Activation of Large Conductance Sodium Channels upon Expression of Amiloride-sensitive Sodium Channel in Sf9 Insect Cells

Abstract

The amiloride-sensitive epithelial sodium channels (ENaC) mediate Na ؉ reabsorption in epithelial tissues including distal nephron, colon, lung, and secretory glands and plays a critical role in pathophysiology of hypertension and cystic fibrosis. The ENaC is a multimeric protein composed of ␣-ENaC, ␤-ENaC, and ␥-ENaC subunits. To study the biochemical properties of the channel, the subunit cDNAs of rat colon ENaC (rENaC) were subcloned into baculoviruses, and the corresponding proteins were expressed in Sf9 insect cells. The functional characteristics of the expressed rENaC were studied in planar lipid bilayers. The results show that expression of ␣-rENaC and ␣␤␥-rENaC in Sf9 insect cells results in the generation of cation-selective large conductance channels. Although the large conductance channels observed in the ␣-rENaC-containing membranes were unaffected by amiloride, the large conductance channels found in ␣␤␥-rENaC complex-containing membranes exhibited voltage-dependent flickering in the presence of micromolar amiloride. Possible implications of these observations are discussed.The highly selective Na ϩ channels (ENaC) 1 located in the apical membranes of epithelial cells of renal tubules, distal colon, lung, and several exocrine glands mediate controlled entry of Na ϩ ions into cells from the luminal or mucosal fluids and exhibit high sensitivity to pyrazine-based K ϩ -sparing diuretics such as amiloride (1-3). Abnormal function of ENaC has been demonstrated in human diseases including hereditary hypertension (Liddle's syndrome) (4), salt-sensitive hypertension (5, 6), and cystic fibrosis (CF) (7,8), indicating the importance of these channels in normal and pathophysiology. In addition, ENaC belongs to newly emerged superfamily of ion channels that include degenerins (9, 10). Rossier and co-workers (11, 12) have first isolated three cDNAs coding for the rat colon ENaC (rENaC) subunits, ␣-rENaC, ␤-rENaC, and ␥-rENaC, and demonstrated that ␣-ENaC is the channel-forming subunit and that ␤-ENaC and ␥-ENaC subunits together greatly increase the channel activity of the ␣-ENaC subunit. Subsequently, highly homologous ENaC subunits from other species were sequenced in several laboratories and established firmly that ENaC is a complex protein formed by the association of these three subunits (13,14). Structurally, all of these subunits contain two transmembrane segments, a large extracellular region and cytoplasmically located relatively short NH 2 and COOH termini, and share nearly 35% amino acid sequence homology (10 -12, 15). However, the number of these three subunits in a fully functional ENaC remains unclear. For example, Firsov et al. (16) reported that the ENaC is a complex of two ␣-ENaC, one ␤-ENaC, and one ␥-ENaC subunits. Snyder et al. (51) have shown that ENaC is a much larger complex formed by three each of the three subunits of ENaC. Based on the kinetic analysis, Berdiev et al. (50) have concluded that four ␣-ENaC subunits together form the conduction pore.It has been shown in the case of CF...

Selective Association of TRPC Channel Subunits in Rat Brain Synaptosomes

“…When used at appropriate multiplicities of infection, individual subunits are properly assembled into a functioning unit in Sf9 cells co-infected with individual recombinant baculoviruses (32)(33)(34)(35). We took advantage of this system to determine whether the TRPC channel proteins co-assemble.…”

Identification of Two Different States of P-glycoprotein in Its Catalytic Cycle

“…Preparation of Pgp-containing Membranes-The Sf9 insect cells were infected with a recombinant baculovirus carrying the human wild-type Pgp cDNA, and the total membrane fraction was prepared as described previously (17). Membranes prepared from Sf9 insect cells infected with a recombinant baculovirus carrying the ␣-subunit cDNA of the amiloride-sensitive sodium channel (25) were used as the control.…”

Characterization of a New Antibody Raised against the NH2 Terminus of P-Glycoprotein

“…The pelleted cells were washed once with ice-cold PBS, and the cellular proteins were precipitated with 6% (w/v) trichloroacetic acid. The precipitated proteins in the cell lysates were dissolved in Laemmli disaggregating buffer and separated on 7.5% SDS-PAGE gels and transferred electrophoretically onto polyvinylidene difluoride membranes (16,17). The immunoblots were developed using the either NH 2 11 or C219 antibody in conjunction with horseradish peroxidase -coupled secondary antibody and the enhanced chemiluminescence assay kit (Amersham, Piscataway, NJ) as described previously (18).…”