Tang CY , Bezanilla F , Papazian DM .

GM30376 NIGMS NIH HHS , GM43459 NIGMS NIH HHS , R01 GM043459-11 NIGMS NIH HHS , R01 GM043459-12 NIGMS NIH HHS , R01 GM043459-13 NIGMS NIH HHS , R01 GM043459-14 NIGMS NIH HHS , R01 GM043459-15 NIGMS NIH HHS , R01 GM043459-15S1 NIGMS NIH HHS , R01 GM043459-16 NIGMS NIH HHS , R01 GM043459-17 NIGMS NIH HHS , R01 GM043459 NIGMS NIH HHS , R37 GM030376 NIGMS NIH HHS , R01 GM030376 NIGMS NIH HHS

	Figure 1. Prepulse hyperpolarization slows activation of eag ionic currents. (A) From a holding potential of −90 mV, 75-ms hyperpolarizing prepulses ranging from −200 to −90 mV were applied in 20- or 30-mV increments, followed by a test pulse to +50 mV. Representative currents evoked by the test pulses are shown. After more negative prepulses, the time course of the ionic current was slower. (B) The time course of the rising phase of the ionic current after prepulses to −200 mV (solid line) and −150 mV (dashed line) cannot be superimposed by shifting the traces along the time axis. The traces were scaled and aligned at either the peak (left) or foot (right) of the rising phase. (C) Fits with a single exponential function (solid lines) to the late phase of activation are shown superimposed on ionic current traces (dashed lines) after prepulses to −90 or −200 mV. (D) Activation time constants at +50 mV obtained from single exponential fits to the late rising phase of the eag ionic current are plotted versus prepulse potential. Data are shown as mean ± SEM, n = 6.
	Figure 2. Extracellular Mg2+ slows activation of eag ionic currents. (A) From a holding potential of −90 mV, 120-ms test pulses from −60 to +60 mV were applied in 20-mV increments in the absence (left) or presence (middle) of 2 mM Mg2+ in the extracellular solution. Note that partial inactivation was sometimes observed in the absence of Mg2+. (Right) Ionic current traces evoked at +40 mV in the absence (dashed line) and presence (solid line) of 2 mM Mg2+ were scaled and overlaid to compare the time course of ionic current activation. (B) Fits with a single exponential function (solid lines) are shown superimposed on the late phase of ionic current activation at +40 mV (dashed lines) in the absence and presence of Mg2+. (C) Activation time constants at +40 mV obtained from single exponential fits to the late rising phase of the eag ionic current in the presence (•) and absence (○) of 2 mM Mg2+ are plotted versus test potential. Data are shown as mean ± SEM, n = 7. In this and subsequent figures, if error bars are not visible, the SEM was smaller than the size of the symbol.
	Figure 3. Extracellular Mg2+ enhances the effect of prepulse hyperpolarization on eag ionic currents. (A) Current traces evoked in the presence of 2 mM Mg2+ by a test pulse to +50 mV after hyperpolarizing prepulses to potentials from −90 to −200 mV are shown. The pulse protocol was the same as in Fig. 1 A. (B) Fits with a single exponential function (solid lines) to the late phase of activation are shown superimposed on ionic current traces (dashed lines) after prepulses to −90 or −200 mV. (C) The time to half maximal current amplitude at +50 mV was measured in the presence (•) and absence (○) of 2 mM Mg2+ and plotted versus prepulse potential. Data are shown as mean ± SEM, n = 5. (D) Activation time constants at +50 mV obtained from single exponential fits to the late rising phase of the eag ionic current in the presence (•) and absence (○) of 2 mM Mg2+ are plotted versus prepulse potential. Data are shown as mean ± SEM, n = 5.
