Identification of a surface charged residue in the S3-S4 linker of the pacemaker (HCN) channel that influences activation gating

Charles A. Henrikson, Tian Xue, Peihong Dong, Dongpei Sang, Eduardo Marban, Ronald A. Li

Research output: Contribution to journalArticle

Abstract

If, encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel family, is a key player in cardiac and neuronal pacing. Although HCN channels structurally resemble voltage-gated K+ (Kv) channels, their structure-function correlation is much less clear. Here we probed the functional importance of the HCN1 S3-S4 linker by multiple substitutions of its residues. Neutralizing Glu235, an acidic S3-S4 linker residue conserved in all hyperpolarization-activated channels, by Ala substitution produced a depolarizing activation shift (V1/2 = -65.0 ± 0.7 versus -70.6 ± 0.7 mV for wild-type HCN1); the charge-reversed mutation E235R shifted activation even more positively (-56.2 ± 0.5 mV). Increasing external Mg2+ mimicked the progressive rightward shifts of E235A and E235R by gradually shifting activation (V1/2 = 1 <3 <10 <30 mM); ΔV1/2, induced by 30 mM Mg2+ was significantly attenuated for E235A (+7.9 ± 1.2 versus +11.3 ± 0.9 mV for wild-type HCN1) and E235R (+3.3 ± 1.4 mV) channels, as if surface charges were already shielded. Consistent with an electrostatic role, the energetic changes associated with ΔV1/2 resulting from various Glu235 substitutions (i.e. Asp, Ala, Pro, His, Lys, and Arg) displayed a strong correlation with their charges (ΔΔG = -2.1 ± 0.3 kcal/mol/charge; r = 0.94). In contrast, D233E, D233A, D233G, and D233R did not alter activation gating. D233C (in C318S background) was also not externally accessible when probed with methanethiosulfonate ethylammonium (MTSEA). We conclude that the S3-S4 linker residue Glu235 influences activation gating, probably by acting as a surface charge.

Original languageEnglish (US)
Pages (from-to)13647-13654
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number16
DOIs
StatePublished - Apr 18 2003
Externally publishedYes

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Pacemakers
Cyclic Nucleotides
Chemical activation
Voltage-Gated Potassium Channels
Viperidae
Static Electricity
Substitution reactions
Surface charge
Mutation
Electrostatics
Electric potential
methanethiosulfonate ethylammonium

ASJC Scopus subject areas

  • Biochemistry

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Identification of a surface charged residue in the S3-S4 linker of the pacemaker (HCN) channel that influences activation gating. / Henrikson, Charles A.; Xue, Tian; Dong, Peihong; Sang, Dongpei; Marban, Eduardo; Li, Ronald A.

In: Journal of Biological Chemistry, Vol. 278, No. 16, 18.04.2003, p. 13647-13654.

Research output: Contribution to journalArticle

Henrikson, Charles A. ; Xue, Tian ; Dong, Peihong ; Sang, Dongpei ; Marban, Eduardo ; Li, Ronald A. / Identification of a surface charged residue in the S3-S4 linker of the pacemaker (HCN) channel that influences activation gating. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 16. pp. 13647-13654.
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abstract = "If, encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel family, is a key player in cardiac and neuronal pacing. Although HCN channels structurally resemble voltage-gated K+ (Kv) channels, their structure-function correlation is much less clear. Here we probed the functional importance of the HCN1 S3-S4 linker by multiple substitutions of its residues. Neutralizing Glu235, an acidic S3-S4 linker residue conserved in all hyperpolarization-activated channels, by Ala substitution produced a depolarizing activation shift (V1/2 = -65.0 ± 0.7 versus -70.6 ± 0.7 mV for wild-type HCN1); the charge-reversed mutation E235R shifted activation even more positively (-56.2 ± 0.5 mV). Increasing external Mg2+ mimicked the progressive rightward shifts of E235A and E235R by gradually shifting activation (V1/2 = 1 <3 <10 <30 mM); ΔV1/2, induced by 30 mM Mg2+ was significantly attenuated for E235A (+7.9 ± 1.2 versus +11.3 ± 0.9 mV for wild-type HCN1) and E235R (+3.3 ± 1.4 mV) channels, as if surface charges were already shielded. Consistent with an electrostatic role, the energetic changes associated with ΔV1/2 resulting from various Glu235 substitutions (i.e. Asp, Ala, Pro, His, Lys, and Arg) displayed a strong correlation with their charges (ΔΔG = -2.1 ± 0.3 kcal/mol/charge; r = 0.94). In contrast, D233E, D233A, D233G, and D233R did not alter activation gating. D233C (in C318S background) was also not externally accessible when probed with methanethiosulfonate ethylammonium (MTSEA). We conclude that the S3-S4 linker residue Glu235 influences activation gating, probably by acting as a surface charge.",
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T1 - Identification of a surface charged residue in the S3-S4 linker of the pacemaker (HCN) channel that influences activation gating

