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TABLE I

Voltage-dependent Gating and Rectification of Cysteine Mutants

Vo
z
Pmin
Rectification
n
WTa  −99 ± 0.8 0.90 ± 0.02 0.076 ± 0.005 1.00 ± 0.004 68 
Q114C  −95 ± 2.2 0.87 ± 0.03 0.063 ± 0.013 0.99 ± 0.007 16 
S117C  −98 ± 3.9 0.93 ± 0.02 0.073 ± 0.013 0.98 ± 0.011 6 
P118C  −93 ± 2.5 0.91 ± 0.01 0.056 ± 0.012 0.98 ± 0.012 7 
Q119C  −106 ± 1.7 0.86 ± 0.02 0.048 ± 0.037 0.98 ± 0.015 3 
A120C  −86 ± 1.9 0.94 ± 0.02 0.041 ± 0.007 0.98 ± 0.009 13 
V121C  −91 ± 1.3 0.91 ± 0.02 0.081 ± 0.013 0.99 ± 0.017 13 
G122C  −94 ± 1.6 0.83 ± 0.02 0.044 ± 0.011 0.85 ± 0.008 17 
S123Cb ND ND ND ND 6 
P126C  −80 ± 2.2 0.73 ± 0.02 0.035 ± 0.011 0.84 ± 0.020 12 
L128C  −94 ± 1.5 0.94 ± 0.11 0.122 ± 0.046 1.12 ± 0.006 4 
V136C  −97 ± 2.0 0.90 ± 0.02 0.069 ± 0.017 0.98 ± 0.012 8 
H138C  −90 ± 2.5 0.93 ± 0.04 0.058 ± 0.013 1.02 ± 0.010 9 
T142C  −90 ± 2.3 0.83 ± 0.04 0.052 ± 0.015 0.98 ± 0.007 5 
R144C  −91 ± 1.1 0.89 ± 0.02 0.058 ± 0.008 1.01 ± 0.007 10 
K149Cb  −28 ± 0.8 0.98 ± 0.01 0.042 ± 0.008 0.87 ± 0.007 19 
H171C  −101 ± 1.5 0.86 ± 0.03 0.069 ± 0.016 1.02 ± 0.009 12 
N304C  −91 ± 3.4 0.80 ± 0.03 0.051 ± 0.025 0.99 ± 0.010 3 
R305C  −92 ± 1.9 0.86 ± 0.05 0.055 ± 0.027 0.98 ± 0.024 3 
Q306C  −95 ± 1.6 0.94 ± 0.04 0.067 ± 0.022 0.98 ± 0.005 5 
I307C  −94 ± 1.8 0.88 ± 0.03 0.045 ± 0.017 0.98 ± 0.008 13 
I308C  −94 ± 1.0 0.95 ± 0.01 0.091 ± 0.006 1.00 ± 0.005 60 
V309C  −90 ± 1.3 0.93 ± 0.03 0.066 ± 0.011 1.01 ± 0.009 12 
F310C  −95 ± 1.2 0.89 ± 0.03 0.107 ± 0.030 0.98 ± 0.017 8 
M311C  −92 ± 0.9 0.94 ± 0.02 0.085 ± 0.017 0.99 ± 0.005 19 
R312C  −95 ± 1.5 0.89 ± 0.01 0.072 ± 0.006 0.93 ± 0.009 26 
K313C  −92 ± 1.0 0.95 ± 0.02 0.076 ± 0.011 1.01 ± 0.005 12 
K314C  −94 ± 0.7 0.91 ± 0.07 0.044 ± 0.023 1.02 ± 0.021 3 
N315C  −85 ± 2.9 0.87 ± 0.02 0.049 ± 0.006 0.98 ± 0.011 8 
F316C  −89 ± 1.0 0.94 ± 0.02 0.063 ± 0.008 0.98 ± 0.010 10 
V409C  −90 ± 0.7 0.93 ± 0.03 0.096 ± 0.011 0.96 ± 0.008 7 
T410C  −96 ± 0.9 0.75 ± 0.05 0.067 ± 0.019 0.88 ± 0.018 5 
P412C  −86 ± 1.4 0.83 ± 0.03 0.043 ± 0.012 0.90 ± 0.023 9 
Y512C  −98 ± 2.1 0.85 ± 0.02 0.000 ± 0.000 0.89 ± 0.007 12 
D513C  −92 ± 1.2 0.71 ± 0.01 0.032 ± 0.009 1.00 ± 0.010 35 
S514C  −98 ± 3.1 0.83 ± 0.02 0.064 ± 0.009 1.00 ± 0.010 10 
I515C  −87 ± 0.8 0.97 ± 0.01 0.029 ± 0.003 0.95 ± 0.006 93 
I516C  −101 ± 2.3 0.88 ± 0.05 0.134 ± 0.020 0.91 ± 0.017 13 
Q517C  −92 ± 1.1 0.94 ± 0.01 0.063 ± 0.008 1.02 ± 0.010 8 
