Shi J , Cui J .

	Scheme S1.
	Scheme S2.
	Scheme S3.
	Figure 1. Intracellular Mg2+ blocks and activates mslo1 channels. (A) mslo1 currents recorded from an inside-out patch with 0 (dark traces) or 10 mM (light traces) [Mg2+]i at [Ca2+]i of 0 (bottom) and 110 μM (top). The voltage protocols are schematically displayed next to the current traces. At 110 μM [Ca2+]i, the holding, test, and repolarizing potentials were −100, 150, and −50 mV, respectively. At 0 [Ca2+]i, they were −50, 200, and −50 mV, respectively. Smooth lines are exponential fits to current traces. The time constant is 0.23 ms at 0 [Mg2+]i, 0.26 ms at 10 mM [Mg2+]i with 110 μM [Ca2+],i and 2.84 ms at 0 [Mg2+]i, 1.72 ms at 10 mM [Mg2+]i with 0 [Ca2+]i. (B) G-V relations of mslo1 channels with 0 (circles) or 10 mM (squares) [Mg2+]i at [Ca2+]i of 0 (open symbols) and 110 μM (closed symbols). Corresponding symbols are also shown in A. The smooth lines are fits with the Boltzmann function, G/Gmax = 1/(1 + exp(−ze(V − V1/2)/kT)), where G is conductance, z is the valence of equivalent charge, e is the elementary charge, V1/2 is the voltage where conductance is half-maximum, k is Boltzmann's constant, and T is the absolute temperature. At 110 μM [Ca2+]i, z = 1.08 and V1/2 = 11.4 mV with 0 [Mg2+]i, and z = 0.92, V1/2 = −52.4 mV with 10 mM [Mg2+]i. At 0 [Ca2+]i, z = 1.01 and V1/2 = 182.6 mV with 0 [Mg2+]i, and z = 1.20, V1/2 = 117.0 mV with 10 mM [Mg2+]i. (C) The response to [Mg2+]i of the peak current at the test potential of 150 mV and the instantaneous tail current at the repolarizing potential of −50 mV. [Ca2+]i was 1 μM. Data points are connected by thin straight lines. (D) Mg2+ block of the peak current at test potentials of 100 and 150 mV. The ratio of the current with internal Mg2+ to that without internal Mg2+, I(Mg)/I(0), from three (at 150 mV) or five (at 100 mV) patches were averaged and plotted versus [Mg2+]i. Error bars in all figures represent the SEM. Smooth lines are fits of the Woodhull model (Woodhull 1973) I(Mg)/I(0) = 1/(1 + [Mg2+]i /KD(0)exp(−2δeV/kT)), where KD(0) = 31.5 mM is the dissociation constant at 0 mV and δ = 0.22 is the fraction of the voltage across the membrane that influences Mg2+ at its binding site, as measured from the intracellular surface.
