Brown AL , Liao Z , Goodman MB .

NS047715 NINDS NIH HHS , R01 NS047715-04 NINDS NIH HHS , R01 NS047715 NINDS NIH HHS

	Figure 1. Single-channel recordings from outside-out patches. (A) MEC-4d/10d only. (B) MEC-4d/10d + MEC-2. (C) MEC-4d/10d + MEC-6. (D) MEC-4d/10d + MEC-2 + MEC-6. In A–D, two sections are shown: the left demonstrates the sensitivity of the channel to amiloride (50 μM), while the right shows the channel behavior in control saline. Vhold = −150 mV. Closed and open states are labeled C and O respectively and indicated by solid lines. To the right of each record is an all-points histogram of the same trace; dotted lines represent Gaussian fits to each peak. Open probability (E) and single-channel conductance (F) of patches containing a single channel. Data are summarized in Table I. Closed triangles represent the example patches shown in A–D. Bars indicate mean values for each isoform. Number of patches indicated in parentheses. *, significantly different from MEC-4d/10d (P < 0.01).
	Figure 2. Example dwell-time histograms of single channel recordings. (A) Dwell-time histogram of the closed states of a single MEC-4d/10d + MEC-2 + MEC-6 channel. (B) Dwell-time histogram of the open states of the same channel as in A. For optimal visual estimation of fit quality (Sigworth and Sine, 1987), dwell times are accumulated into logarithmic bins and displayed with a square-root ordinate. Solid lines are fits to the data with the sum of three exponential functions, dotted lines represent each exponential component.
	Figure 3. Block by divalent cations. (A) Macroscopic I-V curve of an outside-out patch coexpressing MEC-4d/10d, MEC-2, and MEC-6 exposed to varying Ca2+ concentrations. (B) Dose–response curves for blockade by Ca2+ and Mg2+ at −100 mV. The smooth curve is fit to the average values assuming a Hill coefficient of 1: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}I=I_{{\mathrm{max}}}(1+[X^{2+}]/K_{i}')^{-1}\end{equation*}\end{document}. Measured Ki' was 12.9 mM for Ca2+, 7.0 mM for Mg2+. (C) Apparent Ca2+ inhibition constant, Ki' as a function of auxiliary subunits. Vhold = −60 mV. (D) Voltage dependence and variation of block affinity. Each line represents a single patch, with Ki' measured at −60 and −100 mV.
	Figure 4. Activation by extracellular chymotrypsin. (A) Example time course of chymotrypsin-evoked current increase in an oocyte expressing MEC-4d/10d + MEC-2 + MEC-6. a and b indicate when the curves shown in B were collected. Current amplitude was measured every 4 s at −85 mV. (B) Whole-cell I-V relationship before (a) and during chymotrypsin application (b). In all cases, chymotypsin-evoked current was blocked by amiloride (300 μM). (C) Chymotrypsin-evoked increase in the amiloride-sensitive current in the presence and absence of MEC-2 and MEC-6.
	Figure 5. Surface expression and current amplitude as a function of MEC-2 cRNA. (A) Surface expression of myc∷MEC-4d in oocytes coexpressing myc∷MEC-4d, MEC-10d, MEC-2, and MEC-6 (top). The amount of MEC-2 cRNA is indicated below each lane; U indicates uninjected (control) oocytes. Relative surface expression as a function MEC-2 cRNA (bottom), normalized to density with 1 ng MEC-2 cRNA per oocyte. Data represent the results of three independent Western blots. Nonspecific bands are observed in all blots and are visible in uninjected cells. (B) Amiloride-sensitive currents recorded from oocytes analyzed in parallel with those used for Western blotting in A. Stars indicate significance P < 0.01 (Student's t test). Smooth line is fit to the data according to: I = I0 + (Imax − I0)(1 + [MEC-2 cRNA]/EC50)−1. The EC50 is 2.8 ng, IO = 0.48 μA.
	Figure 6. Characterization of cholesterol-binding mutants of MEC-2. (A) Alignment of selected PHB family members. MEC-2 and UNC-24 are coexpressed in vivo. MEC-2 is also represented schematically on the right. In yellow, P134, required for cholesterol binding. In green, C140 and C174 required for palmitoylation (Huber et al., 2006). (B) I-V relationship of channels lacking MEC-2, containing MEC-2 double mutant, and MEC-2 triple mutant. MEC-2(P134S) single mutant currents are essentially indistinguishable from the triple mutant and are omitted for clarity. (C) Average currents recorded from MEC-2 mutant-expressing cells (top). Bars are mean ± SEM. Sample size is indicated in parentheses below each bar. †, P < 0.005, compared with the absence of MEC-2; *, P < 10−6 compared with wild-type MEC-2. Western blot of MEC-2 isoforms (bottom). For clarity, lanes corresponding to each isoform are aligned with the data in A. All lanes were from the same blot (with identical contrast manipulation). Similar results were obtained in a total of three independent experiments. (D) Single-channel activity from an outside-out patch of a cell expressing triple mutant MEC-2. These data were used to measure single-channel conductance. Solid lines indicate zero, one or both channels open; all-points histogram is shown on the right.

