Emerging Concepts in Ion Channel Biophysics

Emerging Concepts in Ion Channel Biophysics

Poster Abstracts

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.

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