Table 1. Effects of Cd 2 on spHCN... | Rockefeller University Press

Table 1.

Effects of Cd²⁺ on spHCN channels containing introduced cysteine residues

Construct	Effect	Control					1 µM Cd²⁺
		V_1/2	k	f_nondecaying	RT_10–50%	n	V_1/2	k	f_nondecaying	RT_10–50%	n
		mV	mV		ms		mV	mV		ms
WT		−63 ± 2	6.7 ± 0.3	0.04 ± 0.01	12 ± 1	3	−65 ± 2	6.3 ± 0.1	0.03 ± 0.01	12.5 ± 0.4	3
S472C		−56 ± 5	5.1 ± 0.5	0.03 ± 0.02	8.8 ± 0.6	3	−53 ± 4	4.7 ± 0.7	0.08 ± 0.03	5.3 ± 0.8^a	3
S473C		−73 ± 2	8.1 ± 0.4	0.09 ± 0.03	23 ± 3	5	−68 ± 2^a	7.8 ± 0.9	0.13 ± 0.05	23 ± 3	5
S474C		−67 ± 2	6.4 ± 0.1	0.03 ± 0.03	21 ± 5	4	−56 ± 5^a	10 ± 2^a	0.24 ± 0.04^a	58 ± 10^a	4
Q476C		−56 ± 4	10 ± 2	0.09 ± 0.03	8.3 ± 0.9	4	−62 ± 3	11 ± 3	0.12 ± 0.06	11.2 ± 0.8	4
R478C		−79 ± 1	6.7 ± 0.3	0.08 ± 0.02	20 ± 2	5	−67 ± 1	7.2 ± 0.6	0.08 ± 0.03	20 ± 1	3
E479C		−63 ± 3	12 ± 1	0.05 ± 0.01	12 ± 1	3	−62 ± 3	10 ± 2	0.09 ± 0.03	11 ± 1	3
K482C		−72 ± 4	10 ± 1	0.09 ± 0.03	20 ± 5	4	−69 ± 4	11 ± 2	0.12 ± 0.03	26 ± 6	4
E485C		−50 ± 1	8 ± 1	0.34 ± 0.11	4.8 ± 0.4	3	−49 ± 1	7.4 ± 0.9	0.37 ± 0.12	8 ± 1	3
F359C		−65 ± 3	8.1 ± 0.3	0.07 ± 0.02	15 ± 3	4	−56 ± 2^a	12 ± 1^a	0.19 ± 0.01	19 ± 5	4
+S472C		−63 ± 2	10 ± 2	0.12 ± 0.06	16 ± 3	4	−68 ± 7	14 ± 4	0.31 ± 0.07	11 ± 1	4
+S473C	LC	−69 ± 2	8.7 ± 0.7	0.08 ± 0.04	16 ± 2	3	−58 ± 8	14 ± 3	0.12 ± 0.04	56 ± 4^a	3
+S474C	LO	−57 ± 4	7.2 ± 0.7	0.20 ± 0.08	24 ± 2	3	ND	ND	0.81 ± 0.06^a	ND	3
+Q476C		−54 ± 5	8 ± 1	0.12 ± 0.01	9 ± 1	3	−54 ± 7	12 ± 3	0.21 ± 0.02	16 ± 4	3
+R478C		−73 ± 3	6.2 ± 0.3	0.04 ± 0.02	14 ± 2	3	−55 ± 2^a	12.6 ± 0.2^a	0.17 ± 0.01	20 ± 1^a	3
+K482C		−61 ± 3	6.3 ± 0.3	0.04 ± 0.01	14 ± 1	3	−36 ± 3^a	19 ± 3^a	0.22 ± 0.03^a	22 ± 3	3
+E485C	LO	−41 ± 5	8.3 ± 0.2	0.31 ± 0.09	4.3 ± 0.2	3	ND	ND	0.84 ± 0.09^a	ND	3
V361C		−53 ± 3	7.0 ± 0.8	0.20 ± 0.07	8.7 ± 0.8	3	−53 ± 3	12 ± 4	0.36 ± 0.08	10 ± 1	3
+S472C	LC	−59 ± 3	7 ± 2	0.21 ± 0.03	11 ± 1	3	−73 ± 4	10 ± 1	0.19 ± 0.02	25 ± 3^a	3
