TABLE II.

Thermodynamic Analysis of Kv4.2 Low-Voltage Inactivation

 τL,-65 (s) f-65 kon,-65 (s−1) koff,-65 (s−1) Kci,−65 n Ω (kT) 
wt 1.28 ± 0.11 0.458 ± 0.032 0.448 ± 0.051 0.368 ± 0.030 0.898 ± 0.130 8  
G309A 0.60 ± 0.05 0.125 ± 0. 012 1.497 ± 0.129 0.208 ± 0.012 0.144 ± 0.016 5  
R311A 0.66 ± 0.26 0.922 ± 0.005 0.193 ± 0.077 2.260 ± 0.885 11.985 ± 0.821 5  
I312A 48.2 ± 8.47 0.428 ± 0.025 0.015 ± 0.005 0.011 ± 0.003 0.763 ± 0.085 5  
L313A 0.17 ± 0.03 0.167 ± 0.009 5.365 ± 0.671 1.085 ± 0.164 0.201 ± 0.013 5  
Y315A 1.10 ± 0.02 0.620 ± 0.032 0.348 ± 0.031 0.566 ± 0.028 1.713 ± 0.248 5  
T316A 0.41 ± 0.01 0.272 ± 0.007 1.791 ± 0.073 0.668 ± 0.015 0.375 ± 0.013 5  
L317A 0.36 ± 0.01 0.305 ± 0.025 1.971 ± 0.133 0.852 ± 0.048 0.446 ± 0.055 5  
K318A 0.91 ± 0.05 0.406 ± 0.017 0.657 ± 0.033 0.450 ± 0.034 0.687 ± 0.048 5  
C320A 1.10 ± 0.07 0.510 ± 0.049 0.452 ± 0.049 0.481 ± 0.075 1.150 ± 0.277 5  
C320S 5.13 ± 0.46 0.425 ± 0.066 0.122 ± 0.024 0.082 ± 0.009 0.859 ± 0.212 6  
A321V 3.50 ± 0.14 0.388 ± 0.039 0.174 ± 0.001 0.113 ± 0.015 0.661 ± 0.110 5  
S322A 1.32 ± 0.07 0.750 ± 0.039 0.187 ± 0.021 0.579 ± 0.063 3.401 ± 0.874 5  
E323A 0.16 ± 0.01 0.124 ± 0.007 5.478 ± 0.162 0.778 ± 0.053 0.142 ± 0.010 6  
G325A 1.26 ± 0.09 0.505 ± 0.028 0.404 ± 0.042 0.407 ± 0.028 1.047 ± 0.117 5  
F326A 0.68 ± 0.13 0.938 ± 0.010 0.100 ± 0.018 1.622 ± 0.440 15.940 ± 2.097 5  
L400A 0.39 ± 0.04 0.141 ± 0.016 2.341 ± 0.244 0.377 ± 0.049 0.167 ± 0.021 6  
V402A 0.56 ± 0.02 0.630 ± 0.027 0.664 ± 0.037 1.148 ± 0.088 1.768 ± 0.219 5  
V404A 0.14 ± 0.01 0.051 ± 0.010 6.783 ± 0.358 0.358 ± 0.055 0.054 ± 0.011 6  
V402I:V404I 17.42 ± 0.46 0.878 ± 0.008 0.007 ± 0.000 0.051 ± 0.002 7.384 ± 0.608 6  
S407A 1.74 ± 0.25 0.738 ± 0.031 0.165 ± 0.026 0.451 ± 0.044 3.079 ± 0.469 7  
N408A 0.72 ± 0.09 0.851 ± 0.037 0.215 ± 0.042 1.381 ± 0.303 7.759 ± 1.900 7  
S410A 0.72 ± 0.10 0.512 ± 0.033 0.735 ± 0.112 0.768 ± 0.124 1.092 ± 0.158 5  
R411A 0.73 ± 0.05 0.552 ± 0.019 0.629 ± 0.077 0.766 ± 0.036 1.240 ± 0.092 3  
I412A 0.60 ± 0.05 0.089 ± 0.006 1.579 ± 0.124 0.154 ± 0.016 0.097 ± 0.007 8  