	Figure 4. Extracellular Mg2+ slows opening of the channel and back transitions between closed states. (A, top) The voltage protocol for the reactivation experiment is shown. From a holding potential of −90 mV, two identical test pulses to +50 mV separated by a variable interpulse interval (Δt) at −90 mV were applied. Test pulse duration was 20 or 70 ms in the absence or presence of 2 mM Mg2+, respectively. The experiment was performed using a nominally K+-free bath solution to eliminate inward tail currents at −90 mV, which would interfere with detecting reactivation after short interpulse intervals. (Bottom) Representative current traces evoked by the reactivation protocol in the absence of Mg2+ (left) were obtained using Δt = 0.5 ms (dashed traces) or 20 ms (solid traces). Representative current traces evoked by the reactivation protocol in the presence of 2 mM Mg2+ (right) were obtained using Δt = 1 ms (dashed traces) or 50 ms (solid traces). (B, left) The late rising phase of currents evoked by the second test pulse (dashed traces) in the presence and absence of Mg2+, as indicated, were fitted with single exponential functions (solid lines). In the representative experiment shown, Δt = 1 ms. (Right) Fitted time constants for the second test pulse were determined for short interpulse intervals (between 0.1 and 1 ms) in the presence (•) and absence (○) of 2 mM Mg2+ and plotted versus Δt. Data are shown as mean ± SEM, n = 3. (C) Tail currents were recorded in the presence (•) or absence (○) of 2 mM Mg2+ by stepping from +50 mV to potentials ranging from −90 to −150 mV. Deactivation time constants were derived from single exponential fits and plotted versus tail potential. (D) Mg2+ slows the time course of the return to the original activation kinetics. Fitted time constants for the second test pulse were determined as shown in B in the presence (•) and absence (○) of 2 mM Mg2+ and plotted versus Δt. Note that the graphs have different scales for both the ordinate and the abscissa. Data are shown as mean ± SEM, n = 3.
	Figure 5. Characterization of eag gating currents. (A) From a holding potential of −90 mV, 70-ms test pulses to the indicated potentials were applied using the cut-open oocyte voltage clamp. Representative gating current traces are shown. (B) After test pulses to various potentials, OFF gating currents were recorded upon return to −90 mV. A single exponential component was fitted to the decaying phase of the OFF gating current to obtain a time constant, which was plotted versus test potential. Data are shown as mean ± SEM, n = 5. (C) Steady state activation curves of eag gating (Q-V, •) and ionic (Po-V, ○) currents. For the Q-V curve, OFF gating currents elicited by 70-ms pulses to the indicated test potentials were integrated to obtain QOFF, which was normalized to the maximal value obtained in each experiment. The Po-V curve was obtained from normalized isochronal tail currents. Data are shown as mean ± SEM, n = 6 and 8 for the Q-V and Po-V curves, respectively. (D) Po-V (left) and Q-V (right) curves obtained in the presence (•) or absence (○) of 2 mM Mg2+ are shown. Data are shown as mean ± SEM, n = 4–6. (E) Ionic or gating currents were elicited by 70-ms pulses to +50 mV, followed by repolarization to a variety of tail potentials. (Left) Time constants from single exponential fits of ionic current tails (○) and OFF gating currents (•) are plotted versus tail potential. Data are shown as mean ± SEM, n = 4 and 3 for ionic tail currents and OFF gating currents, respectively. (Right) A semilogarithmic plot of the same data is shown. The closing valence (Zc), determined from the slope of a linear regression fit of the data, was ∼0.37 for both ionic tail currents and OFF gating currents.
	Figure 7. Slow component of ON gating charge movement is enhanced by Mg2+. (A) From a holding potential of −90 mV, 70-ms test pulses from −30 to +50 mV were applied in 10-mV increments. Time constants, obtained from single exponential components of the decaying phase of ON gating currents recorded in the presence (•) or absence (○) of 2 mM Mg2+, are plotted versus test potential. These time constants describe a fast component of charge movement that is not significantly changed by Mg2+. Data are shown as mean ± SEM, n = 4. (B) From a holding potential of −90 mV, ON gating currents were evoked by test pulses of various durations between 2 and 100 ms to +20, 0, or −20 mV. After each pulse, OFF gating currents were evoked by a return to −90 mV. OFF gating currents, obtained in the presence (•) or absence (○) of 2 mM Mg2+, were integrated to obtain QOFF, which was normalized to the maximal value obtained at the same test potential in the presence or absence of Mg2+ and plotted versus test pulse duration. A slowly developing component of charge movement is more prominent in the presence of Mg2+, especially at smaller depolarizations. Data are shown as mean ± SEM, n = 4.
