SUMMARY
The TRPM8 ion channel is expressed in sensory neurons and is responsible for sensing environmental cues such as cold temperatures and chemical compounds, including menthol and icilin. The channel functional activity is regulated by various physical and chemical factors, and is likely to be pre-conditioned by its molecular composition. Our studies indicate that TRPM8 channel forms a structural-functional complex with the polyester, poly-(R)-3hydroxybutyrate (PHB). We identified by mass spectrometry a number of PHB-modified peptides in the N-terminus of the TRPM8 protein and in its extracellular S3–S4 linker. Removal of PHB by enzymatic hydrolysis, and site-directed mutagenesis of both the serine residues that serve as covalent anchors for PHB and adjacent hydrophobic residues that interact with the methyl groups of the polymer, resulted in significant inhibition of TRPM8 channel activity. We conclude that the TRPM8 channel undergoes post-translational modification by PHB and that this modification is required for its normal function.
The epithelial Na(+) channel (ENaC) plays a key role in the regulation of blood pressure and airway surface liquid volume. ERp29 is a 29-kDa thioredoxin-homologous endoplasmic reticulum (ER) protein that has only a single cysteine instead of the usual thioredoxin CXXC motif. Our group previously demonstrated that ERp29 promotes biogenesis of the cystic fibrosis transmembrane conductance regulator (CFTR). On the basis of similarities of CFTR and ENaC trafficking, we hypothesized that ERp29 would also regulate ENaC biogenesis and functional expression. In epithelial cells, overexpression of wild-type (wt) ERp29 increased ENaC functional expression [amiloride-sensitive short-circuit current (Isc)] in Ussing chamber experiments, as well as the abundance of the cleaved form of γ-ENaC in whole cell lysates. In contrast, siRNA-mediated depletion of ERp29 or overexpression of a mutant ERp29 lacking its single cysteine (C157S ERp29) decreased ENaC functional expression. Cells in which wt ERp29 was overexpressed had a smaller fractional increase in amiloride-sensitive Isc when trypsin was applied to the apical surface to activate uncleaved ENaC, while cells in which C157S ERp29 was overexpressed or ERp29 was depleted had a significantly greater fractional increase in amiloride-sensitive Isc in response to trypsin. Interestingly, these observations were not associated with altered expression of β-ENaC at the apical surface. Instead, ERp29 appeared to promote the interaction of β-ENaC with the Sec24D cargo recognition component of the coat complex II ER exit machinery. Together, these data support the hypothesis that ERp29 directs ENaC toward the Golgi, where it undergoes cleavage during its biogenesis and trafficking to the apical membrane.
ERp29 is an endoplasmic reticulum (ER) 29 kD thioredoxin‐homologous protein that displays chaperone‐like properties. We have previously demonstrated that ERp29 promotes CFTR biogenesis. As the biogenesis of CFTR and ENaC share similar features, we here tested the hypothesis that ERp29 would also regulate ENaC biogenesis and functional expression, as well as further probed ERp29 mechanism. Overexpression of wt ERp29 increased the abundance of the active form of γ‐ENaC, as well as ENaC functional expression. In contrast, ERp29 overexpression of a mutant ERp29 lacking its single Cysteine (C157S ERp29) decreased ENaC functional expression. These observations were not associated with altered expression of β‐ENaC at the apical surface, suggesting that ERp29 may modulate ENaC open probability at the apical surface. ERp29 overexpression promoted the interaction of both ENaC and CFTR with the coat complex II ER exit machinery, whereas C157S ERp29 overexpression decreased this interaction. These data suggest a model where ERp29 may promote ENaC cleavage by directing ENaC to the Golgi. Finally, we also tested the hypothesis that ERp29 subcellular localization is a crucial determinant for its action. We therefore designed a mutant ERp29 containing a KDEL retention motif (ERp29 KDEL) that should be better retained in the ER, and two mutants that would better escape the ER, by mutation and deletion (ERp29 KDEV and ΔKEEL, respectively). Together our findings suggest a key role for ERp29 in the biogenesis of ENaC and CFTR as well as emphasize ERp29 mechanism.
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