cDNA cloning, chromosomal localization and evolutionary analysis of mouse vacuolar ATPase subunit D, Atp6m.

The multi-subunit vacuolar ATPase pump uses ATP hydrolysis to move protons into membrane bound compartments. The pump is involved in a variety of cellular functions, including regulation of cytosolic pH, vesicular transport, endocytosis, secretion, and apoptosis. Here, we describe the cDNA cloning and chromosomal mapping of subunit D of murine V-ATPase. The mouse gene, designated Atp6m, maps to Chromosome 12, in a region of high homology with human chromosome 14q24. Evolutionary analysis of subunit D orthologs in a variety of other species reveals that this is a highly conserved protein that has been under remarkably strong negative selection during evolution, most likely reflecting its critical role in multiple cellular processes.

Prediction of in vivo background in phoswich lung count spectra.

Phoswich scintillation counters are used to detect actinides deposited in the lungs. The resulting spectra, however, contain Compton background from the decay of 40K, which occurs naturally in the striated muscle tissue of the body. To determine the counts due to actinides in a lung count spectrum, the counts due to 40K scatter must first be subtracted out. The 40K background in the phoswich NaI(TI) spectrum was predicted from an energy region of interest called the monitor region (95-129 keV), which is above the 238Pu region (11-32 keV) and the 241Am region (40-82 keV), where photopeaks from 238Pu and 241Am occur. Empirical models were developed to predict the backgrounds in the 238Pu and 241Am regions by testing multiple linear and nonlinear regression models. The initial multiple regression models contain a monitor region variable as well as the variables gender, (weight/height)alpha, and interaction terms. Data were collected from 64 male and 63 female subjects with no internal exposure. For the 238Pu region, the only significant predictor was found to be the monitor region. For the 241Am region, the monitor region was found to have the greatest effect on prediction, while gender was significant only when weight/height was included in a model. Gender-specific models were thus developed. The empirical models for the 241Am region that contain weight/height were shown to have the best coefficients of determination (R2) and the lowest mean squares for error (MSE).

Muscarinic modulation of voltage-dependent Ca2+ channels in insulin-secreting HIT-T15 cells.

Potentiation of insulin secretion from pancreatic beta-cells by acetylcholine requires ongoing cyclic electrical activity initiated by other depolarizing secretagogues. Patch-clamp recordings in glucose-free solutions were made from the clonal beta-cell line HIT-T15 to determine whether the muscarinic agonist bethanechol (BCh) modulated voltage-dependent Ca2+ channels independent of effects on membrane potential. Only high-threshold, dihydropyridine-sensitive (L-type) Ca2+ channels with a mean conductance of 26 pS were observed in cell-attached patches. BCh (100 microM) caused a two- to threefold increase in both fractional open time and mean current of single Ca2+ channels. These changes resulted from a 44% decrease in the longer of two apparent mean closed times and a 25% increase in the mean open time. Similar BCh-stimulated increases in macroscopic Ca2+ currents were recorded in whole cell, perforated-patch recordings. The role of protein kinase C (PKC) in the muscarinic activation of Ca2+ channels was tested using a variety of PKC activators and inhibitors. Acute application of either the active phorbol ester phorbol 12-myristate 13-acetate (PMA) or the membrane-permeable diacylglycerol analog 1,2-didecanoyl-rac-glycerol mimicked the effects of BCh, whereas an inactive phorbol (4 alpha) had no effect. Depletion of PKC activity by chronic exposure to PMA or acute application of the PKC inhibitor staurosporine greatly reduced or abolished muscarinic activation of Ca2+ channels. These results are consistent with muscarinic activation of L-type, voltage-dependent Ca2+ channels mediated in large part by PKC.

Cystic fibrosis transmembrane conductance regulator (CFTR) anion binding as a probe of the pore.

We compared the effects of mutations in transmembrane segments (TMs) TM1, TM5, and TM6 on the conduction and activation properties of the cystic fibrosis transmembrane conductance regulator (CFTR) to determine which functional property was most sensitive to mutations and, thereby, to develop a criterion for measuring the importance of a particular residue or TM for anion conduction or activation. Anion substitution studies provided strong evidence for the binding of permeant anions in the pore. Anion binding was highly sensitive to point mutations in TM5 and TM6. Permeability ratios, in contrast, were relatively unaffected by the same mutations, so that anion binding emerged as the conduction property most sensitive to structural changes in CFTR. The relative insensitivity of permeability ratios to CFTR mutations was in accord with the notion that anion-water interactions are important determinants of permeability selectivity. By the criterion of anion binding, TM5 and TM6 were judged to be likely to contribute to the structure of the anion-selective pore, whereas TM1 was judged to be less important. Mutations in TM5 and TM6 also dramatically reduced the sensitivity of CFTR to activation by 3-isobutyl 1-methyl xanthine (IBMX), as expected if these TMs are intimately involved in the physical process that opens and closes the channel.