I518C  −91 ± 2.0 0.97 ± 0.03 0.072 ± 0.009 1.02 ± 0.005 10 
K519Cb  −114 ± 1.9 0.65 ± 0.01 0.241 ± 0.017 0.61 ± 0.006 56 
K520C  −104 ± 1.5 0.84 ± 0.05 0.077 ± 0.039 1.00 ± 0.019 5 
L521Cc      
P522C  −94 ± 1.6 0.83 ± 0.02 0.115 ± 0.014 0.98 ± 0.005 26 
Y523C  −95 ± 1.7 0.92 ± 0.02 0.124 ± 0.012 0.97 ± 0.006 24 
L524C  −98 ± 1.4 0.88 ± 0.02 0.058 ± 0.009 0.97 ± 0.004 18 
P525C  −98 ± 1.1 0.90 ± 0.03 0.110 ± 0.010 0.97 ± 0.007 6 
E526C  −104 ± 1.5 0.84 ± 0.03 0.050 ± 0.021 0.98 ± 0.011 3 
L527C  −107 ± 2.0 0.87 ± 0.01 0.057 ± 0.017 1.01 ± 0.008 3 
S528C  −107 ± 2.1 0.88 ± 0.04 0.045 ± 0.021 1.01 ± 0.011 6 
W529C  −96 ± 1.7 0.85 ± 0.04 0.047 ± 0.019 0.98 ± 0.009 4 
S530C  −105 ± 2.2 0.85 ± 0.05 0.044 ± 0.028 1.00 ± 0.006 3 
S531C  −106 ± 1.7 0.92 ± 0.01 0.074 ± 0.006 1.00 ± 0.001 2 
Vo
z
Pmin
Rectification
n
WTa  −99 ± 0.8 0.90 ± 0.02 0.076 ± 0.005 1.00 ± 0.004 68 
Q114C  −95 ± 2.2 0.87 ± 0.03 0.063 ± 0.013 0.99 ± 0.007 16 
S117C  −98 ± 3.9 0.93 ± 0.02 0.073 ± 0.013 0.98 ± 0.011 6 
P118C  −93 ± 2.5 0.91 ± 0.01 0.056 ± 0.012 0.98 ± 0.012 7 
Q119C  −106 ± 1.7 0.86 ± 0.02 0.048 ± 0.037 0.98 ± 0.015 3 
A120C  −86 ± 1.9 0.94 ± 0.02 0.041 ± 0.007 0.98 ± 0.009 13 
V121C  −91 ± 1.3 0.91 ± 0.02 0.081 ± 0.013 0.99 ± 0.017 13 
G122C  −94 ± 1.6 0.83 ± 0.02 0.044 ± 0.011 0.85 ± 0.008 17 
S123Cb ND ND ND ND 6 
P126C  −80 ± 2.2 0.73 ± 0.02 0.035 ± 0.011 0.84 ± 0.020 12 
L128C  −94 ± 1.5 0.94 ± 0.11 0.122 ± 0.046 1.12 ± 0.006 4 
V136C  −97 ± 2.0 0.90 ± 0.02 0.069 ± 0.017 0.98 ± 0.012 8 
H138C  −90 ± 2.5 0.93 ± 0.04 0.058 ± 0.013 1.02 ± 0.010 9 
T142C  −90 ± 2.3 0.83 ± 0.04 0.052 ± 0.015 0.98 ± 0.007 5 
R144C  −91 ± 1.1 0.89 ± 0.02 0.058 ± 0.008 1.01 ± 0.007 10 
K149Cb  −28 ± 0.8 0.98 ± 0.01 0.042 ± 0.008 0.87 ± 0.007 19 
H171C  −101 ± 1.5 0.86 ± 0.03 0.069 ± 0.016 1.02 ± 0.009 12 
N304C  −91 ± 3.4 0.80 ± 0.03 0.051 ± 0.025 0.99 ± 0.010 3 
R305C  −92 ± 1.9 0.86 ± 0.05 0.055 ± 0.027 0.98 ± 0.024 3 
Q306C  −95 ± 1.6 0.94 ± 0.04 0.067 ± 0.022 0.98 ± 0.005 5 
I307C  −94 ± 1.8 0.88 ± 0.03 0.045 ± 0.017 0.98 ± 0.008 13 
I308C  −94 ± 1.0 0.95 ± 0.01 0.091 ± 0.006 1.00 ± 0.005 60 
V309C  −90 ± 1.3 0.93 ± 0.03 0.066 ± 0.011 1.01 ± 0.009 12 
F310C  −95 ± 1.2 0.89 ± 0.03 0.107 ± 0.030 0.98 ± 0.017 8 
M311C  −92 ± 0.9 0.94 ± 0.02 0.085 ± 0.017 0.99 ± 0.005 19 
R312C  −95 ± 1.5 0.89 ± 0.01 0.072 ± 0.006 0.93 ± 0.009 26 
K313C  −92 ± 1.0 0.95 ± 0.02 0.076 ± 0.011 1.01 ± 0.005 12 
K314C  −94 ± 0.7 0.91 ± 0.07 0.044 ± 0.023 1.02 ± 0.021 3 
