C]hydroquinone (8 microM). Benzene and phenol were also incubated with mouse microsomes in the presence of trans-dichloroethylene, a CYP2E1 inhibitor, and benzene was incubated with trichloropropene oxide, an epoxide hydrolase inhibitor. These experiments did not indicate significant contributions of enzymes other than CYP2E1. Mathematical model parameters were fitted to rodent data and the model was validated by predicting human data. Model simulations predicted the qualitative behavior of three human time course data sets and explained up to 81% of the total variation in data from incubations of benzene for 16 min with microsomes from nine human individuals. While model predictions did deviate systematically from the data for benzene oxide and trihydroxybenzene, overall model performance in predicting the human data was good. The model should be useful in quantifying human risk due to benzene exposure and explicitly accounts for interindividual variation in CYP2E1 activity. (+info)