A Cl(-) cotransporter selective for NH(4)(+) over K(+) in glial cells of bee retina

Abstract

There appears to be a flux of ammonium (NH(4)(+)/NH(3)) from neurons to glial cells in most nervous tissues. In bee retinal glial cells, NH(4)(+)/NH(3) uptake is at least partly by chloride-dependant transport of the ionic form NH(4)(+). Transmembrane transport of NH(4)(+) has been described previously on transporters on which NH(4)(+) replaces K(+), or, more rarely, Na(+) or H(+), but no transport system in animal cells has been shown to be selective for NH(4)(+) over these other ions. To see if the NH(4)(+)-Cl(-) cotransporter on bee retinal glial cells is selective for NH(4)(+) 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 NH(4)(+), but also of NH(3). To estimate transmembrane fluxes of NH(4)(+), 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 NH(3) was 13 microm s(-1). A numerical model was used to deduce the NH(4)(+) 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 [NH(4)(+)](o); the relation was fitted with a Michaelis-Menten equation with K(m) approximately 7 mM. The inhibition of NH(4)(+) flux by extracellular K(+) appeared to be competitive, with an apparent K(i) of approximately 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 NH(4)(+) with greater affinity than K(+). We conclude that this transporter is functionally selective for NH(4)(+) over K(+) and that the transporter molecule probably has a greater affinity for NH(4)(+) than for K(+).

Figure 1

Speed of the solution changes. (A) The perfusion chamber. A channel was milled in a polymethacrylate slab and a glass cover slip was glued on the bottom. Note the rounded edges of the channel. (B) Speed of solution change. The data points show the change in fluorescence observed through the 40× microscope objective focused on cells on the floor of the chamber during a 120-s perfusion change from standard solution to one containing 1 μg liter⁻¹ fluorescein. The time scale was displaced so that zero coincides with the beginning of the fluorescence change. The solid lines represent the best fits of data points obtained by regressions with simple exponentials {1 − exp(−t/τ₁) and exp[−(t − 120)/τ₂]} whose time constants, τ₁ and τ₂, are given in the figure.

Figure 2

Effect of extracellular ammonium application on pH_i of an isolated bundle of glial cells. (A) The ammonium-induced acidification was slightly inhibited by K⁺ or Rb⁺. For each application of 2 mM NH₄ ⁺, [K⁺]_o was either maintained at its baseline value of 10 mM, changed as indicated, or replaced by 50 mM Rb⁺. (B) The response to a 5-min application of 2 mM NH₄ ⁺ can be divided into five phases, which correspond to different patterns of fluxes (C). This cell had a fairly acid baseline pH_i (≈7.17) so that Phase 1 was prominent. (C) Schemes of fluxes corresponding to four of the phases indicated in B. (Phase 1) Inward flux of NH₃ is greater than ∼1% of inward flux of NH₄ ⁺. The maintenance of intracellular equilibrium NH₄ ⁺ ↔ NH₃ + H⁺ consumes H⁺ ions. (Phase 2) NH₄ ⁺ transmembrane gradient is still inward, while [NH₃]_i slightly exceeds [NH₃]_o. H⁺ ions are shuttled into the cell. (Phase 3) Extrusion of H⁺ ions by pH regulatory mechanisms equals inward flux of NH₄ ⁺. (Phase 4) This phase is approximately the inverse of Phase 1. Phase 5 (not shown) consists almost entirely of H⁺ efflux. (D) Scheme of transmembrane fluxes during ammonium exposure. pH_i changes result from three transmembrane fluxes, F_NH4, F_NH3, and net H⁺ flux through pH regulatory processes (F_reg) (see text).

Figure 2

Effect of extracellular ammonium application on pH_i of an isolated bundle of glial cells. (A) The ammonium-induced acidification was slightly inhibited by K⁺ or Rb⁺. For each application of 2 mM NH₄ ⁺, [K⁺]_o was either maintained at its baseline value of 10 mM, changed as indicated, or replaced by 50 mM Rb⁺. (B) The response to a 5-min application of 2 mM NH₄ ⁺ can be divided into five phases, which correspond to different patterns of fluxes (C). This cell had a fairly acid baseline pH_i (≈7.17) so that Phase 1 was prominent. (C) Schemes of fluxes corresponding to four of the phases indicated in B. (Phase 1) Inward flux of NH₃ is greater than ∼1% of inward flux of NH₄ ⁺. The maintenance of intracellular equilibrium NH₄ ⁺ ↔ NH₃ + H⁺ consumes H⁺ ions. (Phase 2) NH₄ ⁺ transmembrane gradient is still inward, while [NH₃]_i slightly exceeds [NH₃]_o. H⁺ ions are shuttled into the cell. (Phase 3) Extrusion of H⁺ ions by pH regulatory mechanisms equals inward flux of NH₄ ⁺. (Phase 4) This phase is approximately the inverse of Phase 1. Phase 5 (not shown) consists almost entirely of H⁺ efflux. (D) Scheme of transmembrane fluxes during ammonium exposure. pH_i changes result from three transmembrane fluxes, F_NH4, F_NH3, and net H⁺ flux through pH regulatory processes (F_reg) (see text).

