Wang Y , Matthewman C , Han L , Miller T , Miller DM , Bianchi L .

MH078028 NIMH NIH HHS, NIH RO1NS070969 NINDS NIH HHS , NIH RO1NS26115 NINDS NIH HHS , R21MH077302 NIMH NIH HHS, R21 MH077302 NIMH NIH HHS, R01 NS026115 NINDS NIH HHS , P50 MH078028 NIMH NIH HHS, R01 NS070969 NINDS NIH HHS , R01 NS081259 NINDS NIH HHS , R01 NS106951 NINDS NIH HHS

	Figure 1. Selective degeneration of ventral cord cholinergic motor neurons in unc-8(n491) animals. DIC images of L1 larval ventral nerve cords of (A) wild-type versus (B) unc-8(n491) animals. Cholinergic DA and DB motor neurons (asterisks) and P cell precursor (arrowhead) appear swollen in unc-8(n491) versus wild type. DD GABA motor neurons are labeled with punc-25::GFP. Bar, 1 µm. (C and D) DD/VD GABAergic motor neurons are marked with punc-25::dsRed (red) and DA/DB neurons are labeled with unc-129nsp::CFP (blue) in (C) wild-type and (D) unc-8(n491) young adult animals. Insets show linearized view of DA/DB motor neurons. Bars, 25 µm. (E) GABAergic and DA/DB motor neurons were counted in the ventral cord region between DD1 and DD6 (arrowheads in C and D; see also Fig. S1). unc-8(n491) animals (n = 14) contain the full complement of 19 DD/VD GABAergic motor neurons but are missing a significant fraction (∼36%) of the 11 DA/DB motor neurons from this region of the adult ventral nerve cord (wild type; n = 24). ***, P = 3 × E-11 by t test.
	Figure 2. Amiloride-sensitive currents in UNC-8(G387E)–expressing oocytes. (A) Example of ionic currents recorded in an oocyte expressing UNC-8(G387E) perfused with a NaCl physiological solution. Currents were elicited by voltage steps from −160 to +100 mV in 20-mV increments. The holding potential was −30 mV. (B) The same oocyte shown in A was perfused with NaCl physiological solution plus 1 mM amiloride. (C) Amiloride-sensitive currents. Currents obtained by subtracting the amiloride-resistant current from the total current are shown here. Dashed lines represent the zero current level. The outward amiloride-resistant current is likely oocytes endogenous current, which varies in amplitude from batch to batch of oocytes. (D) Current–voltage relationship of amiloride-sensitive currents in UNC-8(G387E)–expressing oocytes (n = 5). Note the strong inward rectification at negative voltages. The solid line represents the amiloride-sensitive current recorded from an oocyte injected with wild-type UNC-8. (E) Amiloride dose–response curve. Currents recorded in amiloride at −100 mV were normalized against the currents recorded in NaCl physiological solution at −100 mV. Data points were fitted by Hill equation, which gave a Ki of 7.8 µM and an n value of 0.5 (n = 6). Data are expressed as mean ± SE.
	Figure 3. UNC-8(G387E) currents are larger in divalent cation–free solution. (A) Example of currents recorded in an oocyte injected with UNC-8(G387E) and perfused with NaCl physiological solution, which contains 1 mM CaCl2 and 2 mM MgCl2. Currents were stimulated by voltage steps from −160 to +100 mV in 20-mV increments. The holding potential was −30 mV. (B) The same oocyte shown in A was perfused with a divalent cation–free NaCl solution containing 1 mM EGTA. Currents were recorded with the same voltage protocol used in A. (C) Currents were recorded from the same oocyte in divalent cation–free EGTA solution plus 1 mM amiloride. Dashed lines represent the zero current level. (D) Current–voltage relationships from noninjected oocytes and oocytes injected with UNC-8 or UNC-8(G387E), and perfused with either the NaCl physiological solution or the divalent cation–free or divalent cation–free plus EGTA NaCl solution, as indicated. The number of oocytes tested is shown in parentheses. (E) Shift in the oocyte resting potential upon switching from NaCl physiological solution (1 mM CaCl2 and 2 mM MgCl2) to divalent cation–free plus EGTA NaCl solution for noninjected oocytes (open squares; n = 9) and oocytes injected with UNC-8(G387E) (closed circles; n = 32). Small symbols and lines correspond to measurement from individual oocytes, and large symbols are means ± SE. (F) Amiloride dose–response curve obtained from oocytes injected with UNC-8(G387E) and perfused with divalent cation–free plus EGTA NaCl solution. Data points were fitted by Hill equation, which gave a Ki of 106 µM and an n value of 1. The inset shows the voltage dependence of amiloride blockade. Data points were fitted using the Woodhull model, which gave a δ of 0.08 (Woodhull, 1973). Data are expressed as mean ± SE (n = 8).
