Electrophysiological characterization of native Na+–HCO3– cotransporter current in bovine parotid acinar cells

摘要

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.