Cyclic nucleotide-gated ion channels are distributed most widely in the neuronal and nonneuronal cell. Great progress has been made in molecular mechanisms of CNG channel gating in recent years since their discovery in 1985(Fesenko et al., 1985). Results of many experiments have indicated that the stoichiometry and assembly of CNG channel subunit affect their property and gating. The substituted cysteine accessibility method (SCAM) has been a very powerful tool in understanding many of the molecular mechanisms underlying their functions. Cite directed mutagenesis has been a great help in elucidating the possible mechanism behind the ligand discrimination among channels expressed in different cell types. In the recent years the advance in computer technology has provided tremendous help in understanding the three dimensional arrangement of proteins by virtue of molecular biology. Most probably this is the perfect time in which molecular biology, biochemistry and computational science has come together to provide some amazing view of membrane proteins. Still crystallography has its own limitations, and electrophysiology serves as an adequate substitute. Most of our understanding about the CNG channels arises from the study of these channels expressed in sensory neurons, viz photoreceptors and olfactory sensory neurons. In my work I have used heterologously expressed homologous CNGA1 subunit from bovine rod receptors as a target. The expression system used was Xenopus leavis oocytes. Though the homologous channels thus expressed vary in several aspects provides a very good tool in studying the structure function relationship of these channels. In the preliminary part of the study an extensive site directed mutagenesis from residue F375 to V424, one at a time, has been performed (SCAM). I have then probed these mutant channels with divalent cations such as Cd2+ and Ni2+ and several methane thiosulfonate compounds to study their accessibility and interaction. The residues from F375 until S399 does not show much effect to these externally applied compounds with few exceptions. One remarkable exception is F380C, which is found to be potentiated by Cd2+ when applied in the open state of the channel inhibited when applied in the closed. Further studies have revealed a locking effect of the channel and thus some insight into the proximity of residues and possible molecular rearrangement while channel passing from closed to open. Another study has revealed the interaction of native Cys505 residues with several other residues in the C-linker domain when are mutated into cysteine. This study has helped to propose a molecular model of C-linker domain. Also it provided some knowledge in the possible rearrangement of C-linker region while channel opens. One another course of study has revealed that the residues from 390 to 400 come closer in the closed state than in the open. The following stretch of residues, from 410 to 420, on the contrary comes closer in the open state than in the closed. My studies suggest that the channel while passing from close to open does not undergo a major translational movement of residues near the S6. Probably the coupled movement of of S6 with pore helix provides enough energy to open the gate.
|Autori:||Nair, Anil V.|
|Titolo:||Structural modification of CNG channels during activation|
|Relatore/i interni:||Torre, Vincent|
|Data di pubblicazione:||18-ott-2007|
|Appare nelle tipologie:||8.1 PhD thesis|