Roles of proton removal and membrane fluidity in Na+- and Cl(-)-dependent uptake of gamma-aminobutyric acid by mouse brain particles.
Proton removal is required for Na+-dependent uptake of GABA into mouse brain microsomal particles (P3) to take place at 0 degrees C and pH 7.3. No temporal coordination was demonstrated to exist between proton removal and GABA uptake processes, ruling out the possibilities that either a proton gradient or proton outflux from the particles is required. Observations on proton dissociation from the particles indicated that the protons are derived from a particulate compartment that is not in pH equilibrium with the bulk solution. Experiments on the effects of temperature on GABA uptake in the presence of 80 mM NaCl alone or with 10 mM triethanolamine (TREA) at pH 7.3 showed that the effects of TREA and temperature were interactive. The relative enhancing effects of TREA on GABA uptake diminished progressively with increasing temperature. The break points in Arrhenius plots obtained in presence and absence of TREA were the same, indicating that the effect of TREA was not on bulk viscosity of the membrane. Measurements made of fluorescence polarization as a function of temperature and of the absorbance-corrected fluorescence using TMA-DPH, a probe believed to be anchored at the lipid-water interface, showed clearly that the characteristic viscosity changes that take place with temperature in the membrane regions through which the probe is distributed were not correlated with the effects of temperature on GABA uptake. It is tentatively concluded that the protons may be attached to the membranes of the P3 particles by strong coulombic interactions in unstirred electrical double layers, possibly both on the inside and outside of particles. The importance of the existence of differences between pH at the surfaces of membranes, possibly both on the inside and outside of the particles, and that of the bulk solutions that bathe them was reiterated.