Y413A 1.22 ± 0.20 0.561 ± 0.022 0.405 ± 0.098 0.529 ± 0.114 1.343 ± 0.132 8  
E323A:V404A 0.17 ± 0.01 0.068 ± 0.020 5.552 ± 0.351 0.402 ± 0.117 0.075 ± 0.024 5 8.74 ± 3.58a 
E323A:S407A 0.46 ± 0.03 0.360 ± 0.037 1.433 ± 0.170 0.784 ± 0.064 0.583 ± 0.090 5 1.19 ± 0.32 
E323A:N408A 0.37 ± 0.03 0.222 ± 0.030 2.178 ± 0.262 0.591 ± 0.049 0.293 ± 0.049 5 4.19 ± 0.08 
E323A:I412A 0.24 ± 0.01 0.056 ± 0.005 4.102 ± 0.163 0.225 ± 0.018 0.060 ± 0.006 5 3.81 ± 0.77 
S322A:V404A 0.21 ± 0.01 0.022 ± 0.002 4.700 ± 0.308 0.105 ± 0.013 0.022 ± 0.002 5 9.27 ± 0.04 
S322A:S407A 3.44 ± 0.21 0.918 ± 0.007 0.024 ± 0.003 0.271 ± 0.016 11.447 ± 0.896 5 1.02 ± 0.34 
S322A:N408A 1.54 ± 0.23 0.897 ± 0.022 0.077 ± 0.022 0.623 ± 0.070 10.611 ± 2.279 5 2.77 ± 0.16 
S322A:I412A 1.57 ± 0.28 0.434 ± 0.034 0.421 ± 0.093 0.296 ± 0.028 0.791 ± 0.104 5 2.15 ± 0.71 
 τL,-65 (s) f-65 kon,-65 (s−1) koff,-65 (s−1) Kci,−65 n Ω (kT) 
wt 1.28 ± 0.11 0.458 ± 0.032 0.448 ± 0.051 0.368 ± 0.030 0.898 ± 0.130 8  
G309A 0.60 ± 0.05 0.125 ± 0. 012 1.497 ± 0.129 0.208 ± 0.012 0.144 ± 0.016 5  
R311A 0.66 ± 0.26 0.922 ± 0.005 0.193 ± 0.077 2.260 ± 0.885 11.985 ± 0.821 5  
I312A 48.2 ± 8.47 0.428 ± 0.025 0.015 ± 0.005 0.011 ± 0.003 0.763 ± 0.085 5  
L313A 0.17 ± 0.03 0.167 ± 0.009 5.365 ± 0.671 1.085 ± 0.164 0.201 ± 0.013 5  
Y315A 1.10 ± 0.02 0.620 ± 0.032 0.348 ± 0.031 0.566 ± 0.028 1.713 ± 0.248 5  
T316A 0.41 ± 0.01 0.272 ± 0.007 1.791 ± 0.073 0.668 ± 0.015 0.375 ± 0.013 5  
L317A 0.36 ± 0.01 0.305 ± 0.025 1.971 ± 0.133 0.852 ± 0.048 0.446 ± 0.055 5  
K318A 0.91 ± 0.05 0.406 ± 0.017 0.657 ± 0.033 0.450 ± 0.034 0.687 ± 0.048 5  
C320A 1.10 ± 0.07 0.510 ± 0.049 0.452 ± 0.049 0.481 ± 0.075 1.150 ± 0.277 5  
C320S 5.13 ± 0.46 0.425 ± 0.066 0.122 ± 0.024 0.082 ± 0.009 0.859 ± 0.212 6  
A321V 3.50 ± 0.14 0.388 ± 0.039 0.174 ± 0.001 0.113 ± 0.015 0.661 ± 0.110 5  
S322A 1.32 ± 0.07 0.750 ± 0.039 0.187 ± 0.021 0.579 ± 0.063 3.401 ± 0.874 5  
E323A 0.16 ± 0.01 0.124 ± 0.007 5.478 ± 0.162 0.778 ± 0.053 0.142 ± 0.010 6  
G325A 1.26 ± 0.09 0.505 ± 0.028 0.404 ± 0.042 0.407 ± 0.028 1.047 ± 0.117 5  
F326A 0.68 ± 0.13 0.938 ± 0.010 0.100 ± 0.018 1.622 ± 0.440 15.940 ± 2.097 5  