Figure 3

Absolute measurement of pH_i during application of 2 mM NH₄ ⁺. (A) Recording of BCECF fluorescence ratio from a bundle of glial cells. Different mixtures of propionate (Prop) and trimethylamine (TMA) were applied during prolonged application of 2 mM ammonium, first with pH_o = 6.90, and then with pH_o = 7.30. (B) In experiments like that of A, pH_i was calculated from responses to application of two mixtures of propionate and TMA and ascribed to the time midway between the two applications (e.g., arrows in A and B correspond to the same point). The data points shown are for an ammonium concentration of 2 mM and the solid line is a linear regression through them. The dashed line simply illustrates that the mean initial pH_i was 7.3. (C) Summary of results for the pH_i measured ∼10 min after application of 2 mM ammonium at pH 6.90. The results for different pairs of weak acids and weak bases are shown separately; for the pair propionate/TMA, a possible dependence on pH_i was examined by comparing the mean values for cells with an initial baseline pH_i < 7.10 (“acid”) and those with baseline pH_i > 7.10 (“alkaline”). Bars show SDs. (D) Absolute pH_i reached during 2 mM NH₄ ⁺ application at pH_os 6.50 (n = 7), 6.90 (n = 6), 7.30 (n = 6), and 7.70 (n = 7). The solid line is a linear regression through the data points (R = 0.996), vertical bars represent ±SD, horizontal bars represent the precision of the adjusted pH_o of the solutions. (E) Recording showing pH_i reached in nigericin 10 μM pH_o 6.90 compared with pH_i reached in NH₄ ⁺ 2 mM pH_o 6.90.

Figure 4

Direct measurement of P_NH3. (A) A bundle of cells was held with a pipette ∼50 μm above the floor of the chamber and perifused for 30 min in pH_o 6.20 to reduce pH_i. The cells were then perifused with 0 Cl⁻ solution containing bumetanide 500 μM for 1 min before 10 mM NH₄ ⁺ _o was substituted for Na⁺ _o. (B) A few minutes after the response of A, the same bundle attached to the pipette was perifused with 10 mM propionate in the same conditions. The propionate-induced pH_i change gave a lower limit for the speed of the change in solution at the cell membrane.

Figure 5

Indirect measurement of NH₃ permeability. (A) Direct measurement of P_MA. The pH_i change induced by 10 mM methylamine (MA) (thick line) was compared with that induced by 10 mM trimethylamine (TMA) (thin line) and to the change in fluorescence on switching to a solution containing 1μg liter⁻¹ fluorescein (dotted line). The microscope was focused on the same bundle for the three recordings. (B) Relative cell membrane permeabilities to neutral forms of propionate (Prop) and MA. A bundle of cells was superfused with 2 mM NH₄ ⁺ so that pH_i was maintained at a value close to pH_o (Fig. 3 D). When 10 mM Prop + 10 mM MA was applied at pH_o 7.2, entry of the neutral form of Prop initially predominated, but as [Prop]_i increased, entry of MA began to predominate and pH_i started to increase. The initial change in pH_i reversed for a value of pH_o between 7.40 and 7.60. (C) Relative cell membrane permeabilities to NH₃ and to the neutral form of Prop. A mixture of 5 mM NH₄ ⁺ and 5 mM Prop was applied to cells superfused with a 0 Cl⁻ solution at pH_o 7.10 (thick trace) or 7.00 (thin trace). The two recordings were from the same bundle of cells. At pH_o 7.10, the initial change in pH_i was an increase, while at pH_o 7.00 it was a decrease.

