HCN channel gating models: a re-evaluation based on how the voltage-sensing and cAMP-sensing domains regulate kinetics

Date created: 
HCN channels
Cyclic nucleotides
Voltage sensor
Voltage clamp
S4 helix

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to rhythmic oscillations in the heart and brain. Upon membrane hyperpolarization, HCN channel pore opening is coupled to inward movement of the S4 helix within the transmembrane voltage sensing domain (VSD, helices S1-S4). The gating pathway is proposed to include an initial voltage-dependent VSD movement step followed by a voltage-independent pore movement step (a cyclic allosteric mechanism). Various other mechanisms influence open state stability: A cytosolic cyclic nucleotide-binding (CNB) fold destabilizes the open state when unliganded (an autoinhibition mechanism), whereas binding of the phospholipid PIP2 to the transmembrane domain stabilizes the open state. After pore opening, the channel undergoes a mode-shift, presumed to include lateral movement of S4 towards S2, forming a more stable open state. Despite the knowledge of open state stabilization mechanisms, it remains unclear how these mechanisms affect the kinetics of the gating pathway. Do these mechanisms apply equally strongly to channel thermodynamics and kinetics? Do they apply under a variety of cellular conditions? And do they regulate the VSD movement step, the pore movement step, or both? In this work I examined both the thermodynamics and kinetics of the activation and deactivation pathways in a variety of HCN channel derivatives. I used two-electrode voltage clamp to determine that while channel thermodynamics follow the predictions of the autoinhibition model, a channel with an unliganded CNB fold has faster activation than a channel with autoinhibition relieved by CNB fold deletion. I propose this fast activation is promoted by a “quickening conformation” of the intact CNB fold. The quickening conformation is independent of PIP2 in both autoinhibited and autoinhibition-free channels. I used voltage clamp fluorometry to determine the speed of a VSD movement during channel deactivation in relation to pore closure. The speed of this VSD movement did not limit the rate of the deactivation pathway at strong depolarizations and showed stronger voltage dependence than pore closure. The speed of this VSD movement was independent of both cAMP binding and mode shift. Together my results clarify the HCN gating mechanisms of cyclic allostery, autoinhibition, PIP2 potentiation and mode shift, and produce novel models of both HCN channel activation and deactivation.

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This thesis may be printed or downloaded for non-commercial research and scholarly purposes. Copyright remains with the author.
Edgar Young
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.