+S473C	LO	−77 ± 2	7.7 ± 0.2	0.07 ± 0.03	23 ± 2	3	−55 ± 1^a	13 ± 1^a	0.61 ± 0.09^a	15 ± 2^a	3
+S474C	LO	−59 ± 4	6.9 ± 0.9	0.22 ± 0.05	12 ± 3	3	ND	ND	0.80 ± 0.01^a	ND	3
+Q476C	LC	−51 ± 3	4 ± 3	0.15 ± 0.06	6.3 ± 0.2	5	−60 ± 2^a	8.2 ± 0.4	0.20 ± 0.04	30 ± 6^a	5
+R478C	LC	−72 ± 2	6.3 ± 0.4	0.02 ± 0.01	9.9 ± 0.8	4	−70 ± 3	8.9 ± 0.8^a	0.26 ± 0.05^a	16 ± 2^a	3
+E479C		−65 ± 4	11 ± 1	0.07 ± 0.02	11 ± 1	4	−68 ± 3	15 ± 5	0.28 ± 0.02^a	11 ± 1	4
+K482C		−63 ± 3	6.2 ± 0.6	0.13 ± 0.04	10 ± 2	3	−47 ± 3^a	10.7 ± 0.8^a	0.42 ± 0.06^a	10 ± 2	3
+E485C	LO	−41 ± 1	8 ± 2	0.51 ± 0.03	3.5 ± 0.7	3	−84 ± 5^a	20 ± 3	0.94 ± 0.01^a	9 ± 2	3
A364C		−53 ± 2	8 ± 1	0.08 ± 0.01	8.2 ± 0.4	3	−56 ± 3	7.9 ± 0.9	0.12 ± 0.03	9.6 ± 0.4	3
+S472C	LO	−47 ± 2	8 ± 1	0.22 ± 0.03	15 ± 3	3	−68 ± 1^a	20 ± 7	0.66 ± 0.01^a	33 ± 7	3
+S473C	LC	−69 ± 5	7.9 ± 0.7	0.17 ± 0.04	15 ± 3	3	−89 ± 3^a	8.6 ± 0.8	0.08 ± 0.04	94 ± 13^a	3
+S474C	LC	−54 ± 8	8.4 ± 0.2	0.19 ± 0.01	10 ± 3	3	−93 ± 6^a	7.6 ± 0.3	0.03 ± 0.02^a	150 ± 10^a	3
+Q476C	LC	−49 ± 3	8.0 ± 0.3	0.14 ± 0.01	7.6 ± 0.5	3	−98 ± 1^a	9.4 ± 0.8	0.08 ± 0.01^a	163 ± 1^a	3
+R478C	LC	−73 ± 2	6.8 ± 0.2	0.06 ± 0.01	13 ± 2	3	−101 ± 2^a	6.0 ± 0.8	0.03 ± 0.02	127 ± 5^a	3
+E479C	LC	−61 ± 3	15 ± 2	0.15 ± 0.03	16 ± 2	5	−75 ± 4^a	22 ± 4	0.20 ± 0.04	40 ± 7^a	5
+K482C	LO	−54 ± 2	8 ± 1	0.14 ± 0.01	12 ± 2	4	ND	ND	0.89 ± 0.02^a	ND	4
+E485C	LO	−32 ± 5	8 ± 1	0.28 ± 0.04	5.5 ± 0.8	4	−32 ± 1	5.6 ± 0.4	0.89 ± 0.01^a	7 ± 1	4
Dimers
6472_6472	LO	−46 ± 1	5.9 ± 0.2	0.11 ± 0.01	ND	4	−69 ± 4^a	21 ± 4^a	0.74 ± 0.01^a	ND	4
WT_6472	LO	−63 ± 1	6.6 ± 0.6	0.15 ± 0.02	ND	6	−60 ± 2	8.0 ± 0.7^a	0.27 ± 0.03^a	ND	6
364C_472C		−63 ± 3	7.2 ± 0.3	0.08 ± 0.02	ND	4	−63 ± 2	8.5 ± 0.4^a	0.12 ± 0.02	ND	4
6476_6476	LC	−61 ± 1	10 ± 2	ND	10 ± 2	3	−108 ± 7^a	8 ± 1	ND	195 ± 13^a	3
WT_6476	LC	−64 ± 1	9 ± 2	ND	10 ± 2	3	−69 ± 2	9.4 ± 0.9	ND	27 ± 5^a	3
