RESUMEN
Urea transport (UT-B) proteins are known to facilitate urea movement across the ruminal epithelium; however, other mechanisms may be involved as well because inhibiting UT-B does not completely abolish urea transport. Of the aquaporins (AQP), which are a family of membrane-spanning proteins that are predominantly involved in the movement of water, AQP-3, AQP-7, and AQP-10 are also permeable to urea, but it is not clear if they contribute to urea transport across the ruminal epithelium. The objectives of this study were to determine (1) the functional roles of AQP and UT-B in the serosal-to-mucosal urea flux (Jsm-urea) across rumen epithelium; and (2) whether functional adaptation occurs in response to increased diet fermentability. Twenty-five Holstein steer calves (n=5) were assigned to a control diet (CON; 91.5% hay and 8.5% vitamin and mineral supplement) or a medium grain diet (MGD; 41.5% barley grain, 50% hay, and 8.5% vitamin and mineral) that was fed for 3, 7, 14, or 21 d. Calves were killed and ruminal epithelium was collected for mounting in Ussing chambers under short-circuit conditions and for analysis of mRNA abundance of UT-B and AQP-3, AQP-7, and AQP-10. To mimic physiologic conditions, the mucosal buffer (pH 6.2) contained no urea, whereas the serosal buffer (pH 7.4) contained 1 mM urea. The fluxes of (14)C-urea (Jsm-urea; 26 kBq/10 mL) and (3)H-mannitol (Jsm-mannitol; 37 kBq/10 mL) were measured, with Jsm-mannitol being used as an indicator of paracellular or hydrophilic movement. Serosal addition of phloretin (1 mM) was used to inhibit UT-B-mediated urea transport, whereas NiCl2 (1 mM) was used to inhibit AQP-mediated urea transport. Across treatments, the addition of phloretin or NiCl2 reduced the Jsm-urea from 116.5 to 54.0 and 89.5 nmol/(cm(2) × h), respectively. When both inhibitors were added simultaneously, Jsm-urea was further reduced to 36.8 nmol/(cm(2) × h). Phloretin-sensitive and NiCl2-sensitive Jsm-urea were not affected by diet. The Jsm-urea tended to increase linearly as the duration of adaptation to MGD increased, with the lowest Jsm-urea being observed in animals fed CON [107.7 nmol/(cm(2) × h)] and the highest for those fed the MGD for 21 d [144.2 nmol/(cm(2) × h)]. Phloretin-insensitive Jsm-urea tended to increase linearly as the duration of adaptation to MGD increased, whereas NiCl2-insensitive Jsm-urea tended to be affected by diet. Gene transcript abundance for AQP-3 and UT-B in ruminal epithelium increased linearly as the duration of MGD adaptation increased. For AQP-7 and AQP-10, gene transcript abundance in animals that were fed the MGD was greater compared with that of CON animals. These results demonstrate that both AQP and UT-B play significant functional roles in urea transport, and they may play a role in urea transport during dietary adaptation to fermentable carbohydrates.
Asunto(s)
Acuaporinas/metabolismo , Bovinos/metabolismo , Dieta/veterinaria , Proteínas de Transporte de Membrana/metabolismo , Rumen/metabolismo , Urea/metabolismo , Animales , Acuaporinas/antagonistas & inhibidores , Acuaporinas/genética , Transporte Biológico , Reactores Biológicos , Carbohidratos de la Dieta/administración & dosificación , Carbohidratos de la Dieta/metabolismo , Suplementos Dietéticos , Grano Comestible , Epitelio/metabolismo , Fermentación , Masculino , Proteínas de Transporte de Membrana/genética , Minerales/administración & dosificación , Membrana Mucosa , Níquel/farmacología , Floretina/farmacología , ARN Mensajero/análisis , Vitaminas/administración & dosificación , Transportadores de UreaRESUMEN
The proper trafficking and localization of cardiac potassium channels is profoundly important to the regulation of the regionally distinct action potentials across the myocardium. These processes are only beginning to be unravelled and involve modulators of channel synthesis and assembly, post-translational processing, various molecular motors and an increasing number of modifying enzymes and molecular anchors. The roles of anchoring proteins, molecular motors and kinases are explored and recent findings on channel internalization and trafficking are presented.