	Figure 2. G-V relations in the presence of 0∼100 mM [Mg2+]i at 0 (A) or 110 μM [Ca2+]i (B). The symbols represent [Mg2+]i at (in mM) 0 (open circle), 0.1 (closed circle), 0.5 (open square), 1 (closed square), 5 (open triangle), 10 (closed triangle), 30 (open diamond), and 100 (closed diamond). G-V relations from a number (n) of patches at each [Mg2+]i are averaged and plotted. Smooth curves are Boltzmann fits to averaged G-V relations. At 0 [Ca2+]i (A), z and V1/2 at various [Mg2+]i (mM) are as follows for 0, 1.17, 186.4 mV (n = 12); for 0.1, 1.30, 177.0 mV (n = 4); for 0.5, 1.26, 169.5 mV (n = 4); for 1, 1.30, 162.7 (n = 4); for 5, 1.36, 142.4 mV (n = 4); for 10, 1.32, 129.6 mV (n = 4); for 30, 1.30, 97.76 mV (n = 7); and for 100, 1.13, 80.1 mV (n = 8), respectively. At 110 μM [Ca2+]i (B), z and V1/2 at various [Mg2+]i (mM) are as follows: for 0, 1.10, 1.2 mV (n = 10); for 0.1, 1.17, 5.8 mV (n = 5); for 0.5, 1.12, −7.6 mV (n = 6); for 1: 1.20, -16.0 (n = 6); for 5, 1.06, −38.2 mV (n = 5); for 10, 1.26, −62.5 mV (n = 4); for 30, 0.97,−70.4 mV (n = 4); and for 100, 0.95, −73.4 mV (n = 4), respectively. (C) Left shifts of G-V relations on the voltage axis caused by various [Mg2+]i at 0 and 110 μM [Ca2+]i. ΔV1/2 = (V1/2 at each [Mg2+]i − V1/2 at 0 [Mg2+]i) obtained from n = 4 patches at 0 [Ca2+]i and n = 6 patches at 110 μM [Ca2+]i are averaged and then plotted. The smooth curves are fits of to data at 0 [Ca2+]i with z = 1.30. For the thick curve, m is fixed at 4, KC = 15.0 mM, KO = 3.6 mM. For the thin curve, m is let free in the fit and is 1.94, KC = 45.7 mM, KO = 2.12 mM. (D) Changes in the activation energy provided by Ca2+ binding as the result of an increased [Ca2+]i from 0 to 110 μM in the absence or presence of 10 mM [Mg2+]i. −ΔΔGCa = (zV1/2 at 0 [Ca2+]i − zV1/2 at 110 μM [Ca2+]i)eN, where e is elementary charge and N is Avogadro's number. Averaged from n = 4 patches.
	Figure 3. Mg2+ effects on mslo3 channels or the channels from coexpression of the mslo1 core and mslo3 tail. (A and D) Schematic representation of mslo3 (A) and the mslo1 core (light) and the mslo3 tail (dark) (D). S0–S6 are transmembrane segments, RCK indicates the RCK domain (Jiang et al. 2001), and CB signifies the lack of calcium bowl. (B) mslo3 currents from an inside-out patch in the absence (top) or presence (bottom) of 10 mM [Mg2+]i. The holding and repolarizing potentials are −50 mV. The test potentials are from −80 to 200 mV with 20-mV increment. [Ca2+]i = 0. (C) G-V relations of mslo3 channels in the absence or presence of 10 mM [Mg2+]i. [Ca2+]i = 0. G-V curves are averaged from n = 6 patches, and then fitted with the Boltzmann equation (smooth lines) with z = 0.76 and V1/2 = 111.3 mV at 0 [Mg2+]i and z = 0.76 and V1/2 = 117.6 mV at 10 mM [Mg2+]i, respectively. (E) Currents from an inside-out patch that coexpresses the mslo1 core and mslo3 tail in the absence (top) or presence (bottom) of 10 mM [Mg2+]i. The voltage protocol is the same as in B except that the test potential stops at 140 mV. [Ca2+]i = 1.1 μM. (F) G-V relations of channels from the coexpression of the mslo1 core and mslo3 tail in the absence or presence of 10 mM [Mg2+]i. [Ca2+]i = 1.1 μM. Smooth lines are fits of the Boltzmann equation with z = 1.14 and V1/2 = 55.9 mV at 0 [Mg2+]i and z = 1.16 and V1/2 = 0.2 mV at 10 mM [Mg2+]i, respectively. n = 4 patches.
