Influence of proteolytic cleavage of ENaC's γ subunit upon Na+ and K+ handling.

The epithelial Na+ channel (ENaC) γ subunit is essential for homeostasis of Na+, K+, and body fluid. Dual γ subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (PO), in vitro. Cleavage proximal to the tract occurs at a furin recognition sequence (143RKRR146, in the mouse γ subunit). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143RKRR146 mutation to 143QQQQ146 (γQ4) in 129/Sv mice would reduce ENaC PO, impair flow-stimulated flux of Na+ (JNa) and K+ (JK) in perfused collecting ducts, reduce colonic amiloride-sensitive short-circuit current (ISC), and impair Na+, K+, and body fluid homeostasis. Immunoblot of γQ4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, γQ4/Q4 male mice on a low Na+ diet did not exhibit altered ENaC PO or flow-induced JNa, though flow-induced JK modestly decreased. Colonic amiloride-sensitive ISC in γQ4/Q4 mice was not altered. γQ4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na+ diet. Blood Na+ and K+ were unchanged on a regular, low Na+, or high K+ diet. These findings suggest that biochemical evidence of γ subunit cleavage should not be used in isolation to evaluate ENaC activity. Furthermore, factors independent of γ subunit cleavage modulate channel PO and the influence of ENaC on Na+, K+, and fluid volume homeostasis in 129/Sv mice, in vivo.NEW & NOTEWORTHY The epithelial Na+ channel (ENaC) is activated in vitro by post-translational proteolysis. In vivo, low Na+ or high K+ diets enhance ENaC proteolysis, and proteolysis is hypothesized to contribute to channel activation in these settings. Using a mouse expressing ENaC with disruption of a key proteolytic cleavage site, this study demonstrates that impaired proteolytic activation of ENaC's γ subunit has little impact upon channel open probability or the ability of mice to adapt to low Na+ or high K+ diets.

Lhs1 dependent ERAD is determined by transmembrane domain context.

Transmembrane proteins have unique requirements to fold and integrate into the endoplasmic reticulum (ER) membrane. Most notably, transmembrane proteins must fold in three separate environments: extracellular domains fold in the oxidizing environment of the ER lumen, transmembrane domains (TMDs) fold within the lipid bilayer, and cytosolic domains fold in the reducing environment of the cytosol. Moreover, each region is acted upon by a unique set of chaperones and monitored by components of the ER associated quality control machinery that identify misfolded domains in each compartment. One factor is the ER lumenal Hsp70-like chaperone, Lhs1. Our previous work established that Lhs1 is required for the degradation of the unassembled α-subunit of the epithelial sodium channel (αENaC), but not the homologous ß- and γENaC subunits. However, assembly of the ENaC heterotrimer blocked the Lhs1-dependent ER associated degradation (ERAD) of the α-subunit, yet the characteristics that dictate the specificity of Lhs1-dependent ERAD substrates remained unclear. We now report that Lhs1-dependent substrates share a unique set of features. First, all Lhs1 substrates appear to be unglycosylated, and second they contain two TMDs. Each substrate also contains orphaned or unassembled TMDs. Additionally, interfering with inter-subunit assembly of the ENaC trimer results in Lhs1-dependent degradation of the entire complex. Finally, our work suggests that Lhs1 is required for a subset of ERAD substrates that also require the Hrd1 ubiquitin ligase. Together, these data provide hints as to the identities of as-yet unconfirmed substrates of Lhs1 and potentially of the Lhs1 homolog in mammals, GRP170.

Salt sensitivity of volume and blood pressure in a mouse with globally reduced ENaC γ-subunit expression.

