Matthewman C , Johnson CK , Miller DM , Bianchi L .

R01 NS106951 NINDS NIH HHS

	Fig. 1. The finger domain contains the most divergent sequence. A: sequence alignment (Clustal Omega, https://www.ebi.ac.uk/Tools/msa/clustalo/) of human acid-sensing ion channel (ASIC) ASIC1a and Caenorhabditis elegans degenerin (DEG) channels MEC-4 and UNC-8. Shaded in gray are transmembrane domains TM1 and TM2; in yellow is the finger domain of MEC-4. B: schematic representation of a DEG/epithelial Na+ channel subunit. Cylinders and arrows represent α-helixes and β-sheets, respectively; lines depict loops. α-Helixes and β-sheets are numbered according to the primary protein sequence. In yellow, the finger domain with the degenerin-specific domains shown as dotted lines. Red dots represent hyperactive MEC-4 mutation A404T and UNC-8 mutation G387E; the gray dot represents mutation A387S in brain liver intestine Na+ channel (BLINaC) (56). Gray-shaded area represents the plasma membrane.
	Fig. 2. MEC-6 exerts its effects on current amplitude via the finger domain. Schematic representations of channel subunits expressed and comparison of mean current amplitudes recorded at −100 mV in physiologic solution (gray columns) and divalent-free solution (black columns). A: UNC-8(d), UNC-8(d) +MEC-2, and UNC-8(d) +MEC-2 +MEC-6. n = 11, 5, and 15 respectively in physiologic solution, and n = 9, 5, and 8 in divalent-free solution. B: MEC-4(d), MEC-4(d) + MEC-2, and MEC-4(d) + MEC-2 + MEC-6; n = 5, 12, and 14 respectively in physiologic solution, and n = 5, 4, and 8 in divalent-free solution. C: UNC-8/MEC-4 A404T (extracellular domain, ECD) chimera, UNC-8/MEC-4 A404T (ECD) + MEC-2, and UNC-8/MEC-4 A404T (ECD) + MEC-2 + MEC-6; n = 11, 11, and 8 respectively in physiologic solution, and n = 11, 14, and 8 in divalent-free solution. D: UNC-8/MEC-4 A404T (finger) chimera, UNC-8/MEC-4 A404T (finger) + MEC-2, and UNC-8/MEC-4 A404T (finger) + MEC-2 + MEC-6; n = 8, 8, and 10 respectively in physiologic solution, and n = 8, 8, and 10 in divalent-free solution. Data are expressed as mean ± SD. Statistical significance (by ANOVA with Bonferroni correction) between MEC-4(d) and MEC-4(d) coexpressed with MEC-2, and MEC-6 in physiologic solution and divalent-free solution and between UNC-8/ME-4 A404T (ECD) and UNC-8/MEC-4 A404T (finger) chimeras alone and coexpressed with MEC-6 is indicated by asterisks. P values are shown above the columns.
	Fig. 3. The finger domain establishes the cell death phenotype. A and B: micrographs of an intact healthy oocyte (noninjected) and a ruptured dead one [injected with UNC-8(G387E)] 3 days after injection. C: we scored cell death by counting the ratio of ruptured oocytes or oocytes with cytoplasmic protrusions over the course of 5 days, starting from day 2 after injection for oocytes injected with UNC-8(d) + MEC-2 + MEC-6 (black open circles, n of experiments was 5, with 10–15 oocytes injected per experiment), MEC-4(A404T) + MEC-2 + MEC-6 (black open squares, n = 2–4, with 10–15 oocytes injected per experiment), UNC-8/MEC-4 A404T (extracellular domain, ECD) + MEC-2 + MEC-6 (blue open triangles n = 2–3, with 10–15 oocytes injected per experiment), and UNC-8/MEC-4 A404T (finger) + MEC-2 + MEC-6 (pink asterisks, n = 2–4, with 10–15 oocytes injected per experiment). Data are expressed as mean ± SE.
	Fig. 4. UNC-8 Ca2+ block is conferred by the finger domain. A: representative whole cell currents recorded from an oocyte expressing UNC-8(d) + MEC-2 +MEC-6 perfused with a divalent cation-free NaCl solution containing 1 mM EGTA. The voltage protocol consisted of a holding potential of −30 mV and steps from −160 to + 100 mV in 20 mV increments. Right column currents represent same oocyte perfused with 500 µM Ca2+. B: same as in A for oocyte expressing MEC-4(A404T) +MEC-2 +MEC-6. Right column represents same oocyte perfused with 500 µM Ca2+. C: Same as in A and B for oocyte expressing UNC-8/MEC-4 (A404T) (finger) chimera +MEC-2 +MEC-6. Right column represents same oocyte perfused with 500 µM Ca2+. D: calcium dose-response curves for oocytes expressing UNC-8(d) +MEC-2 +MEC-6 (n = 10; black open circles, Ki = 7.4 µM), MEC-4 (A404T)+MEC-2+MEC-6 (n = 6) (pink open triangles, Ki = 1002 µM), UNC-8/MEC-4 (A404T) (extracellular, ECD) chimera + MEC-2+MEC-6 (n = 8) (blue open triangles, Ki = 944 µM), and UNC-8/MEC-4 (A404T) (finger) chimera+MEC-2+MEC-6 (n = 9; red asterisk, Ki = 766 µM). Data are expressed as mean ± SE and were fitted with a sigmoidal curve.
	Fig. 5. Block of UNC-8(d) by Mg2+ conferred by the extracellular domain (ECD). A: representative whole cell currents recorded from an oocyte expressing UNC-8(d) perfused with a divalent cation-free NaCl solution. Right column currents represent same oocyte perfused with 500 µM Mg2+. B and C: Same as in A for an oocyte expressing MEC-4(A404T) +MEC-2 +MEC-6 and one expressing UNC-8/MEC-4(A404T) (finger) chimera +MEC-2 +MEC-6. Next column represents same oocytes perfused with 500 µM Mg2+. D: average current remaining at −100 mV after perfusion with 500 µM Mg2+ for UNC-8(d) (n = 6), UNC-8(d) + MEC-2 + MEC-6 (n = 6), MEC-4(d)+MEC-2+MEC-6 (n = 9), MEC-4 (A404T) + MEC-2 + MEC-6 (n = 5), UNC-8/MEC-4 A404T (ECD) chimera + MEC-2 + MEC-6 (n = 4), and UNC-8/MEC-4(A404T) (finger) chimera + MEC-2 + MEC-6 (n = 7). Data are expressed as mean ± SE. Statistical significance (one-way ANOVA with Bonferroni multiple comparison test) between UNC-8(d) or UNC-8(d) + MEC-2 + MEC-6, and all the other experimental groups is indicated by the asterisk. P values are shown above the columns.

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