Asunto(s)
Activación del Canal Iónico , Canales de Potasio/fisiología , Animales , Proteínas Motoras Moleculares/fisiología , Fosforilación , Procesamiento Proteico-Postraduccional , Transporte de ProteínasRESUMEN
Chloride concentration has been shown to have a dramatic impact on protein folding and subsequent tertiary conformation [K.D. Collins, Ions from the Hofmeister series and osmolytes: effects on proteins in solution and in the crystallization process, Methods 34 (2004) 300-311; I. Jelesarov, E. Durr, R.M. Thomas, H.R. Bosshard, Salt effects on hydrophobic interaction and charge screening in the folding of a negatively charged peptide to a coiled coil (leucine zipper), Biochemistry 37 (1998) 7539-7550]. As it is known that Kv channel gating is linked to the stability of the cytoplasmic T1 multimerization domain conformation [D.L. Minor, Y.F. Lin, B.C. Mobley, A. Avelar, Y.N. Jan, L.Y. Jan, J.M. Berger, The polar T1 interface is linked to conformational changes that open the voltage-gated potassium channel, Cell 102 (2000) 657-670; B.A. Yi, D.L. Minor Jr., Y.F. Lin, Y.N. Jan, L.Y. Jan, Controlling potassium channel activities: interplay between the membrane and intracellular factors, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 11016-11023] and that intracellular chloride concentration has been linked to Kv channel kinetics [L.K. Bekar, W. Walz, Intracellular chloride modulates A-type potassium currents in astrocytes, Glia 39 (2002) 207-216; W.B. Thoreson, S.L. Stella, Anion modulation of calcium current voltage dependence and amplitude in salamander rods, Biochim. Biophys. Acta 1464 (2000) 142-150], the objective of the present study was to address how chloride concentration changes affect Kv channel kinetics more closely in an isolated expression system. Initially, no significant chloride concentration-dependent effects on channel steady-state gating kinetics were observed. Only after disruption of the cytoskeleton with cytochalasin-D did we see significant chloride concentration-dependent shifts in gating kinetics. This suggests that the shift in gating kinetics is mediated through effects of intracellular chloride concentration on cytoplasmic domain tertiary conformation as cytoskeletal interaction appears to mask the effect. Furthermore, as cytoskeletal disruption only impacts channel gating kinetics at low physiological intracellular chloride concentrations, these studies highlight the importance of paying close attention to anion concentrations used under experimental conditions.
Asunto(s)
Membrana Celular/metabolismo , Cloruros/metabolismo , Activación del Canal Iónico/fisiología , Canales de Potasio con Entrada de Voltaje/metabolismo , Línea Celular , Membrana Celular/efectos de los fármacos , Cloruros/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/fisiología , Humanos , Líquido Intracelular/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Canales de Potasio con Entrada de Voltaje/efectos de los fármacosRESUMEN
Attempts to attribute ileal brush-border chloride conductance to specific proteins were pursued by screening a porcine intestinal cDNA library. A 0.94-kb clone was identified on expression screening with a monoclonal antibody that inhibited enterocyte brush-border chloride conductance. Further screening approaches led to the isolation of a 3.1-kb full-length sequence called pCLCA1, consistent with the identification of a 2.9-kb transcript through Northern analysis. This sequence had significant homology to the CLCA gene family of calcium-regulated chloride channels, especially to hCLCA1. However, a strong A-kinase consensus phosphorylation site in a predicted cytoplasmic loop of the protein was a notable difference from the hCLCA1 gene product. Several porcine exocrine epithelial tissues, including ileum, trachea, and the major salivary glands express pCLCA1 mRNA. In situ hybridization studies localized the expression of pCLCA1 mRNA to the crypt and villus epithelia of porcine ileum, whereas tracheal expression was observed in both surface epithelium and submucosal glands. In situ expression of pCLCA1 in mouse 3T3 cells induces an ionomycin-dependent chloride conductance activity in these cells.
Asunto(s)
Membrana Celular/química , Polaridad Celular , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Enterocitos/metabolismo , Íleon/metabolismo , Porcinos/genética , Células 3T3 , Secuencia de Aminoácidos , Animales , Canales de Cloruro/química , Canales de Cloruro/genética , Clonación Molecular , Conductividad Eléctrica , Enterocitos/citología , Enterocitos/efectos de los fármacos , Perfilación de la Expresión Génica , Glicosilación , Humanos , Íleon/citología , Íleon/efectos de los fármacos , Hibridación in Situ , Ionomicina/farmacología , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Fosforilación , Filogenia , Conformación Proteica , ARN Mensajero/análisis , ARN Mensajero/genética , Alineación de Secuencia , TransfecciónRESUMEN
A new form of the widely expressed, volume-regulated chloride channel, ClC-2, has been cloned from a pig ileal cDNA library. This ClC-2 homologue, called ClC-2i, has interesting variability within its cDNA sequence, including the deletion of bases that correspond to positions 1326 through 1401 in rat ClC-2 cDNA sequence. This 75 bp deletion corresponds to the complete loss of exon 13 plus the first four bases of exon 14, and involves an atypical intron-exon splice site. Tissue-specific mRNA expression patterns in the pig show variable degrees of exon 13 skipping in ClC-2i. Exon 13 skipping was also observed in rat ClC-2i, albeit at a higher frequency than in the pig in tissues that were examined. A relatively purine-rich 76 bp insertion in the pig genomic sequence of ClC-2i, close to the 3' end of intron 12, may be responsible for the relatively high frequency of exon 13 skipping during the processing of this mRNA.