Hu L , Yang H , Shi J , Cui J .

R01-HL70393 NHLBI NIH HHS , R01 HL070393 NHLBI NIH HHS

	Figure 1. Mutation E374A:E399N reduces Mg2+ sensitivity. (A) Current traces of WT mslo1 channels (left) and E374A:E399N mutant channels (right) recorded at indicated [Mg2+]i and [Ca2+]i. Testing potentials were −20 to 240 mV with 20-mV increments. The holding and repolarizing potentials were −80 and −50 mV, respectively. (B) Mean G-V relations of WT mslo1 (gray symbols) and the mutant channels (black symbols) at 0 or 10 mM [Mg2+]i and 0 [Ca2+]i. G-V relations of these channels are fitted with the Boltzmann relation (solid lines, see MATERIALS AND METHODS). (C) Shift of G-V relations from 0 to 10 mM [Mg2+]i at 0 [Ca2+]i for WT mslo1 and the mutant channels. V1/2 is the voltage where the G-V relation is at half maximum.
	Figure 2. Mg2+ sensitivity of WT mslo1 and mutant channels at high [Mg2+]i. (A) Current traces of WT mslo1 channels (left) and E374A:E399N mutant channels (right) recorded at indicated [Mg2+]i and [Ca2+]i. Testing potentials were −70 to 190 mV with 20-mV increments for WT mslo1 channels at 100 mM [Mg2+]i and 0 [Ca2+]i; and were −20 to 240 mV with 20-mV increments for others. The holding and repolarizing potentials were −80 and −50 mV, respectively. (B) Mean G-V relations of WT mslo1 (gray symbols) and E374A:E399N mutant channels (black symbols) at 0 or 100 mM [Mg2+]i and 0 [Ca2+]i, and fits with the Boltzmann relation (solid lines). (C) G-V shifts at 0 [Ca2+]i from 0 to 10 mM [Mg2+]i (gray bars) and from 0 to 100 mM [Mg2+]i (black bars) of WT mslo1 and mutant channels E374A:E399N, E374Q and E399Q.
	Figure 3. Voltage and Mg2+ dependence of activation time courses of mslo1 channels. (A–E) Left panels, current traces recorded from a single membrane patch in response to depolarizing membrane voltages of 20 mM increments, and fitted with a single exponential function (thin curves). [Mg2+]i was as indicated. Right panels, activation time constants as a function of test potential, and fits with the function τ = Ae−qFV/RT + b (solid curves). (F) Semi-log plots of mean activation time constants as a function of both voltage and [Mg2+]i. Solid lines represent fits to the same function as in A–E.
	Figure 4. Effects of 100 μM [Ca2+]i on channel activation at high [Mg2+]i. (A) Current traces of WT mslo1 channels (left) and E374A:E399N mutant channels (right) recorded at indicated [Mg2+]i and [Ca2+]i. Dashed lines indicate zero current. Testing potentials were −200 to 140 mV with 20-mV increments. The holding and repolarizing potentials were −80 and −100 mV, respectively. (B) Mean G-V relations of WT mslo1 (gray symbols) and E374A:E399N mutant channels (black symbols) at 0 or 100 mM [Mg2+]i and 100 μM [Ca2+]i, and fits with the Boltzmann relation (solid lines). (C) G-V shifts from 0 to 100 mM [Mg2+]i at 100 μM [Ca2+]i of WT mslo1 and mutant channels E374A:E399N, E374Q, and E399Q.
	Figure 5. Mg2+ competes with Ca2+ by binding to Ca2+ binding sites. (A) Current traces of WT mslo1 channels (left) and E374A:E399N mutant channels (right) recorded at indicated [Mg2+]i and [Ca2+]i. Testing potentials were −140 to 180 mV with 20-mV increments. The holding and repolarizing potential were −80 and −50 mV, respectively. (B) Mean G-V relations of WT mslo1 (gray symbols) and the mutant channels (black symbols) at 0 or 10 mM [Mg2+]i and 3.7 μM [Ca2+]i, and fits with the Boltzmann relation (solid lines). (C) G-V shifts from 0 to 10 mM [Mg2+]i at 3.7 μM [Ca2+]i of WT mslo1 and the mutant channels.
	Figure 6. Voltage, Ca2+, and Mg2+-dependent activation of WT and E374A:E399N mutant channels and fittings with the hypothesis 1 model. (A–C) G-V relations of WT mslo1 and E374A:E399N mutant channels at indicated [Mg2+]i and [Ca2+]i. The data were fitted (solid lines) with Eq. 2. The parameters obtained from fittings in A and B are listed in Table I. In the fits in C, KcC = 8 μM and KoC = 1 μM.