Figure 6

pH_i recovery from ammonium-induced acid loads. (A) Analysis of recoveries from acidifications induced by applications of 0.5, 1, 2, 5, and 10 mM NH₄ ⁺ on the same bundle of glial cells. ln([H⁺]_i − [H⁺]_∞) was plotted against t − t _f, where t _f is 45 s after the end of the NH₄ ⁺ superfusion. From linear regressions of the data points, τ_reg was calculated as being (min): 2.74 (correlation coefficient, R = 0.761), 3.96 (R = 0.971), 3.29 (R = 0.978), 3.68 (R = 0.996), and 3.75 (R = 0.998) for the recoveries from the increasing acidifications induced by 0.5, 1, 2, 5, and 10 mM NH₄ ⁺. (B) τ_reg, calculated as in A, as a function of ΔpH_i at the beginning of the recovery. The data are from 17 cell bundles (each represented by a different symbol) for which at least three different [NH₄ ⁺]_o were applied. For each cell bundle, a linear regression was calculated and a line with the mean of their slopes [(0.3 min (pH unit )⁻¹] is shown passing through the barycenter of the points.

Figure 7

pH_i changes as a function of ammonium concentration (for 30-s applications in 0 K⁺). (A) Typical recording of pH_i responses to various NH₄ ⁺ concentrations. Cells were normally superfused with 10 mM K⁺ standard solution that was switched to a 0 K⁺ solution 15 s before each NH₄ ⁺ application. The response to 10 mM propionate (near the end of the experiment) gives a lower limit for the rapidity of the solution changes. On the same cells, after a delay of ≈20 min (“Δt*”), removal of extracellular K⁺ with no ammonium application had no detectable effect on pH_i. (B) Superposition of pH_i responses to NH₄ ⁺ (from A) on a shorter time scale. Dotted lines for 0.5 and 1 mM NH₄ ⁺ are the second (control) responses. The delay between activation of the solenoid valve (t = 0) and the start of the changes in pH_i was ∼10 s. Note that during the NH₄ ⁺ application, pH_i fell more rapidly for 10 mM NH₄ ⁺ than for 20 mM, but that the peak reached after the application (Phase 4) was greater for 20 mM. (C) Calculation of δpH_i/δt(NH₄ ⁺) for each NH₄ ⁺-induced acidification. Mean slopes were measured between 15 and 35 s after the onset of the NH₄ ⁺ application by linear regression. Taking into account the delay for arrival of solutions, this interval corresponds to the last 20 s of the NH₄ ⁺ applications. (D) Mean values for ΔpH_i([NH₄ ⁺]_o) (peak change in pH_i, open symbols) and δpH_i/δt([NH₄ ⁺]_o) (filled symbols) from four experiments similar to that of A. Bars represent ± SEM. The best Michaelis-Menten fits of ΔpH_i([NH₄ ⁺]_o) for [NH₄ ⁺]_o = 0.5, 1, 2, 5, 10, and 20 mM (solid line) and of δpH_i/δt([NH₄ ⁺]_o) for [NH₄ ⁺]_o = 0.5, 1, 2, and 5 mM (dotted line) are shown.

Figure 8

Use of the cell model (Fig. 2 D) to derive inward F_NH4([NH₄ ⁺]_o) from measured pH_i changes induced by brief applications of NH₄ ⁺. (A) Constant inward F_NH4 (ⁱⁿF_NH4) was imposed on the model for 30 s. δpH_i/δt, calculated 15 s after onset, was plotted against ⁱⁿF_NH4. Data are shown for simulations with [NH₄ ⁺]_o set to 2 mM (to fix [NH₃]_o) and P_NH3 = 7, 13, and 19 μm s⁻¹. (B) As in A, δpH_i/δt was plotted versus ⁱⁿF_NH4 for simulations with [NH₄ ⁺]_o = 0.5 mM (triangles), 1 and 2 mM (diamonds), and 5, 10, and 20 mM (circles). P_NH3 = 13 μm s⁻¹. Because increasing [NH₄ ⁺]_o increases [NH₃]_o, δpH_i/δt(ⁱⁿF_NH4) is smaller for higher [NH₄ ⁺]_o. (C) Simulations in which an ⁱⁿF_NH4 was imposed for 30 s and followed by an outward F_NH4 (^outF_NH4). ⁱⁿF_NH4 was set to 6.65 mM min⁻¹ and [NH₃]_o was fixed by setting [NH₄ ⁺]_o = 2 mM; this gave δpH_i/δt = 0.44 pH unit min⁻¹, equal to the mean measured pH_i change induced by 2 mM NH₄ ⁺ in 0 K⁺ for cells with baseline pH_i ≈ 7.4. At t = 30 s, F_NH4 switched instantaneously from ⁱⁿF_NH4 to maximum ^outF_NH4, ^outF_{NH4 max}, and decreased to zero as [NH₄ ⁺]_i decreased to zero (see text). Simulations for ^outF_{NH4 max} equal to 0, −6.65, and −20 mM min⁻¹ show that the rebound acidification after 30 s decreased when ^outF_{NH4 max} increased. (D) Plot of ΔpH_i(ⁱⁿF_NH4) measured as baseline pH_i (7.4) minus the minimal pH_i reached during the rebound acidification after 30 s of influx ⁱⁿF_NH4. Simulations for 0.5 mM (triangles) or 20 mM (circles) NH₄ ⁺ _o (+NH_3o) show that [NH₃]_o has little effect on ΔpH_i(ⁱⁿF_NH4). ^outF_{NH4 max} = 0. (E) ⁱⁿF_NH4([NH₄ ⁺]_o) calculated from δpH_i/δt([NH₄ ⁺]_o) (•) and ΔpH_i([NH₄ ⁺]_o) (○) of Fig. 7 D. The points were fitted by Michaelis-Menten curves (R = 0.963 and 0.994, respectively) with apparent constants K ′_m equal to 5.9 ± 1.3 and 7.8 ± 0.7 mM.