	Figure 4. Cell death and UNC-8(d) permeability properties. (A) Representative photographs of a noninjected oocyte (top), oocyte-injected UNC-8 wild type, and an UNC-8(G387E) (middle and bottom panels, respectively) incubated for 24 h in OR2 solution containing 5 µM calcium and no magnesium. Note the lysis of the oocyte-expressing UNC-8(G387E). (B) Quantification of the ratio of oocytes that are intact after a 24-h treatment in OR2 containing 5 µM calcium. Values are averages of four independent injections with 10 oocytes in each replicate. Amiloride was added to a final concentration of 500 µM. Values are mean ± SE. **, P ≤ 0.01 by ANOVA. (C) Example of currents recorded in a noninjected oocyte (top) and an oocyte injected with UNC-8(G387E) perfused with a solution whose only permeant cation was calcium. Voltage steps were from −160 to −40 mV from a holding potential of 0 mV. (D) Average current amplitudes at −160 recorded from noninjected oocytes and oocytes injected with UNC-8(G387E) perfused with the calcium solution. Note that the oocyte-endogenous Ca2+-activated Cl− current is not activated in either sample (Bianchi et al., 2004). Data are expressed as mean ± SE (n = 8). NS, not statistically different. (E) Average current amplitude recorded at −100 mV in oocytes injected with UNC-8(G387E) perfused the solutions indicated on the x axis. Data are expressed as mean ± SE (n = 12, 12, 12, and 9, respectively).
	Figure 5. UNC-8(G387E) calcium and magnesium sensitivity. (A) Example of ionic currents recorded in an oocyte injected with UNC-8(G387E) and perfused with the divalent cation–free plus EGTA NaCl solution. The voltage protocol from Fig. 3 A was used here. (B) The same oocyte was perfused with the NaCl solution containing 10 µM CaCl2. (C) Calcium dose–response curve. Currents were recorded at −100 mV in oocytes perfused with increasing concentrations of extracellular calcium. Currents in the presence of calcium were normalized for the current recorded in the same oocyte in divalent cation–free plus EGTA NaCl solution. Before normalization, all currents were leak subtracted. Data points were fitted with a sigmoidal curve for a Ki of 6 µM (n = 8). (D) Voltage dependence of calcium block. The Ki for calcium was plotted against the voltage. Data points were fitted using the Woodhull model; the δ value was 0.06 (n = 7). (E) Same as in A, except that the divalent-free solution did not contain EGTA. (F) The same oocyte shown in E was perfused with the NaCl solution containing 500 µM MgCl2. (G) Magnesium dose–response curve. Data points were derived as in C. Fitting with a sigmoidal curve gave a Ki value of 598 µM (n = 11). (H) The Ki for magnesium block was plotted against the voltage. Data points were fitted by the Woodhull model; the δ value was 0.13 (n = 9).
	Figure 6. Calcium and magnesium block UNC-8(A586T) currents. (A) Example of currents stimulated by voltage steps from −160 to +100 mV in an oocyte injected with UNC-8(A586T); the holding potential was −30 mV. The oocyte was perfused with a physiological NaCl solution containing 1 mM CaCl2 and 2 mM MgCl2. (B) Average current–voltage relationships obtained from currents similar to the ones shown in A (n = 6). (C) The same oocyte shown in A was perfused with the divalent cation–free plus EGTA NaCl solution. (D) Average current–voltage relationship obtained from currents similar to the ones shown in C (n = 6).
	Figure 7. UNC-8(H114Y), the trans-suppressor of UNC-(G387E) neuronal swelling, suppresses UNC-8(G387E) currents. (A) Example of currents recorded from an oocyte injected with UNC-8(G387E). (B) Example of currents recorded from an oocyte injected with UNC-8(G387E) plus UNC-8(H11Y). Both oocytes were perfused with the divalent cation–free plus EGTA NaCl solution. Currents were elicited by voltage steps from −160 to +100 mV in 20-mV increments from a holding potential of −30 mV. The dashed line corresponds to the zero current level. (C) Average current amplitudes at −160 mV recorded from oocytes injected with UNC-8(G387E) (n = 23), noninjected oocytes (n = 14), wild-type UNC-8 (n = 17), UNC-8(H114Y) (n = 23), UNC-8(G387E) plus UNC-8(wt) (n = 11), UNC-8(G387E) plus UNC-8(H114Y) (n = 23), and UNC-8(G387E/H114Y) (n = 13). **, P ≤ 0.01 by ANOVA. (D) Mean change in resting potential upon switch from NaCl physiological solution containing 1 mM CaCl2 and 2 mM MgCl2 to the divalent cation–free plus EGTA NaCl solution in oocytes injected with UNC-8(H114Y) (open diamonds; n = 5) and in oocytes injected with UNC-8(G387E) plus UNC-8(H114Y) (closed diamonds; n = 13).
	Figure 8. pH sensitivity of neurotoxic UNC-8 mutants. (A) Example of currents stimulated by voltage steps from −160 to +100 mV in an oocyte injected with UNC-8(G387E); the holding potential was −30 mV. The oocyte was perfused with the divalent cation–free plus EGTA NaCl solution at pH 7.2. (B) The same oocyte shown in A was perfused with the divalent cation–free plus EGTA NaCl solution at pH 6.5. (C and D) Same as in A and B for an oocyte expressing UNC-8(A586T). (E) pH dose–response curves for oocytes expressing UNC-8(G387E) (closed circles; n = 22) and UNC-8(A586T) (closed squares; n = 14), respectively. Before normalization, all currents were leak subtracted. Data points were fitted by sigmoidal curves that gave pH0.5 values of 5.6 and 6.6 for UNC-8(G387E) and UNC-8(A586T), respectively.

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