Silverman WR , Tang CY , Mock AF , Huh KB , Papazian DM .

GM08496 NIGMS NIH HHS , GM43459 NIGMS NIH HHS , R01 GM043459-10 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 , T32 GM008496 NIGMS NIH HHS

	Figure 1. Acidic amino acids within the voltage sensor of eag. (A) A model is shown for the membrane topology of the Drosophila eag K+ channel subunit containing six transmembrane segments (S1–S6) and the P-region. The approximate locations of acidic amino acids within the second and third transmembrane segments are indicated. Italics denote eag-specific acidic residues. (B) Sequence alignment of S2 and S3 segments from voltage-dependent K+ channels, with acidic residues shown in bold. Segments S2 and S3 can be aligned unambiguously due to the presence of highly conserved residues, including three acidic residues (Chandy and Gutman 1995). Members of the eag subfamily differ from other voltage-dependent K+ channels due to the presence of additional acidic amino acids in S2 and S3 (Warmke and Ganetzky 1994). Sequences shown are from eag family members and representative members of Kv1-Kv4 families (Chandy and Gutman 1995; Shi et al. 1997; Ganetzky et al. 1999). Arrows indicate acidic residues that were mutated in this study. Numbering corresponds to the Drosophila eag sequence. Abbreviations used are: d, Drosophila; r, rat; b, bovine; h, human; m, mouse; erg, eag related gene; elk, eag-like K+ channel gene; Sh, Shaker.
	Figure 2. Mg2+ slows activation of wild-type eag. (A) Activation time constants (τ) obtained in the presence (•) or absence (○) of 10 mM Mg2+ have been plotted versus test pulse potential. Currents were evoked by depolarizing from a holding potential of −90 mV to the indicated voltages. A single exponential component was fitted to the late rising phase of ionic currents to derive τ values. Data are shown as mean ± SEM, n = 5. In this and subsequent figures, if error bars are not visible, the SEM was smaller than the size of the symbols. At 0 mV, τ = 100 ± 16.4 ms in the presence of 10 mM Mg2+, and 11.1 ± 0.9 ms in the absence of Mg2+. At +100 mV, τ = 10.0 ± 0.7 ms in the presence of 10 mM Mg2+, and 2.3 ± 0.06 ms in the absence of Mg2+. The data were fitted with single exponential functions (solid curves) to estimate values for τlim at infinite positive voltage (see text). (Inset) Fits with single exponential functions (bold curves) are shown superimposed on current traces evoked by depolarizing to +60 mV in the presence (dashed line) or absence (solid line) of 10 mM Mg2+. Bars: 2 μA and 25 ms. (B) The time to half maximal current amplitude (t1/2) at +60 mV was measured in the presence (•) or absence (○) of 10 mM Mg2+ and plotted versus test potential. Values are shown as mean ± SEM, n = 5. At 0 mV, t1/2 = 100 ± 3.0 ms in the presence of 10 mM Mg2+, and 11.6 ± 0.6 ms in the absence of Mg2+. At +100 mV, t1/2 = 15.8 ± 0.5 ms in the presence of 10 mM Mg2+, and 4.3 ± 0.2 ms in the absence of Mg2+. The data were fitted with single exponential functions (solid curves) to estimate values for t1/2lim at infinite positive voltage (see text).
	Figure 3. Hyperpolarizing prepulses decelerate activation in wild-type eag. (A) From a holding potential of −90 mV, 50 ms prepulses to voltages ranging from −170 to −60 mV were applied in 10 mV increments, followed by a test pulse to +60 mV. Representative currents evoked in the absence (left) or presence (right) of 10 mM Mg2+ are shown. (B, left) Values of τ were obtained from single exponential fits to the late rising phase of currents evoked at +60 mV in the presence (•) or absence (○) of 10 mM Mg2+ and plotted versus prepulse potential. Data are shown as mean ± SEM, n = 5. (B, right) Values of t1/2 at +60 mV were measured in the presence (•) or absence (○) of 10 mM Mg2+ and plotted versus prepulse potential. Values are shown as mean ± SEM, n = 5.
