NH4+ currents across the peribacteroid membrane of soybean. Macroscopic and microscopic properties, inhibition by Mg2+, and temperature dependence indicate a subpicoSiemens channel finely regulated by divalent cations

Abstract

The control of ammonium (NH₄⁺) transport is critical in preventing futile cycles of NH₄⁺/ammonia transport. An unusual nonselective cation channel with subpicoSiemens single-channel conductance permeable to NH₄⁺ had previously been identified in the peribacteroid membrane (PBM) of symbiosomes from soybean (Glycine max) nodules. Here, we investigate the proposed channel mechanism and its control by luminal magnesium. Currents carried by NH₄⁺ were measured in inside-out PBM patches by patch clamp. NH₄⁺ transport corresponding to the physiological direction of net transfer showed time-dependent activation and associated single-channel-like events. These could not be resolved to discrete conductances but had the same selectivity as the total current. The voltage dependence of the steady-state current was affected by temperature consistent with the rate constant of channel opening being reduced with decreased temperature. This resulted in steady-state currents that were more temperature sensitive at voltages where the current was only partially activated. When fully activated, the current reflected more the ion conduction through open channels and had an activation energy of 28.2 kJ mol–¹ (Q10 = 1.51, 8°C–24°C). Increased Mg²⁺ on the symbiosome lumen side blocked the current (ID50 = 351 μM, with 60 mM NH₄⁺). Complete inhibition with 2 mM Mg²⁺ was relieved with a small increase in NH₄⁺ on the lumen side of the membrane (shift of 60–70 mM). With Mg²⁺ the selectivity of the transport for divalent cations increased. From these features, we propose a divalent-dependent feedback regulation of the PBM-nonselective cation channel that could maintain a constant NH₄⁺ gradient across the membrane.