PMID- 12771192 OWN - NLM STAT- MEDLINE DCOM- 20040220 LR - 20181113 IS - 0022-1295 (Print) IS - 1540-7748 (Electronic) IS - 0022-1295 (Linking) VI - 121 IP - 6 DP - 2003 Jun TI - A cysteine scan of the inner vestibule of cyclic nucleotide-gated channels reveals architecture and rearrangement of the pore. PG - 563-82 AB - Cyclic nucleotide-gated (CNG) channels belong to the P-loop-containing family of ion channels that also includes KcsA, MthK, and Shaker channels. In this study, we investigated the structure and rearrangement of the CNGA1 channel pore using cysteine mutations and cysteine-specific modification. We constructed 16 mutant channels, each one containing a cysteine mutation at one of the positions between 384 and 399 in the S6 region of the pore. By measuring currents activated by saturating concentrations of the full agonist cGMP and the partial agonists cIMP and cAMP, we show that mutating S6 residues to cysteine caused both favorable and unfavorable changes in the free energy of channel opening. The time course of cysteine modification with 2-aminoethylmethane thiosulfonate hydrochloride (MTSEA) was complex. For many positions we observed decreases in current activated by cGMP and concomitant increases in current activated by cIMP and cAMP. A model where modification affected both gating and permeation successfully reproduced the complex time course of modification for most of the mutant channels. From the model fits to the time course of modification for each mutant channel, we quantified the following: (a) the bimolecular rate constant of modification in the open state, (b) the change in conductance, and (c) the change in the free energy of channel opening for modification of each cysteine. At many S6 cysteines, modification by MTSEA caused a decrease in conductance and a favorable change in the free energy of channel opening. Our results are interpreted within the structural framework of the known structures of KcsA and MthK. We conclude that: (a) MTSEA modification affects both gating and permeation, (b) the open configuration of the pore of CNGA1 channels is consistent with the structure of MthK, and (c) the modification of S6 residues disrupts the helical packing of the closed channel, making it easier for channels to open. FAU - Flynn, Galen E AU - Flynn GE AD - Department of Physiology and Biophysics, University of Washington, Seattle 98195-7290, USA. FAU - Zagotta, William N AU - Zagotta WN LA - eng GR - R01 EY010329/EY/NEI NIH HHS/United States GR - R01 EY010329-10/EY/NEI NIH HHS/United States GR - EY10329/EY/NEI NIH HHS/United States PT - Journal Article PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, P.H.S. PL - United States TA - J Gen Physiol JT - The Journal of general physiology JID - 2985110R RN - 0 (Cyclic Nucleotide-Gated Cation Channels) RN - 0 (Indicators and Reagents) RN - 0 (Ion Channels) RN - 0 (methanethiosulfonate ethylammonium) RN - 9H154DI0UP (Ethyl Methanesulfonate) RN - E0399OZS9N (Cyclic AMP) RN - K848JZ4886 (Cysteine) SB - IM MH - Amino Acid Sequence MH - Cyclic AMP/metabolism MH - Cyclic Nucleotide-Gated Cation Channels MH - Cysteine/genetics MH - Ethyl Methanesulfonate/*analogs & derivatives/pharmacokinetics MH - Humans MH - Indicators and Reagents/pharmacokinetics MH - Ion Channel Gating/*physiology MH - Ion Channels/chemistry/*physiology MH - Molecular Sequence Data MH - Oocytes MH - Patch-Clamp Techniques MH - Permeability MH - Point Mutation MH - Protein Structure, Secondary PMC - PMC2217351 EDAT- 2003/05/29 05:00 MHDA- 2004/02/21 05:00 PMCR- 2003/12/01 CRDT- 2003/05/29 05:00 PHST- 2003/05/29 05:00 [pubmed] PHST- 2004/02/21 05:00 [medline] PHST- 2003/05/29 05:00 [entrez] PHST- 2003/12/01 00:00 [pmc-release] AID - jgp.200308819 [pii] AID - 200308819 [pii] AID - 10.1085/jgp.200308819 [doi] PST - ppublish SO - J Gen Physiol. 2003 Jun;121(6):563-82. doi: 10.1085/jgp.200308819.