	Figure 6. Extracellular Mg2+ enhances the effect of prepulse hyperpolarization on eag gating currents. (A) The effect of hyperpolarizing prepulses on the gating current of eag in the absence (left) or presence (right) of 2 mM Mg2+. Gating currents were evoked using the same voltage protocol as in Fig. 1, except that leak and capacitative currents were compensated electronically at 0 mV. Representative traces are shown. After more negative prepulses, the time course of the decay phase of the ON gating current was slower. (B) After 75-ms prepulses to potentials between −80 and −180 mV, ON gating currents were evoked by a test pulse to +50 mV. The decay phase of the ON gating currents obtained in the presence (○) or absence (•) of 2 mM Mg2+ was fitted with a single exponential function and plotted versus prepulse potential. Data are shown as mean ± SEM, n = 3.
	Figure 8. Mg2+ does not significantly modulate gating current reactivation time course. Gating currents were recorded using the reactivation protocol shown in Fig. 4. The decay phase of ON gating currents evoked by the second test pulse in the presence (•) or absence (○) of 2 mM Mg2+ were fitted with single exponential functions. Fitted time constants are plotted versus Δt. Data are shown as mean ± SEM, n = 3.
	Figure 9. Δ333–337 activation kinetics are sensitive to Mg2+ but unaltered by prepulse hyperpolarization. (A, left) Ionic currents from the Δ333–337 mutant were evoked by pulsing from a holding potential of −80 mV to voltages from −60 to +80 mV in 20-mV increments. Representative traces, 150 ms in length, are shown. (Right) Currents evoked at +60 mV from Δ333–337 (solid trace) and wild-type eag (dashed trace) have been overlaid. (B) Prepulse hyperpolarization increases the delay but does not alter activation kinetics in Δ333–337 channels. (Left) From a holding potential of −80 mV, 150-ms hyperpolarizing prepulses ranging from −150 to −70 mV were applied in 10-mV increments, followed by a 120-ms test pulse to +50 mV. Representative currents evoked by the test pulses are shown. (Middle) Activation time constants at +50 mV, determined by fitting a single exponential function to the late rising phase, have been plotted as a function of prepulse potential. Data are shown as mean ± SEM, n = 6. (Right) To illustrate the effect of hyperpolarizing prepulses on the delay before current activation, the time to half maximal current amplitude at +50 mV has been plotted as a function of prepulse potential. Data are shown as mean ± SEM, n = 6. (C) Comparison of activation kinetics of Δ333–337 channels in the presence and absence of Mg2+. (Left) Representative currents from Δ333–337 channels, evoked by pulses to +60 or +20 mV, as indicated, in the presence (solid traces) or absence (dashed traces) of 2 mM Mg2+, have been overlaid. (Middle) Activation time constants at +50-mV pulse in the presence (•) or absence (○) of 2 mM Mg2+, determined by fitting a single exponential function to the late rising phase, have been plotted as a function of test potential. Data are shown as mean ± SEM, n = 8. (Right) The time to half maximal current amplitude at +50 mV in the presence (•) or absence (○) of 2 mM Mg2+ has been plotted as a function of prepulse potential. Data are shown as mean ± SEM, n = 8.
