Table 2 Mutant-cycle analysis reveals conformational changes during common gating.

	n	V _1/2 (mV)	z (e⁰)	Δ G_{V= 0} (kJ mol⁻¹)	ΔΔ G_interact (kJ mol⁻¹)
Common gating
WT ClC-1	4	−51.6±1.7	1.05±0.04	−5.22±0.31	—
R300Q	3	−72.7±2.2	0.81±0.03	−5.89±0.22	—
R304Q	4	−64.6±1.2	1.28±0.03	−7.97±0.37	—
R300Q R304Q	4	−71.6±1.4	0.95±0.04	−6.60±0.23	+2.04±0.58^(0.0056)
D265N	3	−22.2±0.8	1.03±0.04	−2.19±0.10
D265N R300Q	4	−49.0±0.8	1.15±0.02	−5.42±0.23	−2.56±0.46^(0.0003)′ (−2.71±0.54^(0.0005))^*
D265N R304Q	4	−66.9±0.9	1.08±0.04	−6.99±0.25	−2.05±0.55^(0.0040) (−2.20±0.44^(0.0005))^†
D265N R300Q R304Q	4	−80.2±1.2	1.08±0.02	−8.33±0.19	−4.75±0.44^(<0.0001)
Protopore gating
WT ClC-1	4	−81.6±1.2	1.26±0.04	−9.92±0.44	—
R300Q	3	−103.8±1.4	1.13±0.02	−11.32±0.15	—
R304Q	4	−83.9±1.2	1.19±0.09	−9.71±0.66	—
R300Q R304Q	4	−96.8±1.9	1.11±0.05	−10.39±0.28	+0.72±0.85
D265N	3	−67.9±1.5	1.05±0.06	−6.93±0.37
D265N R300Q	4	−82.3±1.5	1.21±0.02	−9.63±0.28	−1.30±0.48^(0.0241) (−0.72±0.94)^*
D265N R304Q	4	−93.8±2.0	1.19±0.04	−10.77±0.28	−4.05±0.92^(0.0011) (−2.03±0.69^(0.0147))^†
D265N R300Q R304Q	4	−97.5±1.6	1.13±0.05	−10.73±0.55	−3.33±0.84^(0.0027)

Data show the parameters for fits of equation (1) to the experimental data points derived from n separate experiments. Apparent gating valence (z) was calculated using equation (3), the zero-voltage free energy of gating (ΔG_V=0) was calculated using equation (2). Data are means and s.e.m. Values for the apparent interaction energy between charged residues (ΔΔG_interact) were calculated using equation (4), and are means and linearly propagated values of s.e.m. Bracketed values for ΔΔG_interact were calculated (*) for the R304–D265 interaction on the R300Q background and (†) for the R300–D265 interaction on the R304Q background. P-values comparing the calculated interaction energies against zero, using single value t-tests, are shown in superscript in the far right column. Graphical representations of P_o data and fits of equation (1) are available in Supplementary Fig. S5.

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