AU - Henrikson, Charles A.

AU - Xue, Tian

AU - Dong, Peihong

AU - Sang, Dongpei

AU - Marban, Eduardo

AU - Li, Ronald A.

PY - 2003/4/18

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N2 - If, encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel family, is a key player in cardiac and neuronal pacing. Although HCN channels structurally resemble voltage-gated K+ (Kv) channels, their structure-function correlation is much less clear. Here we probed the functional importance of the HCN1 S3-S4 linker by multiple substitutions of its residues. Neutralizing Glu235, an acidic S3-S4 linker residue conserved in all hyperpolarization-activated channels, by Ala substitution produced a depolarizing activation shift (V1/2 = -65.0 ± 0.7 versus -70.6 ± 0.7 mV for wild-type HCN1); the charge-reversed mutation E235R shifted activation even more positively (-56.2 ± 0.5 mV). Increasing external Mg2+ mimicked the progressive rightward shifts of E235A and E235R by gradually shifting activation (V1/2 = 1 <3 <10 <30 mM); ΔV1/2, induced by 30 mM Mg2+ was significantly attenuated for E235A (+7.9 ± 1.2 versus +11.3 ± 0.9 mV for wild-type HCN1) and E235R (+3.3 ± 1.4 mV) channels, as if surface charges were already shielded. Consistent with an electrostatic role, the energetic changes associated with ΔV1/2 resulting from various Glu235 substitutions (i.e. Asp, Ala, Pro, His, Lys, and Arg) displayed a strong correlation with their charges (ΔΔG = -2.1 ± 0.3 kcal/mol/charge; r = 0.94). In contrast, D233E, D233A, D233G, and D233R did not alter activation gating. D233C (in C318S background) was also not externally accessible when probed with methanethiosulfonate ethylammonium (MTSEA). We conclude that the S3-S4 linker residue Glu235 influences activation gating, probably by acting as a surface charge.

AB - If, encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel family, is a key player in cardiac and neuronal pacing. Although HCN channels structurally resemble voltage-gated K+ (Kv) channels, their structure-function correlation is much less clear. Here we probed the functional importance of the HCN1 S3-S4 linker by multiple substitutions of its residues. Neutralizing Glu235, an acidic S3-S4 linker residue conserved in all hyperpolarization-activated channels, by Ala substitution produced a depolarizing activation shift (V1/2 = -65.0 ± 0.7 versus -70.6 ± 0.7 mV for wild-type HCN1); the charge-reversed mutation E235R shifted activation even more positively (-56.2 ± 0.5 mV). Increasing external Mg2+ mimicked the progressive rightward shifts of E235A and E235R by gradually shifting activation (V1/2 = 1 <3 <10 <30 mM); ΔV1/2, induced by 30 mM Mg2+ was significantly attenuated for E235A (+7.9 ± 1.2 versus +11.3 ± 0.9 mV for wild-type HCN1) and E235R (+3.3 ± 1.4 mV) channels, as if surface charges were already shielded. Consistent with an electrostatic role, the energetic changes associated with ΔV1/2 resulting from various Glu235 substitutions (i.e. Asp, Ala, Pro, His, Lys, and Arg) displayed a strong correlation with their charges (ΔΔG = -2.1 ± 0.3 kcal/mol/charge; r = 0.94). In contrast, D233E, D233A, D233G, and D233R did not alter activation gating. D233C (in C318S background) was also not externally accessible when probed with methanethiosulfonate ethylammonium (MTSEA). We conclude that the S3-S4 linker residue Glu235 influences activation gating, probably by acting as a surface charge.

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