Gating mechanisms in hyperpolarization-activated cyclic nucleotide-gated ion channels

Hyperpolarization-activated cyclic nucleotide-gated 1on channels (HCN) are gated by voltage, that is, they are able to detect changes in voltage and convert that into work for opening and closing (gating) the channel gate. The focus of this thesis was to investigate the gating mechanisms in HCN channels. Results from these experiments revealed that HCN channels have a voltage sensor (S4) similar to K v channels but have reversed gating. This makes up the first half of the thesis. In addition, the outward currents through HCN channels are susceptible to a voltage-dependent block by intracellular Mg +. The experiments relating to the Mg+ block make up the second half of the thesis. HCN channels mediate an inward cation current that contributes to spontaneous rhythmic firing activity in the heart and brain. These channels share homology with depolarization-activated Kv channels, including six transmembrane domains (S 1-S6) and a positively charged S4 segment. The S4 domain has been shown to function as the voltage sensor and undergo a voltage-dependent movement in the Shaker K+ channel. Experiments making up the first half of the thesis, incorporated the substituted cysteine accessibility method in conjunction with two-electrode voltage clamp to test for state dependent movement in the S4 domain of HCNl. Six cysteine mutations (R247C, T249C, 1251C, S253C, L254C, and S261 C) were used to assess S4 movement of the heterologously expressed HCNl channel in Xenopus oocytes. State-dependent accessibility was found for four residues, T249C and S253C from the extracellular solution and