	Figure 4. D278 mutants are insensitive to extracellular Mg2+. (A) Representative current traces for D278V (left) and D278E (right) are shown. From a holding potential of −80 mV, test pulses to +60 mV were applied in the absence (solid lines) or presence (dashed lines) of 10 mM Mg2+. Single exponential fits to the late rising phase are shown superimposed in bold on ionic current traces. (B) Values of t1/2 at +60 mV for D278V (▪), D278E (▴), and wild type (•) were measured in various concentrations of Mg2+ up to 20 mM, expressed as fold change in t1/2, and plotted as a function of Mg2+ concentration. Values are shown as mean ± SEM, n = 3–4. (C) Values of τ (left) and t1/2 (right) for D278V (▪, □), D278E (▴, ▵), and wild type (•, ○) were measured as a function of prepulse potential in the presence (filled symbols) or absence (open symbols) of 10 mM Mg2+. For the mutants, currents were evoked by a test pulse to +60 mV after 250 ms hyperpolarizing prepulses to the indicated potentials. Data for wild-type eag are the same as in Fig. 3 B. Values are shown as mean ± SEM, n = 3–5. (D) The probability of opening as a function of voltage for wild type (○), D278V (□), and D278E (▵) channels was determined from isochronal tail currents in the absence of Mg2+. Values are shown as mean ± SEM, n = 4–5. Po–V curves did not differ significantly in the presence of 10 mM Mg2+ (data not shown).
	Figure 5. Mutations at D327 dramatically reduce sensitivity to Mg2+. (A) Representative current traces for D327A (left) and D327F (right) are shown. From a holding potential of −80 mV, test pulses to +60 mV were applied in the absence (solid lines) or presence (dashed lines) of 10 mM Mg2+. Single exponential fits to the late rising phase are shown superimposed in bold on D327F ionic current traces. (B) Values of t1/2 at +60 mV for D327A (▪), D327F (▴), and wild type (•) were measured in various concentrations of Mg2+ up to 20 mM, expressed as fold change in t1/2, and plotted as a function of Mg2+ concentration. Values are shown as mean ± SEM, n = 3–4. (C) Values of τ (left) for D327F (▴, ▵) and wild type (•, ○), and values of t1/2 (right) for D327A (▪, □), D327F (▴, ▵), and wild type (•, ○) were measured as a function of prepulse potential in the presence (filled symbols) or absence (open symbols) of 10 mM Mg2+. For the mutants, currents were evoked by a test pulse to +60 mV after 250 ms hyperpolarizing prepulses to the indicated potentials. Data for wild-type eag are the same as in Fig. 3 B. Values are shown as mean ± SEM, n = 3–5. For D327F, asterisks indicate τ and t1/2 values that differed significantly in the presence and absence of Mg2+ (*, P < 0.05, ANOVA). (D) The probability of opening as a function of voltage for wild type (○), D327A (□), and D327F (▵) channels was determined from isochronal tail currents in the absence of Mg2+. Values are shown as mean ± SEM, n = 3–5. Po–V curves did not differ significantly in the presence of 10 mM Mg2+ (data not shown).
	Figure 6. D274 mutants retain Mg2+ sensitivity. (A) Representative current traces for D274A (left) and D274E (right) are shown. From a holding potential of −80 mV, test pulses to +60 mV were applied in the absence (solid lines) or presence (dashed lines) of 10 mM Mg2+. (B) Values of t1/2 at +60 mV for D274A (▪), D274E (▴), and wild type (•) were measured in various concentrations of Mg2+ up to 20 mM, expressed as fold change in t1/2, and plotted as a function of Mg2+ concentration. Values are shown as mean ± SEM, n = 3–5. Half maximal Mg2+ concentrations, estimated by fitting rectangular hyperbolae (solid curves) to the data, were 3.5 mM for wild type and 1.2 mM for D274E. (C) Activation kinetics recover from Mg2+ more slowly in D274E channels than in wild-type eag. Cells expressing D274E and wild-type channels were subjected to continuous perfusion and held at −90 mV, with 400 ms test pulses to +60 mV applied at 4.5 s intervals during wash in for both wild type and D274E, and at 4.5 or 9.5 s intervals for wild type and D274E, respectively, during recovery. After establishing a baseline in the absence of Mg2+, cells were perfused with extracellular solution containing 20 mM Mg2+ beginning at t = 0, followed by perfusion of the Mg2+-free solution, beginning at the time indicated by the arrow. Activation kinetics were quantified by measuring the time interval between 10 and 90% of maximal current amplitude. This value was normalized to the longest interval (i.e., slowest kinetics), and plotted as a function of perfusion time. Values for wild type (•) and D274E (▴) are shown as mean ± SEM, n = 5.