Figure 9

Inhibition of inward NH₄ ⁺ flux by external K⁺. (A) Comparison of responses to 2 and 5 mM NH₄ ⁺ in 0 and 10 mM K⁺. For each response, NH₄ ⁺ was applied for 30 s; when applied in 0 K⁺, K⁺ was removed from the superfusate 15 s earlier. (B) Double inverse plot of mean ΔpH_i vs. [NH₄ ⁺]_o (n = 6). Straight lines passing through mean data points intersect the abscissa at [NH₄ ⁺]_o = 5.02 mM (0 K⁺, ○) and 6.90 mM (10 mM K⁺, •). (C) Double inverse plot of ⁱⁿF_NH4 vs. [NH₄ ⁺]_o. ⁱⁿF_NH4 is the mean value during the NH₄ ⁺ application calculated from ΔpH_i of the six experiments of B using the relation of Fig. 8 E. Straight lines passing through mean data points intersect the abscissa for [NH₄ ⁺]_o = 5.16 mM (0 K⁺, ○) and 7.09 mM (10 mM K⁺, •). (D) Intracellular recording from a retinal slice with a microelectrode in the glial compartment. Increasing [K⁺] from 10 to 20 mM in the superfusate induced a depolarization of the cell membranes. When the same increase of [K⁺] was made after superfusing the slice with 5 mM barium for a few minutes, the depolarization was undetectable for at least the first 45 s.

Figure 10

pH_i responses to stepwise increases in [NH₄ ⁺]_o are accounted for by a model of the transport process. (A) Kinetic scheme of the cotransport of Cl⁻ and NH₄ ⁺ or K⁺. The unloaded transporter molecule is symbolized by X; o or i indicate the position of the transporter at the external or internal side of the cell membrane. Binding ions Cl⁻, NH₄ ⁺, and K⁺ are not shown in this scheme for visual simplicity. The three binding constants K _c, K _m, and K _i for Cl⁻, NH₄ ⁺, and K⁺ are unaffected by the side to which the transporter faces. Two kinetic constants, k and g, describe the transit steps of the unloaded and loaded transporter. (B) Experimental response to stepwise increases in [NH₄ ⁺]_o. (C) Simulated response of the cell model of Fig. 2 D including the transporter model of A to stepwise increases in [NH₄ ⁺]_o with affinity for NH₄ ⁺, K _m = 5 mM (continuous line) and 20 mM (dashed line). K _i was 15 mM.

Figure 11

Effects of increasing [K⁺]_o from 10 to 50 mM on the plateau phase pH_i during superfusion with 2 mM or 20 mM NH₄ ⁺. (A) Typical recording. At the beginning and end of the experiment, the cells were superfused with 2 mM NH₄ ⁺ at pH_o 7.50 to provide a pH_i calibration (see Fig. 3 D). (B) Simulated effect of increasing [K⁺]_o. In the model, [K⁺]_i was kept constant when [K⁺]_o was increased. The cell model included the transporter of Fig. 10 A (see online supplemental material for full list of parameter values). K _m was 7 mM and K _i was 10, 15, or 20 mM (superposed traces), the greatest inhibition being for K _i = 10 mM. (C) Simulation with the model modified so that transport of NH₄ ⁺ was inhibited by extracellular K⁺ in a noncompetitive manner. The transporter of Fig. 10 A was modified by removing the effect of increasing [K⁺]_o on the binding step XClo → XClKo (this modification amounts to suppressing this binding step with no modification of the other parameters) and by the addition of an inhibitory step XClNH₄o → XClNH₄Ko, whose affinity constant for K⁺ _o, K _K, was 5, 10, or 15 mM (superposed traces), the greatest inhibition being for K _K = 5 mM.