Mutations in the Pore Region Modify Epithelial Sodium Channel Gating by Shear Stress

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Activity of the epithelial Na؉ channel (ENaC) is modulated by Na ؉ self-inhibition, an allosteric down-regulation of channel open probability by extracellular Na ؉ . We searched for determinants of Na ؉ self-inhibition by analyzing changes in this inhibitory response resulting from specific mutations within the extracellular domains of mouse ENaC subunits. Mutations at ␥Met 438 altered the Na ؉ self-inhibition response in a substitution-specific manner. Fourteen substitutions (Ala, Arg, Asp, Cys, Gln, Glu, His, Ile, Phe, Pro, Ser, Thr, Tyr, and Val) significantly suppressed Na ؉ self-inhibition, whereas three mutations (Asn, Gly, and Leu) moderately enhanced the inhibition. Met to Lys mutation did not alter Na ؉ self-inhibition. Mutations at the homologous site in the ␣ subunit (G481A, G481C, and G481M) dramatically increased the magnitude and speed of Na ؉ self-inhibition. Mutations at the homologous ␤Ala 422 resulted in minimal or no change in Na ؉ self-inhibition. Low, high, and intermediate open probabilities were observed in oocytes expressing ␣G481M␤␥, ␣␤␥M438V, and ␣G481M/ ␤␥M438V, respectively. This pair of residues map to the ␣5 helix in the extracellular thumb domain in the chicken acid sensing ion channel 1 structure. Both residues likely reside near the channel surface because both ␣G481C␤␥ and ␣␤␥M438C channels were inhibited by an externally applied and membrane-impermeant sulfhydryl reagent. Our results demonstrate that ␣Gly 481 and ␥Met 438 are functional determinants of Na ؉ self-inhibition and of ENaC gating and suggest that the thumb domain contributes to the channel gating machinery.Maintenance of body fluid volume homeostasis requires a collaborative interaction of many Na ϩ transport mechanisms. Na ϩ transport in epithelia that line the late distal convoluted tubule, connecting tubule, and collecting tubule relies on apical Na ϩ entry through epithelial Na ϩ channels (ENaC self-inhibition (4, 6 -8). However, detailed elements regarding its mechanism have not been revealed.A logical place to search for structural elements associated with Na ϩ self-inhibition is the large extracellular domain (ECD) that connects the two transmembrane domains (M1 and M2) within each ENaC subunit. The ECD likely exists as well structured subdomains with 16 conserved Cys residues. We recently reported that point mutations at multiple ␣ and ␥ ECD Cys residues blunted Na ϩ self-inhibition, and certain double or triple mutations rendered ENaC insensitive to high concentration of extracellular Na ϩ . These results suggest that multiple Cys residues are required to establish the proper tertiary structure permitting this allosteric regulation (9). In addition, the N-terminal portion of ECD contains ␥His 239 , a previously identified residue critical for Na ϩ self-inhibition, as well as defined protease cleavage sites (4, 10 -12). Various proteases have been shown to regulate ENaC activity, in part, by interfering with Na ϩ self-inhibition (6, 7, 13). The resolved high resolution structure of the chicken acidsensing i...

Section: Substitutions At ␣Glysupporting

confidence: 73%

Novel Determinants of Epithelial Sodium Channel Gating within Extracellular Thumb Domains

Maarouf

Sheng

Chen

et al. 2009

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“…Previous studies from our group have suggested that TM2 and adjacent residues have important roles in the response of ENaC to LSS as both the magnitude and kinetics of the LSS response were altered by substitutions introduced within this region (43,44). Interestingly, the magnitude of the LSS response of the MEC-4d/MEC-10 channel was not altered by replacing the TM2 and C terminus of MEC-10 with the corresponding region of MEC-4 (MEC-10-4TM2C chimera) (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…For example, ENaC is activated by shear stress in native kidney tubules, endothelial cells, and heterogeneous expression systems (26,41,42). ENaC gating in response to LSS was altered by mutations introduced at key sites within the large ECD and transmembrane helices (43)(44)(45)(46). We have proposed a model where LSS-induced motions within the extracellular region of ENaC lead to movement of the transmembrane helices where the channel gate resides, transitioning the channel to a higher P o state (22).…”

Section: Discussionmentioning

confidence: 99%

Activation of the Caenorhabditis elegans Degenerin Channel by Shear Stress Requires the MEC-10 Subunit

