FaPIP1 IS MORE SENSITIVE TO pH THAN FaPIP2

MERCEDES MARQUEZ; KARINA ALLEVA; GABRIELA AMODEO

Cluj-Napoca,

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

Gheorghe Benga Foundation

In previous work, we reported the cloning of two strawberry fruit aquaporins showing differential behavior show differential water permeability (Pf) when expressed at Xenopus oocytes: high (FaPIP2;1) and low (FaPIP1;1). Also the co-expression of both subtypes resulted in an enhancement of water permeability, showing Pf values that exceeds their individual contribution. Moreover, this high water permeability is modulated by cytosolic pH, must block flow of water when the acid pHint. On the contrary, when FaPIP2;1 is injected alone shows only partial inhibition (Alleva et al., 2010). Both FaPIP, like the rest of the PIPs have highly conserved histidines have been described to trigger a pH inhibitory response, shutting down PIPs when cytosolic medium is acidified (Tournaire-Roux et al., 2003). We ask: Are PIP1 and PIP2 different in their pH sensitivity?  Because of, the low permeability of  FaPIP1;1, unable to regulation study. For this reason, we designed a FaPIP2 inactivated mutant to coinject whit FaPIP1;1. Materials and Methods. Oocyte expression system was used to analyze osmotic water permeability. Defolliculated Xenopus oocytes were injected with different mass ratio of cRNA encoding FaPIP1, FaPIP2, and a FaPIP2 inactivated mutant S121A). Osmotic water permeability (Pf) was determined by measuring the rate of oocyte swelling induced by transferring oocytes to the same solution diluted fivefold with distilled water. Oocyte internal (cytosolic) pH was modified following an already described protocol (Tournaire-Roux et al. 2003). Briefly, oocytes were pre-incubated for 15 min in a solution of different pHs (5,8-7,6). Results and Discussion. When FaPIP1 and FaPIP2  are co-injected and tested at different internal pH the inhibition response behaves differently than the obtained by injecting only FaPIP2 RNAm, regardless of the quantities or injected mass proportions the EC50 parameter remains unchanged. Oocytes that expressed FaPIP2 when assay at different pH resulted in an inhibition of the water permeability at acidic pH with an EC50 of 6,0. When FaPIP2 and FaPIP1 where expressed the observed EC50 was 6,4. But when FaPIP1 was co-expressed with the inactive mutant of FaPIP2 S121A, the EC50 observed was 6,8. This way,  we can indirect study about  regulation of PIP1. Conclusions. FaPIP1 seems to be more sensitive to pH than FaPIP2, and co-injection response would be the result from the addition of both individual activities.