Epithelial localization of a reptilian Na+/H+ exchanger homologous to NHE-1.

Basolateral membranes of turtle (Pseudemys scripta) colon epithelial cells exhibit robust Na+/H+ exchange activity that can be activated by cell shrinkage and is blocked by amiloride [M. A. Post and D. C. Dawson. Am. J. Physiol. 262 Cell Physiol. 31):C1089-C1094, 1992]. The colonic epithelium actively absorbs Na+ and secretes K+ and HCO3-, but the role of basolateral Na+/H+ exchange, if any, in transepithelial transport is unknown. The current studies were undertaken to identify the gene product(s) responsible for the observed basolateral Na+/H+ exchange activity and to determine the cellular localization of the reptilian Na+/H+ exchange protein. We cloned and sequenced partial-length cDNAs that are likely to encode a reptilian homologue of the mammalian NHE-1 Na+/H+ exchanger isoform. The partial-length cDNAs were > 80% identical to mammalian NHE-1 homologues at the nucleotide level and recognized a transcript (approximately 5.8-6.0 kb) in RNA isolated from turtle colon, small intestine, stomach, kidney, urinary bladder, heart, and liver. In situ hybridization showed that mRNA encoding the reptile homologue of NHE-1 was expressed predominantly in the epithelial cells of these tissues. Immunofluorescent localization of the reptilian Na+/H+ exchanger protein using an antibody raised against a human NHE-1 fusion protein confirmed that protein expression paralleled abundant mRNA expression in epithelial cells of turtle stomach and colon, as well as in some nephron segments, and showed that the reptile NHE-1 homologue was localized exclusively to the basolateral membranes of these cells. The relatively high level of NHE-1 expression in epithelial cells, particularly those of the colon and stomach, suggests that NHE-1 function is important for the maintenance or regulation of ion transport processes that occur in these cell types.

Selective block of specific K(+)-conducting channels by diphenylamine-2-carboxylate in turtle colon epithelial cells.

1. The conduction and gating properties of K(+)-conducting channels were studied in isolated turtle colon cells in an attempt to identify the single channels responsible for specific components of the macroscopic conductance of the basolateral membrane. Three types of Ca(2+)-activated channel were identified, two of which were selective for K+ over Na+ and a third which was selective for monovalent cations over anions, but did not discriminate between K+ and Na+. 2. One of the K(+)-selective channels was a large-conductance 'maxi' K+ channel. A second was characterized by a lower conductance and pronounced inward rectification. 3. The inward-rectifying K+ channel was selectively blocked by diphenylamine-2-carboxylate (DPC). Neither the maxi K+ channel nor a previously identified K(+)-selective channel thought to be activated by cell swelling was affected by this compound. DPC also blocked the non-selective cation channel. 4. An inward-rectifying, DPC-sensitive current was prominent in whole cell-recordings, and DPC blocked basolateral K+ currents in colonic cell layers apically permeabilized with amphotericin-B. In addition, the compound blocked active Na+ absorption. 5. The selective block of a class of epithelial K+ channels by DPC may be a useful tool for determining the contribution of this specific subpopulation to macroscopic conductance and transepithelial salt transport.

Basolateral K conductance: role in regulation of NaCl absorption and secretion.

In this review we explore the possible role of basolateral K conductance (gK) in the regulation of salt absorption and secretion. This inquiry is prompted by a growing body of evidence which, taken together, suggests that basolateral gK is very labile and that alterations in basolateral gK may be a key feature in both stimulatory and inhibitory regulatory mechanisms. We first consider the role of basolateral gK in relation to models for salt absorption and secretion, particularly in relation to the maintenance of cellular charge balance and the obligatory coupling between the apical and basolateral membranes that is produced by transcellular current flow. Next, we review some of the experimental evidence that suggests that changes in basolateral gK are associated with transport regulation. The cellular mechanisms that are known to impact on K channel regulation are considered in a general way, and finally, we consider the use of integrated models for understanding possible coordinate regulation of apical and basolateral cell membranes.