	Figure 10. Ionic current activation kinetics in the L342H mutant are insensitive to both Mg2+ and prepulse hyperpolarization. (A, left and middle) Effect of hyperpolarizing prepulses on L342H currents. The voltage protocol was the same as in Fig. 1. Representative currents evoked by the test pulses in the presence or absence of 2 mM Mg2+, as indicated, are shown. (Right) Activation time constants (top) and time to half maximal current (bottom) in the presence (•) or absence (○) of Mg2+ have been plotted as a function of prepulse potential. Data are shown as mean ± SEM, n = 6. (B, left and middle) Currents were evoked in the presence or absence of 2 mM Mg2+, as indicated, by pulses from a holding potential of −90 mV. The voltage protocol was the same as in Fig. 2. (Right) Activation time constants (top) and time to half maximal current (bottom) in the presence (•) or absence (○) of 2 mM Mg2+ have been plotted as a function of test potential. Data are shown as mean ± SEM, n = 6. (C) L342H channels were subjected to the reactivation protocol shown in Fig. 4. Fitted time constants for the late rising phase of currents evoked by the second test pulse were determined for short interpulse intervals (between 0.1 and 1 ms) in the presence (•) and absence (○) of 2 mM Mg2+ and plotted versus Δt. Data are shown as mean ± SEM, n = 5. (D) Fitted time constants for the second test pulse were determined for interpulse intervals between 0.1 and 10 ms in the presence (•) and absence (○) of 2 mM Mg2+ and plotted versus Δt. Data are shown as mean ± SEM, n = 5.
	Figure 11. Gating currents recorded from L342H channels are insensitive to Mg2+ and prepulse hyperpolarization. (A) L342H gating currents were evoked by test pulses to +50 ms after prepulses to hyperpolarized potentials between −180 and −90 mV in the presence or absence of Mg2+, as indicated. The voltage protocol was the same as in Fig. 6. (B) The decay phase of ON gating currents evoked at +50 mV was fitted with a single exponential function to derive a time constant, which was normalized to the value obtained after a prepulse to −180 mV, and plotted versus prepulse potential. wild-type eag (▿, ▾) and L342H (○, •) in the absence (○, ▿) or presence (•, ▾) of 2 mM Mg2+. Data are shown as mean ± SEM, n = 3.
	Figure 14. Computer simulation of the modulation of eag ionic and gating currents by prepulse hyperpolarization and extracellular Mg2+. (A) Simulated effect of prepulse hyperpolarization on ionic (left) and gating (right) currents. A prepulse of −90 mV (dashed line) or −200 mV (solid line) is applied before a test pulse to +50 mV. At the end of the test pulse, the membrane is repolarized to −90 mV. The reversal potential is set at −80 mV. An enlarged view of ON gating currents is provided (right inset) to show the effect of the prepulse on the decay kinetics. (B) Simulated ionic (left) and gating (right) currents in the presence (solid line) and absence (dashed line) of 2 mM Mg2+. From a holding potential of −90 mV, a test to pulse to +50 mV is applied, followed by repolarization to −90 mV. The reversal potential is set at −80 mV. An enlarged view of ON gating currents is provided (right inset) to show the effect of Mg2+ on the decay kinetics. (C) The predicted steady state Po-V curve in the presence (•) or absence (○) of 2 mM Mg2+ is shown.
	Figure 12. In L342H, the slow component of ON gating charge movement is not significantly modulated by Mg2+. The voltage protocol was the same as in Fig. 7 B. OFF gating currents, recorded in the presence (•) or absence (○) of 2 mM Mg2+ at the indicated test potentials, were integrated to obtain QOFF, which was normalized to the maximal value obtained at the same test potential in the presence or absence of Mg2+ and plotted versus test-pulse duration. Data are shown as mean ± SEM, n = 3.
	Figure 13. A qualitative, sequential model for activation gating in eag channels. The model is modified from the class D model proposed by Zagotta et al. 1994b for the Shaker K+ channel. Values for rate constants (s−1; top number) and valences (bottom number) used in the simulation in the absence of Mg2+ are shown above the arrow for forward transitions and below the arrow for reverse transitions. Values for those parameters changed in the presence of 2 mM Mg2+ are shown in bold type in the brackets. The simulation employed a total gating charge of 10.5 e0 per channel, a reasonable value compared to the charge per channel (∼12 e0) estimated experimentally for Shaker K+ channels and skeletal muscle Na+ channels (Schoppa et al. 1992; Hirschberg et al. 1995).

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