364C_476C	LC	−58 ± 2	8.5 ± 0.7	ND	8.9 ± 0.6	4	−76 ± 1^a	11 ± 1^a	ND	40 ± 2^a	4
6482_6482	LO	−55 ± 3	7 ± 1	0.10 ± 0.01	ND	3	ND	ND	1.0 ± 0.01^a	ND	3
WT_6482	LO	−65 ± 2	8.2 ± 0.5	0.06 ± 0.02	ND	6	−63 ± 3	11 ± 2	0.25 ± 0.03^a	ND	6
364C_482C	LO	−67 ± 1	10 ± 1	0.12 ± 0.01	ND	5	−63 ± 2^a	19 ± 3^a	0.39 ± 0.04^a	ND	5

Construct	Effect	Control					1 µM Cd²⁺
		V_1/2	k	f_nondecaying	RT_10–50%	n	V_1/2	k	f_nondecaying	RT_10–50%	n
		mV	mV		ms		mV	mV		ms
WT		−63 ± 2	6.7 ± 0.3	0.04 ± 0.01	12 ± 1	3	−65 ± 2	6.3 ± 0.1	0.03 ± 0.01	12.5 ± 0.4	3
S472C		−56 ± 5	5.1 ± 0.5	0.03 ± 0.02	8.8 ± 0.6	3	−53 ± 4	4.7 ± 0.7	0.08 ± 0.03	5.3 ± 0.8^a	3
S473C		−73 ± 2	8.1 ± 0.4	0.09 ± 0.03	23 ± 3	5	−68 ± 2^a	7.8 ± 0.9	0.13 ± 0.05	23 ± 3	5
S474C		−67 ± 2	6.4 ± 0.1	0.03 ± 0.03	21 ± 5	4	−56 ± 5^a	10 ± 2^a	0.24 ± 0.04^a	58 ± 10^a	4
Q476C		−56 ± 4	10 ± 2	0.09 ± 0.03	8.3 ± 0.9	4	−62 ± 3	11 ± 3	0.12 ± 0.06	11.2 ± 0.8	4
R478C		−79 ± 1	6.7 ± 0.3	0.08 ± 0.02	20 ± 2	5	−67 ± 1	7.2 ± 0.6	0.08 ± 0.03	20 ± 1	3
E479C		−63 ± 3	12 ± 1	0.05 ± 0.01	12 ± 1	3	−62 ± 3	10 ± 2	0.09 ± 0.03	11 ± 1	3
K482C		−72 ± 4	10 ± 1	0.09 ± 0.03	20 ± 5	4	−69 ± 4	11 ± 2	0.12 ± 0.03	26 ± 6	4
E485C		−50 ± 1	8 ± 1	0.34 ± 0.11	4.8 ± 0.4	3	−49 ± 1	7.4 ± 0.9	0.37 ± 0.12	8 ± 1	3
F359C		−65 ± 3	8.1 ± 0.3	0.07 ± 0.02	15 ± 3	4	−56 ± 2^a	12 ± 1^a	0.19 ± 0.01	19 ± 5	4
+S472C		−63 ± 2	10 ± 2	0.12 ± 0.06	16 ± 3	4	−68 ± 7	14 ± 4	0.31 ± 0.07	11 ± 1	4
+S473C	LC	−69 ± 2	8.7 ± 0.7	0.08 ± 0.04	16 ± 2	3	−58 ± 8	14 ± 3	0.12 ± 0.04	56 ± 4^a	3
+S474C	LO	−57 ± 4	7.2 ± 0.7	0.20 ± 0.08	24 ± 2	3	ND	ND	0.81 ± 0.06^a	ND	3
+Q476C		−54 ± 5	8 ± 1	0.12 ± 0.01	9 ± 1	3	−54 ± 7	12 ± 3	0.21 ± 0.02	16 ± 4	3
+R478C		−73 ± 3	6.2 ± 0.3	0.04 ± 0.02	14 ± 2	3	−55 ± 2^a	12.6 ± 0.2^a	0.17 ± 0.01	20 ± 1^a	3
+K482C		−61 ± 3	6.3 ± 0.3	0.04 ± 0.01	14 ± 1	3	−36 ± 3^a	19 ± 3^a	0.22 ± 0.03^a	22 ± 3	3
+E485C	LO	−41 ± 5	8.3 ± 0.2	0.31 ± 0.09	4.3 ± 0.2	3	ND	ND	0.84 ± 0.09^a	ND	3
