Peptide block of constitutively activated Na+ channels in Liddle's disease

Abstract

Hypertension is a multifactorial disorder that results in an increased risk of cardiovascular and end-stage renal disease. Liddle's disease represents a specific hypertensive disease and expresses itself in the human population as an autosomal dominant trait. Recent experimental evidence indicates that patients with Liddle's disease have constitutively active amiloride-sensitive Na+ channels and that these channels are phenotypically expressed in lymphocytes obtained from normal and affected members of the original Liddle's kindred. Linkage analysis indicates that this disease results from a deletion of the carboxy-terminal region of the beta-subunit of a recently cloned epithelial Na+ channel (ENaC). We report the successful immunopurification and reconstitution of both normal and constitutively active lymphocyte Na+ channels into planar lipid bilayers. These channels display all of the characteristics typical of renal Na+ channels, including sensitivity to protein kinase A phosphorylation. We demonstrate that gating of normal Na+ channels is removed by cytoplasmic trypsin digestion and that the constitutively active Liddle's Na+ channels are blocked by a beta- or gamma-ENaC carboxy-terminal peptide in a GTP-dependent fashion.

Peptide Inhibition of Constitutively Activated Epithelial Na+ Channels Expressed in Xenopus Oocytes

“…Physical addition of the missing regions for both subunits of the truncation Liddle's mutations could achieve successful correction of currents in vitro, both at the macroscopic and single channel levels. Combined with similar observations obtained from patch clamping on human B lymphocytes (41), dual-electrode voltage clamping on oocytes expressing ENaC (26), and planar lipid bilayer incorporation experiments (32,33), our results provide an experimental basis for understanding inherent ENaC function. Our results also support the idea that different Liddle's mutations, e.g.…”

“…This finding was confirmed in planar lipid bilayers reconstituted with the Na ϩ channel complex immunopurified from lymphocytes derived from patients affected with Liddle's disease (32). Unresolved questions concern the molecular mechanism underlying peptide inhibition of ENaC and whether peptides comprising the missing segments of the intracellular C-terminal tails of the ␤ or ␥ subunit can inhibit basal-activated Na ϩ currents caused by both single and double truncation mutations (␣␤ T ␥-rENaC, ␣␤␥ T -rENaC, and ␣␤ T ␥ T - rENaC) and missense mutations (␣␤ Y ␥-and ␣␤␥ Y -rENaC) identified in Liddle's syndrome cases.…”

“…More likely, however, is that the decrease in the active channel number per patch seen subsequent to peptide addition is due to a decrease in the fraction of the electrically detected rENaC per unit of cell surface, simply by a reduction in P o . Because the C-terminal domains of the ␤-and ␥-ENaC subunits contain phosphorylation sites for protein kinase A and protein kinase C (8), in vivo the C-terminal regions may interact with some target for protein kinase A-or protein kinase C-mediated phosphorylation on rENaC (subunits or associated cytoskeletal elements) and therefore regulate the activity of rENaC by altering its phosphorylation state (32,46). The enhanced sensitivity to the activating effects (via protein kinase A) of normal endogenous cAMP levels in affected lymphocytes of Liddle's disease (41) is consistent with this possibility.…”

“…The differences between the regulation of wt ENaC and Liddle's mutants have not been explored in detail. However, like other cation channels (29 -31), the activity of truncated Liddle's ␤-ENaC mutants immunopurified from human lymphocytes were also inhibited by synthesized peptides when reconstituted into planar lipid bilayers (32).…”

“…We had previously demonstrated in planar lipid bilayer studies that truncation of the C termini of ␤-or ␥-rENaC led to increased channel P o (32,33). We wished to test further the hypothesis that the C-terminal peptides of these subunits could function as inhibitory peptides of rENaC expressed in Xenopus oocytes.…”