N315C  −85 ± 2.9 0.87 ± 0.02 0.049 ± 0.006 0.98 ± 0.011 8 
F316C  −89 ± 1.0 0.94 ± 0.02 0.063 ± 0.008 0.98 ± 0.010 10 
V409C  −90 ± 0.7 0.93 ± 0.03 0.096 ± 0.011 0.96 ± 0.008 7 
T410C  −96 ± 0.9 0.75 ± 0.05 0.067 ± 0.019 0.88 ± 0.018 5 
P412C  −86 ± 1.4 0.83 ± 0.03 0.043 ± 0.012 0.90 ± 0.023 9 
Y512C  −98 ± 2.1 0.85 ± 0.02 0.000 ± 0.000 0.89 ± 0.007 12 
D513C  −92 ± 1.2 0.71 ± 0.01 0.032 ± 0.009 1.00 ± 0.010 35 
S514C  −98 ± 3.1 0.83 ± 0.02 0.064 ± 0.009 1.00 ± 0.010 10 
I515C  −87 ± 0.8 0.97 ± 0.01 0.029 ± 0.003 0.95 ± 0.006 93 
I516C  −101 ± 2.3 0.88 ± 0.05 0.134 ± 0.020 0.91 ± 0.017 13 
Q517C  −92 ± 1.1 0.94 ± 0.01 0.063 ± 0.008 1.02 ± 0.010 8 
I518C  −91 ± 2.0 0.97 ± 0.03 0.072 ± 0.009 1.02 ± 0.005 10 
K519Cb  −114 ± 1.9 0.65 ± 0.01 0.241 ± 0.017 0.61 ± 0.006 56 
K520C  −104 ± 1.5 0.84 ± 0.05 0.077 ± 0.039 1.00 ± 0.019 5 
L521Cc      
P522C  −94 ± 1.6 0.83 ± 0.02 0.115 ± 0.014 0.98 ± 0.005 26 
Y523C  −95 ± 1.7 0.92 ± 0.02 0.124 ± 0.012 0.97 ± 0.006 24 
L524C  −98 ± 1.4 0.88 ± 0.02 0.058 ± 0.009 0.97 ± 0.004 18 
P525C  −98 ± 1.1 0.90 ± 0.03 0.110 ± 0.010 0.97 ± 0.007 6 
E526C  −104 ± 1.5 0.84 ± 0.03 0.050 ± 0.021 0.98 ± 0.011 3 
L527C  −107 ± 2.0 0.87 ± 0.01 0.057 ± 0.017 1.01 ± 0.008 3 
S528C  −107 ± 2.1 0.88 ± 0.04 0.045 ± 0.021 1.01 ± 0.011 6 
W529C  −96 ± 1.7 0.85 ± 0.04 0.047 ± 0.019 0.98 ± 0.009 4 
S530C  −105 ± 2.2 0.85 ± 0.05 0.044 ± 0.028 1.00 ± 0.006 3 
S531C  −106 ± 1.7 0.92 ± 0.01 0.074 ± 0.006 1.00 ± 0.001 2 
a

WT refers to C212S, the background for all cysteine point mutants.

b

Mutants with Vo shifted >20 mV or rectification changed by >20% from wild type.

c

Mutants for which no current could be detected.

Effects of cysteine mutagenesis. Voltage-dependent gating parameters for wild type and cysteine point mutants: midpoint of the voltage–activation curve (Vo), effective gating charge (z), and minimum open probability (Pmin). Rectification is quantified as described in materials and methods and normalized to wild type, which is slightly inwardly rectifying. Values greater than one indicate inward rectification with respect to wild type; values less than one indicate outward rectification with respect to wild type. Experiments were carried out in slightly asymmetric solution conditions, such that the expected rectification is 1.04 according to the Goldman-Hodgkin-Katz current equation.

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