ENaC channels in oocytes from Xenopus laevis and their regulation by Shroom1 protein.

MARCELO OZU, GABRIELA I. MARINO, BASILIO A. KOTSIAS, YANINA A. ASSEF.

San Francisco, California, EEUU

Congreso; 54 Meeting Biophysical Society USA; 2010

Biophysical Society

ENaC channels in oocytes from Xenopus laevis and their regulation by Shroom1 protein. Marcelo Ozu, Gabriela I. Marino, Basilio A. Kotsias, Yanina A. Assef. Laboratorio de Canales Iónicos, Instituto de Investigaciones Médicas Alfredo Lanari, University of Buenos Aires, IDIM- CONICET, Argentina.   Shroom is a family of related proteins linked to the actin cytoskeleton. One of them, Shroom1 (APX) is constitutively expressed in X. oocytes and is required for the expression of amiloride sensitive sodium channels (ENaC) through an unknown mechanism (Staub et al., J Cell Biol 1992;119: 1497-506; Zuckerman et al., J Biol Chem 1999;274: 23286-95).   Oocytes from X. laevis were injected  with 2 ng of α, β, and γ mENaC and 25 ng of Shroom1 sense or antisense oligonucleotides. After 24–48 h  the oocytes were studied with a voltage clamp  technique (HP= 0 mV, 500 msec pulses, -160 to + 40 mV). Amiloride-sensitive Na currents (INa (amil)) was determined by subtracting the residual current in the presence of 10 µM amiloride from the base line.   Co-injection with ENaC and sense oligonucleotide for Shroom1 showed INa (amil) whereas a marked reduction in these currents was seen in oocytes co-injected with  antisense  (pulse -160 mV, INa (amil)  = -6.00 ± 1.97 μA and -0.21± 0.08 μA respectively (n=12).   We studied INa (amil) in oocytes expressing a DEG mutant β-mENaC subunit (β-S518K) which has a Po of nearly 1. This mutant had enhanced INa (amil) in comparison with the WT ENaC: pulse -160 mV: -10.8 ± 1.79 μA, however, these currents were also blocked  when co-injected with Shroom1 antisense to the same extent that with the wild type ENaC: -0.44 ± 0.21 μA (n=9).   We suggest that Shroom1-dependent ENaC inhibition may be mediated through an effect on the number of channels inserted in the membrane.   Acknowledgements: ENaC cDNAs were generously provided by Dr M. Carattino (Pittsburgh, Pa). The set for oocytes was a gift of Dr C. Peracchia (Rochester, NY). Supported by PICT 38181 and UBACYT MO35 grants.