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Emerging Concepts in Ion Channel Biophysics
Poster Abstracts
100
30-POS
Board 30
Molecular Mechanism Coupling the S4 Voltage-sensor to the Pore Domain in HCN
Channels.
Galen E. Flynn
, William N. Zagotta.
University of Washington, Seattle, WA, USA.
Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are both voltage- and
ligand-activated cation channels that functionally contribute to pace-making activity in cardiac
and neuronal cells. HCN channels are members of the voltage-gated K+ channel superfamily.
However, HCN channels are unique in that they are activated by hyperpolarizing voltages as
well as by the direct binding of cyclic nucleotides. Recently, a cryo-EM 3D structure of the
human HCN1 channel revealed that the voltage-sensing domains (VSDs) and pore domains
(PDs) of a single subunit are juxtapose in the tetrameric complex and not swapped between
subunits as observed for Kv1.2 channels (Lee & MacKinnon, 2017, Long et al., 2005). This
arrangement of transmembrane domains begs the questions: 1) how are the VSD and PD electro-
mechanically coupled and 2) what is the role of the S4-S5 linker in voltage-dependent activation
of HCN channels? To address these questions, site-directed mutagenesis was used to perturb the
S4-S5 linker region of spHCN. Excised inside/out patch-clamp techniques were used to record
macroscopic currents from spHCN channels heterologously expressed in Xenopus oocytes.
Conductance-voltage relationships, measured in the absence or presence of saturating
concentrations of full agonist cAMP or partial agonist cGMP, were fit with a modified Horrigan
and Aldrich (2002) allosteric model. Major findings were: 1) the S4-S5 linker was not required
for voltage-dependent activation or cyclic nucleotide-dependent modulation, 2) the S4C-term
was required for voltage-dependent activation, 3) the S5N-term was involved in pore opening,
and 4) both the S4C-term and the C-terminus acted as auto-inhibitory domains on the pore.
These findings provide new insights into the molecular mechanism of voltage-dependent
activation in HCN channels.