Effects of Cd2+ on the epithelial Na+ channel (ENaC) investigated by experimental and modeling studies

Abstract

The function of the epithelial Na+ channel from the apical membrane of many Na+ transporting epithelia is modulated by various chemical compounds from the extracellular space, such as heavy metals, protons or chloride ions. We have studied the effect of extracellular Cd2+ on the function of the epithelial Na+ channel (ENaC) in heterologously expressed Xenopus laevis oocytes and Na+-transporting epithelia. We assayed channel function as the amiloride-sensitive sodium current (I(Na)). Cd2+ rapidly and voltage-independently inhibited INa in oocytes expressing αβγ Xenopus ENaC (xENaC). The extracellular Cd2+ inhibited Na+ transport and showed no influence on ENaC trafficking, as revealed by concomitant measurements of the transepithelial current, conductance and capacitance in Na+-transporting epithelia. Instead, amiloride inhibition was noticeably diminished in the presence of Cd2+ on the apical membrane. Using molecular modeling approaches, we describe the amiloride binding sites in rat and xENaC structures, and we present four putative binding sites for Cd2+. These results indicate that ENaC functions as a sensor for external Cd2+.

Epithelial Sodium Channel Inhibition by Amiloride Addressed with THz Spectroscopy and Molecular Modeling

“…In a previous study [ 29 ], we modeled xENaC by using as a template the structure of cASIC1 in complex with psalmotoxin 1 at low pH, namely the 4FZ0 structure [ 30 ]. In the present study, the model of xENaC was updated using the most recent template structures deposited in PDB [ 31 ].…”

“…As in the case of cASIC1, this appears as an intermediate site. The amiloride binding site described by mutagenesis studies is located deeper into the pore, involving residues αS514, βG547 and γG539 (by homology with the residues in rat ENaC [ 29 , 33 ]). At the end of the MD simulation, in the structure validated by THz spectroscopy, amiloride interacts with several residues located as deep into the pore as the selectivity filter.…”

“…Initially, amiloride is inserted between helices TM1 and TM2a from β subunit and helix TM2a from γ subunit, close to the corresponding fenestration (state 1). Then it assumes a more central position at the same level within the pore (state 2), where it interacts with αS514, one of the residues experimentally identified as being important for amiloride binding [ 29 , 33 ]. In the following states (states 3 and 4), amiloride descends into the pore, accessing more profound rings of residues.…”

“…In the following states (states 3 and 4), amiloride descends into the pore, accessing more profound rings of residues. It loses the interaction with αS514, but starts interacting with βG547 and γG539, two other residues important for amiloride binding [ 29 , 33 ]. In state 4, the amiloride additionally interacts with residues βG551, βG552 and βS553 that represent the selectivity filter in β subunit and with γG544, a residue from the selectivity filter in γ subunit.…”