	Figure 8. D319N is insensitive to extracellular Mg2+. (A) Representative current traces for D319N are shown. From a holding potential of −80 mV, test pulses to +60 mV were applied in the absence (solid lines) or presence (dashed lines) of 10 mM Mg2+. Single exponential fits to the late rising phase are shown superimposed in bold on ionic current traces. (B) Values of τ (left) and t1/2 (right) for D319N (▪, □) and wild type (•, ○) were measured as a function of prepulse potential in the presence (▪, •) or absence (○, □) of 10 mM Mg2+. For D319N, currents were evoked by a test pulse to +60 mV after 250 ms hyperpolarizing prepulses to the indicated potentials. Data for wild-type eag are the same as in Fig. 3 B. Values are shown as mean ± SEM, n = 3–5. (C) The probability of opening as a function of voltage for wild type (○) and D319N (□) was determined from isochronal tail currents in the absence of Mg2+. Values are shown as mean ± SEM, n = 3–5. Po–V curves did not differ significantly in the presence of 10 mM Mg2+ (data not shown).
	Figure 7. Hyperpolarizing prepulses reveal Mg2+-sensitive transitions in D284 mutants. (A) Representative traces for D284N (left) and D284A (right) are shown. From a holding potential of −80 mV and in the absence (solid lines) or presence (dashed lines) of 10 mM Mg2+, 250 ms prepulses to −150 mV were applied, followed by test pulses to +60 mV. Single exponential fits to the late rising phase are shown superimposed in bold on ionic current traces. (B) Values of τ (left) and t1/2 (right) for D284N were measured as a function of prepulse potential in the presence (filled squares) or absence (open squares) of 10 mM Mg2+. Currents were evoked by a test pulse to +60 mV after 250 ms hyperpolarizing prepulses to the indicated potentials. Values are shown as mean ± SEM, n = 5. For D284N, asterisks indicate τ and t1/2 values that differed significantly in the presence and absence of Mg2+ (*, P < 0.05; **, P < 0.01; ANOVA). (C) Values of τ (left) and t1/2 (right) for D284A were measured as a function of prepulse potential in the presence (▴) or absence (▵) of 10 mM Mg2+. Currents were evoked by a test pulse to +60 mV after 250 ms hyperpolarizing prepulses to the indicated potentials. Values are shown as mean ± SEM, n = 5. (D) The probability of opening as a function of voltage for wild type (○), D284N (□), and D284A (▵) channels was determined from isochronal tail currents in the absence of Mg2+. Values are shown as mean ± SEM, n = 3–4. Po–V curves did not differ significantly in the presence of 10 mM Mg2+ (data not shown).
	Figure 9. Model for the packing arrangement of transmembrane segments S2 (blue), S3 (red), and S4 (green) in the voltage sensor of K+ channels. The model contains structural constraints (dotted lines) inferred from characterization of the Mg2+-binding site in eag (this study) and from second site suppressor analysis of Shaker (Papazian et al. 1995; Tiwari-Woodruff et al. 1997). The transmembrane segments are shown in α-helical conformation with fully extended side chains. A yellow circle denotes a bound Mg2+ ion in eag. Side chains of pertinent residues have been labeled according to Shaker (white) and, in some cases, eag (yellow) numbering. Side chains of other residues are not shown. According to analysis of Shaker channels, the pictured conformation would represent the activated conformation of the voltage sensor (Tiwari-Woodruff et al. 2000). The model was generated using the programs InsightII and WebLab Viewer Lite (Molecular Simulations).

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