xShroom1 regulates the number of ENaC channels inserted in the membrane of oocytes from Xenopus laevis

KOTSIAS BA.; ASSEF YA.; OZU M.; MARINO GI.; GALIZIA L.

Pacific Grove, California

Conferencia; 2011 APS Conference-7th International Symposium on Aldosterone and the ENaC/Degenerin Family; 2011

American Physiological Society

Shroom is a family of proteins linked to the actin cytoskeleton. We studied its effect upon the currents through ENaC channels. Oocytes (X. laevis) were injected with α, β, and γ mENaC and xShroom1 sense or antisense oligonucleotides and amiloride-sensitive Na+ currents (INa amil)were measured. We observed a strong reduction in INa amil in oocytes co-injected with the xShroom1 antisense, thus, the inward conductances (Ginward) (-160 to 0 mV) were 36± 12 μS and 1.80 ±.50 μS with xShroom1 sense and antisense respectively (n=18). The same results were obtained in oocytes expressing a DEG mutant β-mENaC subunit (β-S518K) which has a Po of nearly 1. The Ginward were 65 ± 10 μS and 1.80 ± 2.0 μS for oocytes injected with xShroom1 sense or xShroom1 antisense (n=16). Addition of low (20 ng/ml) concentration of trypsin which activates the membrane-resident ENaC channels led to a slow increase in INa amil in oocytes with xShroom1 sense (n=22). Trypsin had no effect on the currents generated by most of the oocytes coinjected with ENaC and xShroom1 antisense. The same results were obtained with high trypsin concentration (2 μg/ml, 2.5 min). In addition, fluorescence positive staining of plasma membrane in the oocytes expressing α,β and γ mENaC and xShroom1 sense were observed but not in oocytes coinjected with ENaC and xShroom1 antisense oligonucleotides. These data are consistent with the idea that the reduced INaamil in oocytes with blocked expression of xShroom1 is most probably due to a lack of functional ENaC channels in the plasma membrane. Acknowledgements: ENaC cDNAs were provided by Dr M. Carattino (Pittsburgh, Pa) and the set for oocytes was a gift of Dr C. Peracchia (Rochester, NY).