Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, C127

Oral Communications

Volumetric analysis in Xenopus laevis oocytes expressing the rat γ-aminobutyric acid transporter GAT1 and the not functional mutant Q291N

M. Santacroce1, M. Castagna1, V. F. Sacchi1

1. Department of Molecular Sciences Applied to Biosystems, Università degli Studi of Milan, Milan, Italy.

Water transport across membranes is a fundamental process for cells. Currently accepted water transport pathways are the lipid bilayer itself and specialized proteins, the aquaporins. Recently it has been shown that several cotransporters, such as SLGT1 and GAT1 mediate a water influx across membranes (Loo et al., 1999; Lapointe et al., 2002). Some observations support the idea that local osmotic gradients built up immediately after cotransport activity are fully responsible for the cell swelling (Lapointe et al., 2002). An alternative hypothesis suggests a direct coupling of water, ion and solute (Zeuthen et al., 1996). γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in mammalian brain and the GABA transporter GAT-1, belonging to the neurotransmitter sodium symporter family (NSS), is an integral membrane protein responsible for the reuptake of GABA from the synaptic cleft. Glutamine 291 is a strictly conserved residue in all members of the NSS family. It has been previously published that Q291 mutants cannot transport GABA or give rise to currents even though they are targeted to the plasma membrane (Mari et al., 2006). In order to better understand water transport in cotransporters we verified whether the not functional mutant GAT1 Q291N is able to mediate water transport. To this aim, we performed six independent volumetric analysis experiments in hypoosmotic conditions (ΔmOsm = 167) using Xenopus laevis oocytes expressing the wild type (wt) and the mutated protein (Dorr et al., 2007). The expression of GAT1 on oocyte surface was confirmed by radiolabelled aminoacid transport experiments. In each experiment volumetric variations were measured in three groups of 7-14 oocytes (GAT1, GAT1 Q291N and control oocytes) and the mean permeability factors (Pf) of each group were calculated. The Pf of GAT1 expressing oocytes ranged from 1.41×10-3 ± 1.93×10-4 cm/s to 1.99×10-3 ± 7.74×10-5 cm/s (mean ± S.E.); the Pf of Q291N ranged from 1.33×10-3 ± 6.44×10-5 cm/s to 1.75×10-3 ± 7.27×10-5 cm/s (mean ± S.E.); the Pf of control oocytes ranged from 7.30×10-4 ± 4.57×10-5 cm/s to 1.46×10-3 ± 5.63×10-5 cm/s (mean ± S.E.). As expected, in five experiments out of six the Pf of GAT1 was higher than the Pf of control oocytes (p<0.05 Student's t-test). Interestingly, in five experiments out of six also the Pf of GAT1 Q291N was significantly higher than control Pf value (p<0.05). Our experiments thus showed that both wt protein and mutant mediate a water influx across Xenopus oocyte membrane. In conclusion though at the moment we cannot choose for one of the above described hypothesis regarding water transport through cotransporters, our results indicate that, under particular conditions, a water influx occurs both when the cotransport mechanism can take place (GAT1 wt) and when it cannot (GAT1 Q291N).

Where applicable, experiments conform with Society ethical requirements