Oregon Health & Science University
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 Mg2+. The experiments relating to the Mg2+ 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 L254C and S261 C from the intracellular solution. These results suggest that the role ofS4 as the voltage sensor in HCN channels is conserved. HCN channels are also gated by cyclic nucleotides, specifically cAMP, which binds to a cyclic nucleotide binding domain (CNBD) located in the HCN-C terminus. The binding of cAMP relieves a tonic inhibition of channel opening caused by the CNBD. All HCN channels have a CNBD, but different HCN isoforms exhibit varying levels of sensitivity to cAMP modulation. In an attempt to find the structural basis for this difference in sensitivity, the Cterminus was deleted from HCNl and a nonmammalian HCN channel, spHCN, whose currents inactivate during low intracellular levels of cAMP. HCNl channels do not inactivate are relatively insensitive to changes in cAMP concentrations. The C-terminus deleted spHCN channel appeared similar to the C-terminus-deleted HCNl channel, suggesting that differences in gating between spHCN and HCNl are due to differences in the C-terminus or interactions between the C-terminus and the rest of the channel. The second half of the thesis builds upon the fact that HCN channels are activated by membrane hyperpolarization mediating time-dependent, inward-rectifying currents, gated by the movement of the voltage sensor, S4. However, inward rectification of the HCN currents is not only observed in the time-dependent HCN currents, but also in the instantaneous HCN tail currents. Experiments, included in the second half of the thesis, show that intracellular Mg2+ functions as a voltage-dependent blocker of HCN channels, acting to reduce the instantaneous outward currents. The affinity of HCN channels for Mg2+ is in the physiological range, with Mg2+ binding with an IC 50 of 0.53 mM at +50 m V in HCN2 channels and an ICso of 0.82 mM at +50 m V for HCNI. channels. Mg2+ block was also found to be voltage dependent with an effective electrical distance for the Mg2+ binding site of0.19. Removing a cysteine in the selectivity filter reduced the affinity for Mg 2+ suggesting that this residue forms part of the binding site deep within the pore. Mg2+ is also able to block both the time-dependent and time-independent outward currents in HCN channels. Mg2+ is proposed to act as an 'extrinsic' gating mechanism which complements theprimary S4 mediated voltage-dependent gating of an HCN channel.
Neuroscience Graduate Program
School of Medicine
Vemana, Sriharsha, "Gating mechanisms in hyperpolarization-activated cyclic nucleotide-gated ion channels" (2007). Scholar Archive. 834.