	Figure 7. Voltage, Ca2+, and Mg2+-dependent activation of WT and E374A:E399N mutant channels and fittings with the hypothesis 2 model. (A–F) G-V relations of WT mslo1 and E374A:E399N mutant channels at indicated [Mg2+]i and [Ca2+]i. The data were fitted (solid lines) with Eq. 3. The parameters obtained from fittings in A–C are listed in Table II.
	Figure 8. Voltage, Ca2+, and Mg2+-dependent activation of WT and E374A:E399N mutant channels and fittings with the hypothesis 3 model. (A–F) G-V relations of WT mslo1 and E374A:E399N mutant channels at indicated [Mg2+]i and [Ca2+]i. The data were fitted (solid lines) with Eq. 4. The parameters obtained from fittings in A-C are listed in Table III.
	Figure 9. Voltage and Mg2+-dependent activation of WT, 5D5N + D367A + E399N, and 5D5N + D362A:D367A + E399N mutant channels. (A) Current traces of WT mslo1 channels (left), 5D5N + D367A + E399N mutant channels (middle), and 5D5N + D362A:D367A + E399N mutant channels (right) recorded at indicated [Mg2+]i. Testing potentials were −200 to 140 mV with 20-mV increments. The holding and repolarizing potentials were −80 and −120 mV, respectively. (B) Mean G-V relations of 5D5N + D367A + E399N (left) and 5D5N + D362A:D367A + E399N mutant channels (right) in the presence of 0–100 mM [Mg2+]i and fittings with the hypothesis 3 model. The symbols represent [Mg2+]i (in mM) at 0 (open circle), 0.1 (closed circle), 1 (open triangle), 10 (closed triangle), 30 (open diamond), and 100 (closed diamond). The data were fitted (solid lines) with Eq. 4. The parameters were fixed with the numbers in Table III, except L0, which were set free. In the fits, L0 for 5D5N + D367A + E399N and 5D5N + D362A:D367A + E399N is 3516 and 4908, respectively.
	Figure 10. Voltage and Ca2+-dependent activation of WT, 5D5N + D367A + E399N, and 5D5N + D362A:D367A + E399N mutant channels. (A) Current traces of WT mslo1 channels (left), 5D5N + D367A + E399N mutant channels (middle), and 5D5N + D362A:D367A + E399N mutant channels (right) recorded at indicated [Ca2+]i. Dashed lines indicate zero current. Testing potentials for WT at 0.1 mM [Ca2+]i were −150 to 190 mV with 20-mV increments. Testing potentials for WT at 1, 10, 30, and 100 mM [Ca2+]i were −200 to 140 mV with 20-mV increments. Testing potentials for WT at 0 [Ca2+]i and 5D5N + D367A + E399N and 5D5N + D362A:D367A + E399N mutant channels were −20 to 240 mV with 20-mV increments. The holding and repolarizing potentials were −80 and −120 mV, respectively. (B) Mean G-V relations of 5D5N + D367A + E399N mutant channels (left) and 5D5N + D362A:D367A + E399N mutant channels (right) in the presence of 0–100 mM [Ca2+]i The symbols represent [Ca2+]i (in mM) at 0 (open circle), 0.1 (inverted open triangle), 1 (inverted closed triangle), 10 (open diamond), 30 (closed diamond) and 100 (open square). G-V relations of these channels are fitted with the Boltzmann relation (solid lines, see MATERIALS AND METHODS). (C) V1/2 versus [Mg2+]i (closed symbols) or [Ca2+]i (open symbols) for WT (circle), E374A:E399N (diamond), 5D5N + D367A + E399N (triangle), and 5D5N + D362A:D367A + E399N (inverted triangle) mutant channels.
	Figure 11. Effects of Mg2+ on the membrane surface potential due to surface charge screening. (A) Surface potentials at 1, 10, and 100 mM [Mg2+]i as a function of surface charge density. The calculation is based on the Grahame equation (Grahame, 1947). The solution contains 140 mM K+. (B) Change of surface potential as [Mg2+]i increases from 1 to 10 mM and from 10 to 100 mM.

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