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UBC Theses and Dissertations

The unique pore and selectivity filter of HCN channels Macri, Vincenzo S

Abstract

Hyperpolarization-activated Cyclic Nucleotide-modulated (HCN) channels are similar in structure and function to potassium channels. In both, changes in membrane voltage produce directionally similar movement of positively charged residues in the voltage sensor to alter the pore structure at the intracellular side and gate ion flow. Both classes of channels also allow mainly potassium ions to flow, are blocked by cesium ions, and are activated by extracellular potassium. However, HCN channels open when hyperpolarized, whereas most potassium channels open when depolarized. Thus, electromechanical coupling between the voltage sensor and gate is opposite. A key determinant of this coupling is the intrinsic stability of the pore. In potassium channels, the closed, and not the open, pore is more stable, however this it not known for HCN channels. HCN channels are also significantly permeable to sodium despite containing the GYG potassium channel signature selectivity filter sequence. In potassium channels, the selectivity filter sequence is ‘T/S-V/I/L/T-GYG’, which forms a row of four binding sites through which dehydrated potassium ions flow. In HCN channels, the equivalent residues are ‘C-I-GYG’, but whether they form four similarly arrayed cation binding sites is not known. In this thesis, we show using the mammalian HCN2 channel, that the stabilities of the open and closed pore are similar, the voltage sensor must apply force to close the pore, and that the interactions between the pore and voltage-sensor are weak. Furthermore, our data suggest that the conserved cysteine of the selectivity filter does not form a fourth binding site for permeating ions, which prevents it from contributing to either permeation or associated gating functions of the selectivity filter.

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