V361C		−53 ± 3	7.0 ± 0.8	0.20 ± 0.07	8.7 ± 0.8	3	−53 ± 3	12 ± 4	0.36 ± 0.08	10 ± 1	3
+S472C	LC	−59 ± 3	7 ± 2	0.21 ± 0.03	11 ± 1	3	−73 ± 4	10 ± 1	0.19 ± 0.02	25 ± 3^a	3
+S473C	LO	−77 ± 2	7.7 ± 0.2	0.07 ± 0.03	23 ± 2	3	−55 ± 1^a	13 ± 1^a	0.61 ± 0.09^a	15 ± 2^a	3
+S474C	LO	−59 ± 4	6.9 ± 0.9	0.22 ± 0.05	12 ± 3	3	ND	ND	0.80 ± 0.01^a	ND	3
+Q476C	LC	−51 ± 3	4 ± 3	0.15 ± 0.06	6.3 ± 0.2	5	−60 ± 2^a	8.2 ± 0.4	0.20 ± 0.04	30 ± 6^a	5
+R478C	LC	−72 ± 2	6.3 ± 0.4	0.02 ± 0.01	9.9 ± 0.8	4	−70 ± 3	8.9 ± 0.8^a	0.26 ± 0.05^a	16 ± 2^a	3
+E479C		−65 ± 4	11 ± 1	0.07 ± 0.02	11 ± 1	4	−68 ± 3	15 ± 5	0.28 ± 0.02^a	11 ± 1	4
+K482C		−63 ± 3	6.2 ± 0.6	0.13 ± 0.04	10 ± 2	3	−47 ± 3^a	10.7 ± 0.8^a	0.42 ± 0.06^a	10 ± 2	3
+E485C	LO	−41 ± 1	8 ± 2	0.51 ± 0.03	3.5 ± 0.7	3	−84 ± 5^a	20 ± 3	0.94 ± 0.01^a	9 ± 2	3
A364C		−53 ± 2	8 ± 1	0.08 ± 0.01	8.2 ± 0.4	3	−56 ± 3	7.9 ± 0.9	0.12 ± 0.03	9.6 ± 0.4	3
+S472C	LO	−47 ± 2	8 ± 1	0.22 ± 0.03	15 ± 3	3	−68 ± 1^a	20 ± 7	0.66 ± 0.01^a	33 ± 7	3
+S473C	LC	−69 ± 5	7.9 ± 0.7	0.17 ± 0.04	15 ± 3	3	−89 ± 3^a	8.6 ± 0.8	0.08 ± 0.04	94 ± 13^a	3
+S474C	LC	−54 ± 8	8.4 ± 0.2	0.19 ± 0.01	10 ± 3	3	−93 ± 6^a	7.6 ± 0.3	0.03 ± 0.02^a	150 ± 10^a	3
+Q476C	LC	−49 ± 3	8.0 ± 0.3	0.14 ± 0.01	7.6 ± 0.5	3	−98 ± 1^a	9.4 ± 0.8	0.08 ± 0.01^a	163 ± 1^a	3
+R478C	LC	−73 ± 2	6.8 ± 0.2	0.06 ± 0.01	13 ± 2	3	−101 ± 2^a	6.0 ± 0.8	0.03 ± 0.02	127 ± 5^a	3
+E479C	LC	−61 ± 3	15 ± 2	0.15 ± 0.03	16 ± 2	5	−75 ± 4^a	22 ± 4	0.20 ± 0.04	40 ± 7^a	5
+K482C	LO	−54 ± 2	8 ± 1	0.14 ± 0.01	12 ± 2	4	ND	ND	0.89 ± 0.02^a	ND	4
+E485C	LO	−32 ± 5	8 ± 1	0.28 ± 0.04	5.5 ± 0.8	4	−32 ± 1	5.6 ± 0.4	0.89 ± 0.01^a	7 ± 1	4
Dimers
6472_6472	LO	−46 ± 1	5.9 ± 0.2	0.11 ± 0.01	ND	4	−69 ± 4^a	21 ± 4^a	0.74 ± 0.01^a	ND	4
WT_6472	LO	−63 ± 1	6.6 ± 0.6	0.15 ± 0.02	ND	6	−60 ± 2	8.0 ± 0.7^a	0.27 ± 0.03^a	ND	6
364C_472C		−63 ± 3	7.2 ± 0.3	0.08 ± 0.02	ND	4	−63 ± 2	8.5 ± 0.4^a	0.12 ± 0.02	ND	4
