Identification of a surface charged residue in the S3-S4 linker of the pacemaker (HCN) channel that influences activation gating.
I(f), 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 Glu(235), an acidic S3-S4 linker residue conserved in all hyperpolarization-activated channels, by Ala substitution produced a depolarizing activation shift (V(12) = -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 Mg(2+) mimicked the progressive rightward shifts of E235A and E235R by gradually shifting activation (V(12) = 1 < 3 < 10 < 30 mm); Delta V(12) induced by 30 mm Mg(2+) 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 Delta V(12) resulting from various Glu(235) substitutions (i.e. Asp, Ala, Pro, His, Lys, and Arg) displayed a strong correlation with their charges (Delta Delta G = -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 Glu(235) influences activation gating, probably by acting as a surface charge.
Henrikson
Charles A
CA
Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Xue
Tian
T
Dong
Peihong
P
Sang
Dongpei
D
Marban
Eduardo
E
Li
Ronald A
RA
D000409:Alanine / Q000737:chemistry*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D054815:Cyclic Nucleotide-Gated Cation Channels; D004594:Electrophysiology; D018698:Glutamic Acid / Q000737:chemistry; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008564:Membrane Potentials; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins; D009865:Oocytes / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D014981:Xenopus
D054815:Cyclic Nucleotide-Gated Cation Channels; C578666:Hcn1 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D009419:Nerve Tissue Proteins; D015221:Potassium Channels; D018698:Glutamic Acid; D008274:Magnesium; D000409:Alanine; D011188:Potassium