Anishkin, Microbial mechanosensation. 2005, Pubmed

Anishkin, Microbial mechanosensation. 2005, Pubmed
Bengrine, Indirect activation of the epithelial Na+ channel by trypsin. 2007, Pubmed , Xenbase
Bianchi, The neurotoxic MEC-4(d) DEG/ENaC sodium channel conducts calcium: implications for necrosis initiation. 2004, Pubmed , Xenbase
Brown, Gain-of-function mutations in the MEC-4 DEG/ENaC sensory mechanotransduction channel alter gating and drug blockade. 2007, Pubmed , Xenbase
Caldwell, Serine protease activation of near-silent epithelial Na+ channels. 2004, Pubmed
Cantor, Lipid composition and the lateral pressure profile in bilayers. 1999, Pubmed
Chalfie, The identification and suppression of inherited neurodegeneration in Caenorhabditis elegans. 1990, Pubmed
Chelur, The mechanosensory protein MEC-6 is a subunit of the C. elegans touch-cell degenerin channel. 2002, Pubmed , Xenbase
Chillarón, An intracellular trafficking defect in type I cystinuria rBAT mutants M467T and M467K. 1997, Pubmed , Xenbase
Chraïbi, Protease modulation of the activity of the epithelial sodium channel expressed in Xenopus oocytes. 1998, Pubmed , Xenbase
de Weille, Dependence of the acid-sensitive ion channel, ASIC1a, on extracellular Ca(2+) ions. 2001, Pubmed
Draganov, Rabbit serum paraoxonase 3 (PON3) is a high density lipoprotein-associated lactonase and protects low density lipoprotein against oxidation. 2000, Pubmed
Driscoll, The mec-4 gene is a member of a family of Caenorhabditis elegans genes that can mutate to induce neuronal degeneration. 1991, Pubmed
Gandhavadi, Structure, composition, and peptide binding properties of detergent soluble bilayers and detergent resistant rafts. 2002, Pubmed
Goodman, Pressure polishing: a method for re-shaping patch pipettes during fire polishing. 2000, Pubmed
Goodman, MEC-2 regulates C. elegans DEG/ENaC channels needed for mechanosensation. 2002, Pubmed , Xenbase
Harel, Structure and evolution of the serum paraoxonase family of detoxifying and anti-atherosclerotic enzymes. 2004, Pubmed
Huang, A stomatin-like protein necessary for mechanosensation in C. elegans. 1995, Pubmed
Huang, Gene interactions affecting mechanosensory transduction in Caenorhabditis elegans. 1994, Pubmed
Huber, Podocin and MEC-2 bind cholesterol to regulate the activity of associated ion channels. 2006, Pubmed
Hughey, Epithelial sodium channels are activated by furin-dependent proteolysis. 2004, Pubmed , Xenbase
Jasti, Structure of acid-sensing ion channel 1 at 1.9 A resolution and low pH. 2007, Pubmed
Johansson, Detection of tactile stimuli. Thresholds of afferent units related to psychophysical thresholds in the human hand. 1979, Pubmed
Kellenberger, Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure. 2002, Pubmed
Lai, Sequence and transmembrane topology of MEC-4, an ion channel subunit required for mechanotransduction in Caenorhabditis elegans. 1996, Pubmed
Levina, Protection of Escherichia coli cells against extreme turgor by activation of MscS and MscL mechanosensitive channels: identification of genes required for MscS activity. 1999, Pubmed
Lewis, Divalent cation effects on acetylcholine-activated channels at the frog neuromuscular junction. 1984, Pubmed
Mannsfeldt, Stomatin, a MEC-2 like protein, is expressed by mammalian sensory neurons. 1999, Pubmed
Markin, Thermodynamics of mechanosensitivity. 2004, Pubmed
Martinez-Salgado, Stomatin and sensory neuron mechanotransduction. 2007, Pubmed
Moe, Assessment of potential stimuli for mechano-dependent gating of MscL: effects of pressure, tension, and lipid headgroups. 2005, Pubmed
Niemelä, Assessing the nature of lipid raft membranes. 2007, Pubmed
O'Hagan, The MEC-4 DEG/ENaC channel of Caenorhabditis elegans touch receptor neurons transduces mechanical signals. 2005, Pubmed
Planès, Regulation of the epithelial Na+ channel by peptidases. 2007, Pubmed , Xenbase
Porter, Cholesterol modification of hedgehog signaling proteins in animal development. 1996, Pubmed
Schild, Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation. 1997, Pubmed , Xenbase
Sedensky, A stomatin and a degenerin interact in lipid rafts of the nervous system of Caenorhabditis elegans. 2004, Pubmed
Sigworth, Data transformations for improved display and fitting of single-channel dwell time histograms. 1987, Pubmed
Vallet, Cell-surface expression of the channel activating protease xCAP-1 is required for activation of ENaC in the Xenopus oocyte. 2002, Pubmed , Xenbase
Waldmann, A proton-gated cation channel involved in acid-sensing. 1997, Pubmed , Xenbase
Wetzel, A stomatin-domain protein essential for touch sensation in the mouse. 2007, Pubmed
Yeung, Analysis of active-site amino-acid residues of human serum paraoxonase using competitive substrates. 2005, Pubmed
Zhang, MEC-2 is recruited to the putative mechanosensory complex in C. elegans touch receptor neurons through its stomatin-like domain. 2004, Pubmed , Xenbase
Zhang, Combinatorial marking of cells and organelles with reconstituted fluorescent proteins. 2004, Pubmed