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

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 (pH_i) in isolated bundles of glial cells using a fluorescent indicator. These changes in pH_i 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 pH_i 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 pH_i 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⁺.