Shi

Luke

Miedel

et al. 2016

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Mechanotransduction in Caenorhabditis elegans touch receptor neurons is mediated by an ion channel formed by MEC-4, MEC-10, and accessory proteins. To define the role of these subunits in the channel's response to mechanical force, we expressed degenerin channels comprising MEC-4 and MEC-10 in Xenopus oocytes and examined their response to laminar shear stress (LSS). Shear stress evoked a rapid increase in whole cell currents in oocytes expressing degenerin channels as well as channels with a MEC-4 degenerin mutation (MEC-4d), suggesting that C. elegans degenerin channels are sensitive to LSS. MEC-10 is required for a robust LSS response as the response was largely blunted in oocytes expressing homomeric MEC-4 or MEC-4d channels. We examined a series of MEC-10/MEC-4 chimeras to identify specific domains (amino terminus, first transmembrane domain, and extracellular domain) and sites (residues 130 -132 and 134 -137) within MEC-10 that are required for a robust response to shear stress. In addition, the LSS response was largely abolished by MEC-10 mutations encoded by a touch-insensitive mec-10 allele, providing a correlation between the channel's responses to two different mechanical forces. Our findings suggest that MEC-10 has an important role in the channel's response to mechanical forces.

“…Cell-attached Patch Clamp Measurements-1.5-4 h after microinjection of ENaC-expressing oocytes with either K-gluconate or K-ZMP and after removal of their vitelline membranes, single channel patch-clamp recordings in the cell-attached mode were performed using identical bath and pipette solutions with LiCl as the predominant salt, as previously described (33). Cell-attached recordings were generally performed at ϩ60 mV (pipette potential) except when gathering data for I-V plots to determine single-channel conductance.…”

Section: Methodsmentioning

confidence: 99%

Epithelial Sodium Channel Inhibition by AMP-activated Protein Kinase in Oocytes and Polarized Renal Epithelial Cells

Carattino

Edinger

Grieser

et al. 2005

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135

165

The epithelial Na ؉ channel (ENaC) regulates epithelial salt and water reabsorption, processes that require significant expenditure of cellular energy. To test whether the ubiquitous metabolic sensor AMP-activated kinase (AMPK) regulates ENaC, we examined the effects of AMPK activation on amiloride-sensitive currents in Xenopus oocytes and polarized mouse collecting duct mpkCCD c14 cells. Microinjection of oocytes expressing mouse ENaC (mENaC) with either active AMPK protein or an AMPK activator inhibited mENaC currents relative to controls as measured by two-electrode voltageclamp studies. Similarly, pharmacological AMPK activation or overexpression of an activating AMPK mutant in mpkCCD c14 cells inhibited amiloride-sensitive short circuit currents. Expression of a degenerin mutant ␤-mENaC subunit (S518K) along with wild type ␣ and ␥ increased the channel open probability (P o ) to ϳ1. However, AMPK activation inhibited currents similarly with expression of either degenerin mutant or wild type mENaC. Single channel recordings under these conditions demonstrated that neither P o nor channel conductance was affected by AMPK activation. Moreover, expression of a Liddle's syndrome-type ␤-mENaC mutant (Y618A) greatly enhanced ENaC whole cell currents relative to wild type ENaC controls and prevented AMPKdependentinhibition.ThesefindingsindicatethatAMPKdependent ENaC inhibition is mediated through a decrease in the number of active channels at the plasma membrane (N), presumably through enhanced Nedd4-2-dependent ENaC endocytosis. The AMPK-ENaC interaction appears to be indirect; AMPK did not bind ENaC in cells, as assessed by in vivo pull-down assays, nor did it phosphorylate ENaC in vitro. In summary, these results suggest a novel mechanism for coupling ENaC activity and renal Na ؉ handling to cellular metabolic status through AMPK, which may help prevent cellular Na ؉ loading under hypoxic or ischemic conditions.The maintenance of transmembrane ion and solute gradients is a crucial process that permits normal cellular functioning, viability, and coordinated transepithelial transport but also consumes a substantial portion of total cellular energy (1). Under conditions of metabolic stress, the expression and activity of many membrane transport proteins including ENaC 1 are inhibited, thereby limiting the dissipation of ionic gradients and preserving the cellular energy required to maintain them (2). However, the cellular mechanisms that link membrane transport to energy production and metabolic status have remained elusive.AMPK is a ubiquitous metabolic-sensing Ser/Thr kinase that exists as a heterotrimer composed of a catalytic ␣ subunit and regulatory ␤ and ␥ subunits. Its activity increases during conditions of metabolic stress, in response to elevated intracellular AMP:ATP ratios (3). A parallel activation pathway involves phosphorylation of the ␣ subunit by a recently identified upstream LKB1 kinase complex at a Thr residue in its activation loop (4). The earliest discovered substrates of AMPK were important ...