Passive water and urea permeability of a human Na+–glutamate cotransporter expressed in Xenopus oocytes

摘要

The human Na⁺-glutamate transporter (EAAT1) was expressed in Xenopus laevis oocytes. The passive water permeability, Lp, was derived from volume changes of the oocyte induced by changes in the external osmolarity. Oocytes were subjected to two-electrode voltage clamp. In the presence of Na⁺, the EAAT1-specific (defined in Discussion) Lp increased linearly with positive clamp potentials, the Lp being around 23 % larger at +50 mV than at –50 mV. l-Glutamate increased the EAAT1-specific Lp by up to 40 %. The K0.5 for the glutamate-dependent increase was 20 ± 6 μm, which is similar to the K0.5 value for glutamate activation of transport. The specific inhibitor dl-threo-β-benzyloxyaspartate (TBOA) reduced the EAAT1-specific Lp to 72 %. EAAT1 supported passive fluxes of [¹⁴C]urea and [¹⁴C]glycerol. The [¹⁴C]urea flux was increased in the presence of glutamate. The data suggest that the permeability depends on the conformational equilibrium of the EAAT1. At positive potentials and in the presence of Na⁺ and glutamate, the pore is enlarged and water and urea penetrate more readily. The Lp was larger when measured with urea or glycerol as osmolytes as compared with mannitol. Apparently, the properties of the pore are not uniform along its length. The outer section may accommodate urea and glycerol in an osmotically active form, giving rise to larger water fluxes. The physiological role of EAAT1 for water homeostasis in the central nervous system is discussed.