	Figure 4. Mg2+ dependence of mslo1 currents. (A) Average normalized G-V relations at 0 [Ca2+]i and the following [Mg2+]i: 0, 0.1, 0.5, 1, 5, 10, 30, and 100 mM were transformed to dose–response curves as displayed (data at 0 and 0.1 mM [Mg2+]i is not shown on the logarithm scale). Alternating closed and open circles represent the dose–response curves (ascending right to left) at different voltages between 40 and 200 mV in 20-mV increments. Smooth curves represent fits to the Hill equation (G/Gmax = Amp(Mg)/(1 + (Kd/[Mg2+]i)nH) + Amp(0)), where nH is Hill coefficient, Kd is the apparent Mg2+ dissociation constant, Amp(0) = G/Gmax at 0 [Mg2+]i, and Amp(Mg) is the Mg2+-dependent component of G/Gmax. (B–D) The amplitudes, Hill coefficient, and apparent dissociation constant determined from fits to the Hill equation are plotted versus voltage. Open symbols represent parameters determined from the fits in A. Closed symbols represent the mean values from four experiments. In D, solid lines are fits with the function Kd(V) = Kd(0)exp(zeV/kT). The thin line fits the results determined in A, Kd(0) = 378.8 mM and z = 0.77. The thick line fits the mean results from four experiments, Kd(0) = 242.3 mM and z = 0.77.
	Figure 5. The effects of [KCl]i and high [Ca2+]i on the activation of mslo1 channels. (A) G-V relations of mslo1 channels with or without 20 mM KCl added to the basal internal solution (BIS; materials and methods). The total [Cl−]i is 22 mM and 2 mM, respectively. The total [K+]i is 162 and 142 mM, respectively. [Ca2+]i = 110 μM. The data was averaged from the results of n = 4 patches. Smooth lines are Boltzmann fits to data. With BIS, z = 1.29 and V1/2 = 1.1 mV. With 20 mM [KCl]i added to BIS, z = 1.21 and V1/2 = 7.2 mV. (B) G-V relations of mslo1 channels at 110 μM or 10.1 mM [Ca2+]i. Smooth lines are Boltzmann fits to data. At 110 μM [Ca2+]i, z = 1.09 and V1/2 = 1.0 mV. At 10.1 mM [Ca2+]i, z = 1.06 and V1/2 = −75.1 mV.
	Figure 6. Effects of intracellular Mg2+ on Ca2+-dependent activation. (A) V1/2 of G-V relations at various [Ca2+]i in the presence or absence of 10 mM [Mg2+]i. The smooth lines are fits of to data. Each data point is averaged from n = ∼4–8 patches. (B) The difference in V1/2 in the presence or absence of 10 mM [Mg2+]i. At each [Ca2+]i, ΔV1/2 = (V1/2 at 10 mM [Mg2+]i − V1/2 at 0 [Mg2+]i), averaged from n = 4–6 patches. (C) and D) [Ca2+]i dependence of normalized conductance in the absence (C) or presence (D) of 10 mM [Mg2+]i. Each data point is averaged from n = 4–8 patches. Alternating open and closed circles represent the curves (ascending right to left) at different voltages between −40 and 100 mV (C) or between −80 and 60 mV (D) in 20-mV increments. Dark or light smooth curves represent fits to the Hill equation or to (Fig. 2), respectively. (E and F) Hill coefficient (E) and apparent Kd (F) obtained from fits of data in the presence or absence of 10 mM [Mg2+]i to the Hill equation.
	Figure 7. The ratio of conductance with or without intracellular Mg2+. The conductance, G = γPo, in the presence of 5, 3, or 1 mM [Mg2+]i versus that in the absence of Mg2+ are plotted against [Ca2+]i (top to bottom), where γ is the single-channel conductance. Results at three physiological voltages (50, 0, and −50 mV) are displayed. The horizontal straight line indicates the ratio of 1. Po is computed from ∼5 with parameters described in Fig. 2 legends and Fig. 2 footnote. γ is computed from the Woodhull model and the parameters described in Fig. 1 legend. We assume that Ca2+ is equivalent to Mg2+ in blocking the channel (Cox et al. 1997b) and in activating the channel by binding to the low affinity Mg2+/Ca2+ sites (Fig. 5). Therefore, even in the absence of Mg2+, the low affinity Mg2+/Ca2+ sites are occupied by Ca2+ and contribute to activation; similarly, the channel is blocked by Ca2+. At 3 mM [Mg2+]i (middle) we also computed the ratio of conductance without considering the competition of Mg2+ at the high affinity Ca2+ sites (thin curves).

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