A Cl? Cotransporter Selective for Nh4+ over K+ in Glial Cells of Bee Retina

摘要

There appears to be a flux of ammonium (NH4+/NH3) from neurons to glial cells in most nervous tissues. In bee retinal glial cells, NH4+/NH3 uptake is at least partly by chloride-dependant transport of the ionic form NH4+. Transmembrane transport of NH4+ has been described previously on transporters on which NH4+ replaces K+, or, more rarely, Na+ or H+, but no transport system in animal cells has been shown to be selective for NH4+ over these other ions. To see if the NH4+-Cl? cotransporter on bee retinal glial cells is selective for NH4+ over K+ we measured ammonium-induced changes in intracellular pH (pHi) in isolated bundles of glial cells using a fluorescent indicator. These changes in pHi result from transmembrane fluxes not only of NH4+, but also of NH3. To estimate transmembrane fluxes of NH4+, it was necessary to measure several parameters. Intracellular pH buffering power was found to be 12 mM. Regulatory mechanisms tended to restore intracellular [H+] after its displacement with a time constant of 3 min. Membrane permeability to NH3 was 13 μm s?1. A numerical model was used to deduce the NH4+ flux through the transporter that would account for the pHi changes induced by a 30-s application of ammonium. This flux saturated with increasing [NH4+]o; the relation was fitted with a Michaelis-Menten equation with K m ≈ 7 mM. The inhibition of NH4+ flux by extracellular K+ appeared to be competitive, with an apparent K i of ～15 mM. A simple standard model of the transport process satisfactorily described the pHi changes caused by various experimental manipulations when the transporter bound NH4+ with greater affinity than K+. We conclude that this transporter is functionally selective for NH4+ over K+ and that the transporter molecule probably has a greater affinity for NH4+ than for K+.