6476_6476	LC	−61 ± 1	10 ± 2	ND	10 ± 2	3	−108 ± 7^a	8 ± 1	ND	195 ± 13^a	3
WT_6476	LC	−64 ± 1	9 ± 2	ND	10 ± 2	3	−69 ± 2	9.4 ± 0.9	ND	27 ± 5^a	3
364C_476C	LC	−58 ± 2	8.5 ± 0.7	ND	8.9 ± 0.6	4	−76 ± 1^a	11 ± 1^a	ND	40 ± 2^a	4
6482_6482	LO	−55 ± 3	7 ± 1	0.10 ± 0.01	ND	3	ND	ND	1.0 ± 0.01^a	ND	3
WT_6482	LO	−65 ± 2	8.2 ± 0.5	0.06 ± 0.02	ND	6	−63 ± 3	11 ± 2	0.25 ± 0.03^a	ND	6
364C_482C	LO	−67 ± 1	10 ± 1	0.12 ± 0.01	ND	5	−63 ± 2^a	19 ± 3^a	0.39 ± 0.04^a	ND	5

Standard G-V curve parameters, proportions of the nondecaying tail current (f_nondecaying), and the rise times between 10 and 50% (RT₁₀_–_50%)of all the constructs tested in this study. G-V curves were constructed from the start of the tail currents at +50 mV after 500-ms hyperpolarizing pulses ranging from −120 to +50 mV and fit to a Boltzmann function for the half-activation voltage (V_1/2) and slope factor (k) (see Materials and methods). Proportion of the nondecaying tail current (f_nondecaying) was measured by fitting the tail current to a double-exponential function and extracting the fraction of the constant term. RT₁₀_–_50% was measured from fits of the current trace to either a single- or double-exponential function within the 500 ms of the hyperpolarizing pulse to −120 mV. All values are expressed as mean ± sem. Parameters that have not been determined are indicated as ND. Values for double mutants in the presence of Cd²⁺, which are significantly different from the corresponding values for both the respective single S4–S5 and C-linker mutants, are shown in bold and underlined. The double mutants that we consider to form lock-open or lock-closed bridges in the presence of Cd²⁺ are indicated.

Significant difference from the value in the absence of Cd²⁺ (unpaired t test; P < 0.05).

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