| Popis: |
In squid nerves the Na(+)-Ca(2+) exchanger is up-regulated by ATP and phosphoarginine (PA). ATP regulation involves drastic alterations in the Na(+)(i), H(+)(i) and Ca(2+)(i) interactions with the large intracellular cytoplasmic loop of the exchanger protein. In this work we explored the mechanisms associated with PA regulation in intracellular dialysed squid axons and squid optic nerve membrane vesicles. Dialysed axons were used to measure the four modes of exchange fluxes (Na(+)(o)-Ca(2+)(i) or forward exchange, Ca(2+)(o)-Na(+)(i) or reverse exchange, Ca(2+)(o)-Ca(2+)(i) exchange and Na(+)(o)-Na(+)(i) exchange) under controlled intra- and extracellular conditions. Inside-out membrane vesicles allowed measurement of the Na(+)-gradient-dependent (45)Ca(2+) uptake (forward mode) as influenced by ligands and digestion with chymotrypsin from the intracellular side. The results show that, unlike ATP, PA regulation does not affect the H(+)(i), Na(+)(i) and Ca(2+)(i) interactions with the intracellular 'regulatory' loop, but increases the affinity of the intracellular transport sites, preferentially for Ca(2+)(i) (about 20-fold) over Na(+)(i) (50%); i.e. PA favours the forward mode over the other exchange modes. Intracellular chymotrypsin digestion removed ATP regulation while leaving modulation by PA unmodified. Western blot analysis suggested that chymotrypsin disrupts the large intracellular loop. Together these results indicate that ATP and PA regulations are associated with different structures inside and outside the exchanger protein. Based on these observations we expanded our previous model for metabolic regulation of the Na(+)-Ca(2+) exchanger by adding to the original 'ATP region' a new zone, the 'PA region', related to the intracellular transport sites for Na(+)(i) and Ca(2+)(i). This new model is able to explain most previous and present results. |
