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

ASSEF Y.; MARINO G.; GALIZIA L. ; KOTSIAS BA.

Baltimore

Congreso; 55th Biophysical Society Annual Meeting; 2011

Biophysical Society

Shroom is a family of proteins linked to the actin cytoskeleton. We studied its effect upon the currents through ENaC channels (INa amil) in X. laevis oocytes injected with α, β, and γ mENaC and xShroom1 sense or antisense oligonucleotides. We observed a strong reduction in INa amil with the xShroom1 antisense: inward conductances (Ginward) (-160 to 0 mV) were 36 ± 12 µS and 1.80 ±.50 µS with xShroom1 sense and antisense, respectively. Similar results were obtained in oocytes expressing a mutant β-mENaC subunit (β-S518K) with an open probability of 1 (Ginward 65 ± 10 µS and 1.80 ± 2.0 µS for oocytes with xShroom1 sense or xShroom1 antisense, respectively. The negative effects of xShroom1 antisense can not be reversed with forskolin which reduced the rate of ENaC retrieval: Ginward: 124 ± 27 µS and 7.0 ± 1.9 µS with xShroom1 sense or xShroom1 antisense. Trypsin in the range of ng/ml activates the membrane-resident ENaC channels (Bengrine et al.2007), being this effect dependent on activation of G-proteins. Addition of 20 ng/ml of trypsin led to a slow increase in INa amil with xShroom1 sense and it had no effect in most of the oocytes coinjected with ENaC and xShroom1 antisense (2 out of 20). Trypsin were (was?) without effects on the endogenous conductances. These data are consistent with the idea that the reduced INa amil when xShroom1 is blocked 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). Supported by grants MO35 (UBA) and PICT 38181 (ANPCyT).