INTERACTION PIP1-PIP2: INVOLVEMENT OF LOOP A?

CINTIA JOZEFKOWICZ; PABLO ROSI; GABRIELA AMODEO; KARINA ALLEVA

Cluj Napoca

Congreso; The First World Congress on Water Channel Proteins; 2011

GheorgheBenga Fundation

Introduction: The Beta vulgaris plasma membrane (PM) expresses aquaporins that as far as from now have shown to have apparent different transport activity in Xenopus oocytes: BvPIP1;1, BvPIP2;1 and BvPIP2;2. Both BvPIP1;1 and BvPIP2;1 gives low oocyte PM osmotic water permeability (Pf) while BvPIP2;2 gives high Pf. In the literature, the present accepted paradigm is that PIP1 aquaporins have low water transport capacity in comparison with PIP2, which show high water transport; many reports as well show that PIP1-PIP2 co-expression present much higher Pf than the expected for PIP2 alone. This aspect of PIP1 functionality and/or trafficking is still under investigation, and the purpose of our work points to elucidate red beet PIPs behavior considering the above-mentioned framework. Materials and Methods. We assayed BvPIP and mutants functionality by means of heterologous expression in Xenopus oocytes. Pf was calculated after measuring the rate of oocyte swelling induced by transferring oocytes to ND96 diluted five-fold. Results and Discussion: In order to test if the low Pf detected for oocytes injected with BvPIP1;1 or BvPIP2;1 was a consequence of cRNA degradation, we synthetized both cRNA with a polyA tailing and ARCA capping. Under these conditions Pf of oocyte PM expressing BvPIP2;1 was compatible with water transport through active aquaporins; no changes where found for BvPIP1;1. PIP1-PIP2 coexpression experiments show that while BvPIP2;2 was able to interact with BvPIP1;1, BvPIP2;1 shows no functional interaction. BvPIP2;1 amino acid sequence showed differences in the loopA in comparison with the same loop of most PIP2. In order to investigate the influence of this motif in PIP1-PIP2 interaction we tested two mutants. Functional studies showed that both are able to transport water and, interestingly, preliminary results indicates these two mutated channels would affect Pf when co-expressed with BvPIP1;1 in oocytes. Conclusion: BvPIP2;1 is an active aquaporin with limitations to functionally interact with BvPIP1;1. Mutations in BvPIP2;1 loop A reverse this situation suggesting the involvement of this loop in the PIP2-PIP1 interaction.