Electrophysiological characterization of native Na+-HCO3- cotransporter current in bovine parotid acinar cells.
Using patch-clamp and molecular biological techniques, we identified and characterized membrane currents most likely generated by an electrogenic Na+-HCO3- cotransporter (NBCe) in acutely dissociated bovine parotid acinar (BPA) cells. When BPA cells were dialysed with a N-methyl-D-glucamine (NMDG)-glutamate-rich pipette solution, switching a Na-glutamate-rich, nominally HCO3--free bath solution to the one containing 25 mM HCO3-, but not Cl-, elicited a whole-cell current with a linear current-voltage relation. The HCO3- evoked current was abolished by total replacement of extracellular Na+ (Na+o) with NMDG or by 0.5 mM 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS), and was only partially supported by Li+o, but not by K+o, Cs+o, and cholineo. The reversal potential shift of DIDS (0.5 mM)-sensitive current induced by a change of [Na+]o corresponded to an apparent coupling ratio of HCO3- to Na+ of 2. RT-PCR analysis showed the presence of transcripts of NBCe1-B, but not NBCe1-A in BPA cells. Electrophysiological and pharmacological properties of whole-cell currents recorded from HEK293 cells transfected with the NBCe1-B, which was cloned from BPA cells resembled those of the native currents. Non-invasive measurements of membrane potential changes in the cell-attached patch configuration indicated that an NBCe activity is present in intact unstimulated BPA cells bathed in a 25 mM HCO3--containing solution. Collectively, these results not only suggest that an NBCe is present, functional and may be mediated, at least in part, by NBCe1-B in BPA cells, but also provide the first electrophysiological characterization of transport properties of NBCe expressed in native exocrine glands.
Yamaguchi
Souichirou
S
Laboratory of Physiology, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan.
Ishikawa
Toru
T
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D005905:Glyburide / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D008094:Lithium / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D009544:Niflumic Acid / Q000494:pharmacology; D010306:Parotid Gland / Q000187:drug effects / Q000502:physiology*; D010693:Phloretin / Q000494:pharmacology; D011188:Potassium / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D027982:Sodium-Bicarbonate Symporters / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D001639:Bicarbonates; D007473:Ion Channels; D012333:RNA, Messenger; D027982:Sodium-Bicarbonate Symporters; D009544:Niflumic Acid; D008094:Lithium; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium; D010693:Phloretin; D005905:Glyburide