Mechanosensitivity of an epithelial Na+ channel in planar lipid bilayers: release from Ca2+ block.

A family of novel epithelial Na+ channels (ENaCs) have recently been cloned from several different tissues. Three homologous subunits (alpha, beta, gamma-ENaCs) from the core conductive unit of Na(+)-selective, amiloride-sensitive channels that are found in epithelia. We here report the results of a study assessing the regulation of alpha,beta,gamma-rENaC by Ca2+ in planar lipid bilayers. Buffering of the bilayer bathing solutions to [Ca2+] < 1 nM increased single-channel open probability by fivefold. Further investigation of this phenomenon revealed that Ca2+ ions produced a voltage-dependent block, affecting open probability but not the unitary conductance of ENaC. Imposing a hydrostatic pressure gradient across bilayers containing alpha,beta,gamma-rENaC markedly reduced the sensitivity of these channels to inhibition by [Ca2+]. Conversely, in the nominal absence of Ca2+, the channels lost their sensitivity to mechanical stimulation. These results suggest that the previously observed mechanical activation of ENaCs reflects a release of the channels from block by Ca2+

Gating of amiloride-sensitive Na(+) channels: subunit-subunit interactions and inhibition by the cystic fibrosis transmembrane conductance regulator.

In search of the structural basis for gating of amiloride-sensitive Na(+) channels, kinetic properties of single homo and heterooligomeric ENaCs formed by the subunits with individual truncated cytoplasmic domains were studied in a cell-free planar lipid bilayer reconstitution system. Our results identify the N-terminus of the alpha-subunit as a major determinant of kinetic behavior of both homooligomeric and heterooligomeric ENaCs, although the carboxy-terminal domains of beta- and gamma-ENaC subunits play important role(s) in modulation of the kinetics of heterooligomeric channels. We also found that the cystic fibrosis transmembrane conductance regulator (CFTR) inhibits amiloride-sensitive channels, at least in part, by modulating their gating. Comparison of these data suggests that the modulatory effects of the beta- and gamma-ENaC subunits, and of the CFTR, may involve the same, or closely related, mechanism(s); namely, "locking" the heterooligomeric channels in their closed state. These mechanisms, however, do not completely override the gating mechanism of the alpha-channel

Point mutations in alpha bENaC regulate channel gating, ion selectivity, and sensitivity to amiloride.

We have generated two site-directed mutants, K504E and K515E, in the alpha subunit of an amiloride-sensitive bovine epithelial Na+ channel, alpha bENaC. The region in which these mutations lie is in the large extracellular loop immediately before the second membrane-spanning domain (M2) of the protein. We have found that when membrane vesicles prepared from Xenopus oocytes expressing either K504E or K515E alpha bENaC are incorporated into planar lipid bilayers, the gating pattern, cation selectivity, and amiloride sensitivity of the resultant channel are all altered as compared to the wild-type protein. The mutated channels exhibit either a reduction or a complete lack of its characteristic burst-type behavior, significantly reduced Na+:K+ selectivity, and an approximately 10-fold decrease in the apparent inhibitory equilibrium dissociation constant (Ki) for amiloride. Single-channel conductance for Na+ was not affected by either mutation. On the other hand, both K504E and K515E alpha bENaC mutants were significantly more permeable to K+, as compared to wild type. These observations identify a lysine-rich region between amino acid residues 495 and 516 of alpha bENaC as being important to the regulation of fundamental channel properties

A biologic function for an "orphan" messenger: D-myo-inositol 3,4,5,6-tetrakisphosphate selectively blocks epithelial calcium-activated chloride channels.

Inositol phosphates are a family of water-soluble intracellular signaling molecules derived from membrane inositol phospholipids. They undergo a variety of complex interconversion pathways, and their levels are dynamically regulated within the cytosol in response to a variety of agonists. Relatively little is known about the biological function of most members of this family, with the exception of inositol 1,4,5-trisphosphate. Specifically, the biological functions of inositol tetrakisphosphates are largely obscure. In this paper, we report that D-myo-inositol 3,4,5,6-tetrakisphosphate (D-Ins(3,4,5,6)P4) has a direct biphasic (activation/inhibition) effect on an epithelial Ca(2+)-activated chloride channel. The effect of D-Ins(3,4,5,6)P4 is not mimicked by other inositol tetrakisphosphate isomers, is dependent on the prevailing calcium concentration, and is influenced when channels are phosphorylated by calmodulin kinase II. The predominant effect of D-Ins(3,4,5,6)P4 on phosphorylated channels is inhibitory at levels of intracellular calcium observed in stimulated cells. Our findings indicate the biological function of a molecule hitherto considered as an "orphan" messenger. They suggest that the molecular target for D-Ins(3,4,5,6)P4 is a plasma membrane Ca(2+)-activated chloride channel. Regulation of this channel by D-Ins(3,4,5,6)P4 and Ca2+ may have therapeutic implications for the disease states of both diabetic nephropathy and cystic fibrosis

Sodium selectivity of semicircular canal duct epithelial cells

<p>Abstract</p> <p>Background</p> <p>Sodium absorption by semicircular canal duct (SCCD) epithelial cells is thought to contribute to the homeostasis of the volume of vestibular endolymph. It was previously shown that the epithelial cells could absorb Na⁺under control of a glucocorticoid hormone (dexamethasone) and the absorptive transepithelial current was blocked by amiloride. The most commonly-observed target of amiloride is the epithelial sodium channel (ENaC), comprised of the three subunits α-, β- and γ-ENaC. However, other cation channels have also been observed to be sensitive in a similar concentration range. The aim of this study was to determine whether SCCD epithelial cells absorb only Na⁺or also K⁺through an amiloride-sensitive pathway. Parasensory K⁺absorption could contribute to regulation of the transduction current through hair cells, as found to occur via vestibular transitional cells [S. H. Kim and D. C. Marcus. Regulation of sodium transport in the inner ear. <it>Hear.Res</it>. doi:10.1016/j.heares.2011.05.003, 2011].</p> <p>Results</p> <p>We determined the molecular and functional expression of candidate cation channels with gene array (GEO GSE6197), whole-cell patch clamp and transepithelial recordings in primary cultures of rat SCCD. α-, β- and γ-ENaC were all previously reported as present. The selectivity of the amiloride-sensitive transepithelial and cell membrane currents was observed in Ussing chamber and whole-cell patch clamp recordings. The cell membrane currents were carried by Na⁺but not K⁺, but the Na⁺selectivity disappeared when the cells were cultured on impermeable supports. Transepithelial currents across SCCD were also carried exclusively by Na⁺.</p> <p>Conclusions</p> <p>These results are consistent with the amiloride-sensitive absorptive flux of SCCD mediated by a highly Na⁺-selective channel, likely αβγ-ENaC. These epithelial cells therefore absorb only Na⁺via the amiloride-sensitive pathway and do not provide a parasensory K⁺efflux from the canals via this pathway. The results further provide caution to the culture of epithelial cells on impermeable surfaces.</p