L400A 0.39 ± 0.04 0.141 ± 0.016 2.341 ± 0.244 0.377 ± 0.049 0.167 ± 0.021 6  
V402A 0.56 ± 0.02 0.630 ± 0.027 0.664 ± 0.037 1.148 ± 0.088 1.768 ± 0.219 5  
V404A 0.14 ± 0.01 0.051 ± 0.010 6.783 ± 0.358 0.358 ± 0.055 0.054 ± 0.011 6  
V402I:V404I 17.42 ± 0.46 0.878 ± 0.008 0.007 ± 0.000 0.051 ± 0.002 7.384 ± 0.608 6  
S407A 1.74 ± 0.25 0.738 ± 0.031 0.165 ± 0.026 0.451 ± 0.044 3.079 ± 0.469 7  
N408A 0.72 ± 0.09 0.851 ± 0.037 0.215 ± 0.042 1.381 ± 0.303 7.759 ± 1.900 7  
S410A 0.72 ± 0.10 0.512 ± 0.033 0.735 ± 0.112 0.768 ± 0.124 1.092 ± 0.158 5  
R411A 0.73 ± 0.05 0.552 ± 0.019 0.629 ± 0.077 0.766 ± 0.036 1.240 ± 0.092 3  
I412A 0.60 ± 0.05 0.089 ± 0.006 1.579 ± 0.124 0.154 ± 0.016 0.097 ± 0.007 8  
Y413A 1.22 ± 0.20 0.561 ± 0.022 0.405 ± 0.098 0.529 ± 0.114 1.343 ± 0.132 8  
E323A:V404A 0.17 ± 0.01 0.068 ± 0.020 5.552 ± 0.351 0.402 ± 0.117 0.075 ± 0.024 5 8.74 ± 3.58a 
E323A:S407A 0.46 ± 0.03 0.360 ± 0.037 1.433 ± 0.170 0.784 ± 0.064 0.583 ± 0.090 5 1.19 ± 0.32 
E323A:N408A 0.37 ± 0.03 0.222 ± 0.030 2.178 ± 0.262 0.591 ± 0.049 0.293 ± 0.049 5 4.19 ± 0.08 
E323A:I412A 0.24 ± 0.01 0.056 ± 0.005 4.102 ± 0.163 0.225 ± 0.018 0.060 ± 0.006 5 3.81 ± 0.77 
S322A:V404A 0.21 ± 0.01 0.022 ± 0.002 4.700 ± 0.308 0.105 ± 0.013 0.022 ± 0.002 5 9.27 ± 0.04 
S322A:S407A 3.44 ± 0.21 0.918 ± 0.007 0.024 ± 0.003 0.271 ± 0.016 11.447 ± 0.896 5 1.02 ± 0.34 
S322A:N408A 1.54 ± 0.23 0.897 ± 0.022 0.077 ± 0.022 0.623 ± 0.070 10.611 ± 2.279 5 2.77 ± 0.16 
S322A:I412A 1.57 ± 0.28 0.434 ± 0.034 0.421 ± 0.093 0.296 ± 0.028 0.791 ± 0.104 5 2.15 ± 0.71 

From the time constant of onset of low-voltage inactivation at −65 mV (τL,-65) and the fraction of non-inactivated channels, (f-65) on-rates (kon,-65) and off-rates (koff,-65) were calculated for wild-type (wt) and mutant channels to define their apparent affinity (Kci,-65) for the closed-inactivated state. With double mutant cycle analysis based on these affinity values, coupling coefficients (Ω) were determined for pairs of amino acids.

a

SEM values based on linear and independent error propagation.

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