Two K+ channel types, muscarinic agonist-activated and inwardly rectifying, in a Cl- secretory epithelium: the avian salt gland.

Patches of membrane on cells isolated from the nasal salt gland of the domestic duck typically contained two types of K+ channel. One was a large-conductance ("maxi") K+ channel which was activated by intracellular calcium and/or depolarizing membrane voltages, and the other was a smaller-conductance K+ channel which exhibited at least two conductance levels and displayed pronounced inward rectification. Barium blocked both channels, but tetraethylammonium chloride and quinidine selectively blocked the larger K+ channel. The large K+ channel did not appear to open under resting conditions but could be activated by application of the muscarinic agonist, carbachol. The smaller channels were open under resting conditions but the gating was not affected by carbachol. Both of these channels reside in the basolateral membranes of the Cl- secretory cells but they appear to play different roles in the life of the cell.

Exogenous ATP-stimulated calcium uptake in isolated rat intestinal epithelial cells.

ATP in the extracellular medium is known to stimulate Ca uptake into avian intestinal epithelial cells. We have now demonstrated a similar effect of ATP in mammalian intestinal epithelial cells and have further characterized this effect. Exogenous ATP increased 45Ca uptake 2-6 fold in isolated rat small intestinal epithelial cells, with a maximal effect at 1 mM and an ED50 of 290 microM. A strict structural requirement for nucleotide-stimulated 45Ca uptake was observed. ADP was much less effective than ATP and gamma-thio-ATP, and 5'-AMP, cyclic AMP, adenosine, non-adenine nucleotides, non-hydrolyzable ATP analogs and ATP analogs with ring substitutions at the 8 position were inactive. Prenylamine (100 microM) completely inhibited ATP-stimulated 45Ca uptake, while verapamil (100 microM) had only a small effect. In the intact intestine, ATP increased short-circuit current (Isc) when added to the mucosal side of the tissue. This effect was reduced by 10 microM and abolished by 100 microM prenylamine. The effect of ATP on Isc was markedly reduced in Cl-free solutions and in reduced-Ca solutions. Serosal and mucosal addition of the nonhydrolyzable ATP analog, beta, gamma-methylene-ATP, and serosal addition of ATP had little or no effect on Isc. The similarities between the effects of ATP in isolated cells and in the intact intestine suggest that the effect of ATP on Isc may be at least partially mediated through stimulation of Ca uptake into the epithelial cells.

Single potassium channels blocked by lidocaine and quinidine in isolated turtle colon epithelial cells.

The patch-clamp technique for recording single-channel currents across cell membranes was applied to single turtle colon epithelial cells isolated with hyaluronidase. With electrodes fabricated from Corning #7052 glass, high-resistance seals were consistently formed to these cells. In on-cell patches with low K (2.5 mM) in the pipette and high K (114.5 mM) in the bath, outward K currents were recorded that had a slope conductance of 17 pS and a reversal potential greater than -70 mV. Currents through this K channel were blocked by lidocaine, quinidine, and barium. These agents also block a cell swelling-induced K conductance identified by macroscopic current measurements in the basolateral membranes of the intact colonic epithelium, suggesting that the 17 pS K channel identified by single-channel recording in isolated turtle colon cells may be responsible for this macroscopically defined K conductance.

Effects of phenothiazines on phosphate incorporation and chloride uptake in isolated intestinal epithelial cells.

Isolated, intact epithelial cells from rat ileum synthesized a variety of radiolabeled compounds when 32Pi was added to the incubation medium. One of these, referred to as P-0.7 because of its Rf value in the thin layer chromatography system used to separate phosphorylated compounds, was found in the extracellular medium but not in extracts of cells. Synthesis of P-0.7 was inhibited by phenothiazines, with an EC50 value of 205 microM for trifluoperazine. Phenothiazines also inhibited uptake into isolated cells, with EC50 values of 288 and 359 microM for trifluoperazine and chlorpromazine, respectively. Effects of the phenothiazines on chloride uptake were not mimicked by the local anesthetic lidocaine (1 mM), the calcium channel blocker verapamil (0.5 mM), SITS or furosemide, or by removal of sodium or calcium from the medium.