RESUMO
KirBac channels are prokaryotic homologs of mammalian inwardly rectifying potassium (Kir) channels, and recent structures of KirBac3.1 have provided important insights into the structural basis of gating in Kir channels. In this study, we demonstrate that KirBac3.1 channel activity is strongly pH-dependent, and we used x-ray crystallography to determine the structural changes that arise from an activatory mutation (S205L) located in the cytoplasmic domain (CTD). This mutation stabilizes a novel energetically favorable open conformation in which changes at the intersubunit interface in the CTD also alter the electrostatic potential of the inner cytoplasmic cavity. These results provide a structural explanation for the activatory effect of this mutation and provide a greater insight into the role of the CTD in Kir channel gating.
Assuntos
Proteínas de Bactérias/química , Magnetospirillum/química , Canais de Potássio Corretores do Fluxo de Internalização/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ativação do Canal Iônico/fisiologia , Magnetospirillum/genética , Magnetospirillum/metabolismo , Mutação de Sentido Incorreto , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Estrutura Terciária de ProteínaRESUMO
Protein-lipid interactions regulate many membrane protein functions. Using a multiscale approach that combines coarse-grained and atomistic molecular dynamics simulations, we have predicted the binding site for the anionic phospholipid phosphatidylinositol 4,5-bisphosphate (PIP(2)) on the Kir2.2 inwardly rectifying (Kir) potassium channel. Comparison of the predicted binding site to that observed in the recent PIP(2)-bound crystal structure of Kir2.2 reveals good agreement between simulation and experiment. In addition to providing insight into the mechanism by which PIP(2) binds to Kir2.2, these results help to establish the validity of this multiscale simulation approach and its future application in the examination of novel membrane protein-lipid interactions in the increasing number of high-resolution membrane protein structures that are now available.
Assuntos
Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Animais , Sítios de Ligação , Galinhas , Simulação de Dinâmica Molecular , Canais de Potássio Corretores do Fluxo de Internalização/química , Ligação ProteicaRESUMO
We previously reported that TREK-1 gating by internal pH and pressure occurs close to or within the selectivity filter. These conclusions were based upon kinetic measurements of high-affinity block by quaternary ammonium (QA) ions that appeared to exhibit state-independent accessibility to their binding site within the pore. Intriguingly, recent crystal structures of two related K2P potassium channels were also both found to be open at the helix bundle crossing. However, this did not exclude the possibility of gating at the bundle crossing and it was suggested that side-fenestrations within these structures might allow state-independent access of QA ions to their binding site. In this addendum to our original study we demonstrate that even hydrophobic QA ions do not access the TREK-1 pore via these fenestrations. Furthermore, by using a chemically reactive QA ion immobilized within the pore via covalent cysteine modification we provide additional evidence that the QA binding site remains accessible to the cytoplasm in the closed state. These results support models of K2P channel gating which occur close to or within the selectivity filter and do not involve closure at the helix bundle crossing.
Assuntos
Espaço Intracelular/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Compostos de Amônio Quaternário/metabolismo , Animais , Humanos , Modelos Moleculares , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Domínios Poros em Tandem/química , Conformação Proteica , Compostos de Amônio Quaternário/química , Homologia Estrutural de Proteína , XenopusRESUMO
KirBac channels are prokaryotic homologs of mammalian inwardly rectifying (Kir) potassium channels, and recent crystal structures of both Kir and KirBac channels have provided major insight into their unique structural architecture. However, all of the available structures are closed at the helix bundle crossing, and therefore the structural mechanisms that control opening of their primary activation gate remain unknown. In this study, we engineered the inner pore-lining helix (TM2) of KirBac3.1 to trap the bundle crossing in an apparently open conformation and determined the crystal structure of this mutant channel to 3.05 Å resolution. Contrary to previous speculation, this new structure suggests a mechanistic model in which rotational 'twist' of the cytoplasmic domain is coupled to opening of the bundle-crossing gate through a network of inter- and intrasubunit interactions that involve the TM2 C-linker, slide helix, G-loop and the CD loop.