An electrophysiological analysis of conformational changes occurring during the gating of CNG channels

In my thesis I have addressed the problem of understanding the relation between the structure and function of CNG channels by combining electrophysiological experiments with molecular modeling. In particular I focussed my attention into two regions present in the Al subunit: the putative C-helix of the CNBD and the pore region. I have used molecular biology to construct mutant channels and electrophysiology to analyse their properties. I have heterologously expressed mutant channels in Xenopus laevis oocites and studied their properties in excised-patches under voltage-clamp conditions. I have mutated one by one all residues in the C-helix and in the pore region and probed the effect of sulfhydryl reagents on mutant channels. My experiments indicate that when cGMP is bound to the CNBD, the stretch ofresidues from Leu583 to Asn610 is likely to form an alpha helix. In addition I have identified the residues, in which the C-helices are in close contact in the open state. Therefore the conformation of the CNBD of CNGAl channels is different from that determined in HCN2 channels (Zagotta et al., 2003) and is likely to be a dimer of dimers as suggested (Scott et al., 2001 and Higgins et al., 2002). In the absence of cGMP, C-helices are likely to kink and bend at variable angles. My experiments also indicate that in CNG channels the S6 transmembrane segment does not have the large movement during gating as observed in K+ channels (Jiang et al., 2002) and that the gating is primarily localised at the pore level. I suggest that the gating of CNG channels is caused by a fine movement producing a confonnational rearrangement of the pore walls. The movement of the pore helix is likely to be initiated by a translation of the S6 domain mediated by hydrophobic interaction between the Phelix and the S6 domain.