The epithelial Na+ channel (ENaC) promotes the absorption of Na+ in the aldosterone-sensitive distal nephron, colon, and respiratory epithelia. Deletion of genes encoding subunits of ENaC results in early postnatal mortality. Here, we present the initial characterization of a mouse with dramatically suppressed expression of the ENaC γ-subunit. We used this hypomorphic (γmt) allele to explore the importance of this subunit in homeostasis of electrolytes and body fluid volume. At baseline, γ-subunit expression in γmt/mt mice was markedly suppressed in the kidney and lung, whereas electrolytes resembled those of littermate controls. Aldosterone levels in γmt/mt mice exceeded those seen in littermate controls. Quantitative magnetic resonance measurement of body composition revealed similar baseline body water, lean tissue mass, and fat tissue mass in γmt/mt mice and controls. γmt/mt mice exhibited a more rapid decline in body water and lean tissue mass in response to a low-Na+ diet than the controls. Replacement of drinking water with 2% saline selectively and transiently increased body water and lean tissue mass in γmt/mt mice relative to the controls. Lower blood pressures were variably observed in γmt/mt mice on a high-salt diet compared with the controls. γmt/mt also exhibited reduced diurnal blood pressure variation, a "nondipping" phenotype, on a high-Na+ diet. Although ENaC in the renal tubules and colon works to prevent extracellular fluid volume depletion, our observations suggest that ENaC in other tissues may participate in regulating extracellular fluid volume and blood pressure.NEW & NOTEWORTHY A mouse with globally suppressed expression of the epithelial Na+ channel γ-subunit showed enhanced sensitivity to dietary salt, including a transient increase in total body fluid, reduced blood pressure, and reduced diurnal blood pressure variation when given a dietary NaCl challenge. These results point to a role for the epithelial Na+ channel in regulating body fluid and blood pressure beyond classical transepithelial Na+ transport mechanisms.

Interactions between intersubunit transmembrane domains regulate the chaperone-dependent degradation of an oligomeric membrane protein.

In the kidney, the epithelial sodium channel (ENaC) regulates blood pressure through control of sodium and volume homeostasis, and in the lung, ENaC regulates the volume of airway and alveolar fluids. ENaC is a heterotrimer of homologous α-, ß- and γ-subunits, and assembles in the endoplasmic reticulum (ER) before it traffics to and functions at the plasma membrane. Improperly folded or orphaned ENaC subunits are subject to ER quality control and targeted for ER-associated degradation (ERAD). We previously established that a conserved, ER lumenal, molecular chaperone, Lhs1/GRP170, selects αENaC, but not ß- or γ-ENaC, for degradation when the ENaC subunits were individually expressed. We now find that when all three subunits are co-expressed, Lhs1-facilitated ERAD was blocked. To determine which domain-domain interactions between the ENaC subunits are critical for chaperone-dependent quality control, we employed a yeast model and expressed chimeric α/ßENaC constructs in the context of the ENaC heterotrimer. We discovered that the ßENaC transmembrane domain was sufficient to prevent the Lhs1-dependent degradation of the α-subunit in the context of the ENaC heterotrimer. Our work also found that Lhs1 delivers αENaC for proteasome-mediated degradation after the protein has become polyubiquitinated. These data indicate that the Lhs1 chaperone selectively recognizes an immature form of αENaC, one which has failed to correctly assemble with the other channel subunits via its transmembrane domain.

Proteolytic Cleavage of the ENaC γ Subunit - Impact Upon Na + and K + Handling.

The ENaC gamma subunit is essential for homeostasis of Na + , K + , and body fluid. Dual subunit cleavage before and after a short inhibitory tract allows dissociation of this tract, increasing channel open probability (P O ), in vitro . Cleavage proximal to the tract occurs at a furin recognition sequence ( 143 RKRR 146 in mouse). Loss of furin-mediated cleavage prevents in vitro activation of the channel by proteolysis at distal sites. We hypothesized that 143 RKRR 146 mutation to 143 QQQQ 146 ( Q4 ) in 129/Sv mice would reduce ENaC P O , impair flow-stimulated flux of Na + (J Na ) and K + (J K ) in perfused collecting ducts, reduce colonic amiloride-sensitive short circuit current (I SC ), and impair Na + , K + , and body fluid homeostasis. Immunoblot of Q4/Q4 mouse kidney lysates confirmed loss of a band consistent in size with the furin-cleaved proteolytic fragment. However, Q4/Q4 male mice on a low Na + diet did not exhibit altered ENaC P O or flow-induced J Na , though flow-induced J K modestly decreased. Colonic amiloride-sensitive I SC in Q4/Q4 mice was not altered. Q4/Q4 males, but not females, exhibited mildly impaired fluid volume conservation when challenged with a low Na + diet. Blood Na + and K + were unchanged on a regular, low Na + , or high K + diet. These findings suggest that biochemical evidence of gamma subunit cleavage should not be used in isolation to evaluate ENaC activity. Further, factors independent of gamma subunit cleavage modulate channel P O and the influence of ENaC on Na + , K + , and fluid volume homeostasis in 129/Sv mice, in vivo .