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

OZU M.; MARINO G.; KOTSIAS BA.; ASSEF Y.

San Franciso, United States

Congreso; 54th Biophysical Society Annual Meeting; 2010

Biophysical Society

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 the voltage clamp technique (HP= 0 mV, 500 msec pulses, -160 to + 40 mV). Amiloride-sensitive Na currents (INa (amil)) was were determined by subtracting the residual current in the presence of 10 µM amiloride from the basal current. Co-injection with ENaC and Shroom1 sense 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.