Hyperpolarization-activated cyclic nucleotide-gated (HCN1-4) channels are activated by the hyperpolarization of the membrane potential and further regulated by the direct binding of cyclic AMP (cAMP). The binding of cAMP to the cytoplasmic Cyclic Nucleotide Binding Domain (CNBD) increases channel open probability and speeds activation kinetics. cAMP modulation of HCN channels controls heart rate and working memory networks in prefrontal cortex, but also chronic pain at the level of the peripheral nervous system. Thus, a detailed description of the cAMP conformational changes in HCN channels is crucial for understanding several physiological functions, as well as diseases affecting cardiac and neuronal functions. The cAMP-induced conformational changes propagate from the CNBD to the pore through the C-linker region. We and others have previously described, at the atomic level, the conformational changes at the level of the CNBD. The goal of the present work is to describe the propagation of the movements through the C-linker to the pore. To this end, I have constructed a chimeric channel by fusing the C-linker/CNBD of HCN4 to the prokaryotic pore domain of KcsA (hereafter K-H4). Isothermal Titration Calorimetry (ITC) measurements demonstrated that K-H4 is able to bind to cAMP with a KD value of 2μM, which agrees with the one previously published for the isolated C-linker/CNBD fragment of HCN4 and Differential Scanning Calorimetry (DSC) indicated that cAMP causes conformational changes in the chimeric protein. Recently, Double Electron Electron Resonance (DEER) experiments showed that the binding of cAMP at the level of the CNBD domain of K-H4 causes relevant conformational changes in the C-linker region which transits from a dimer of dimers conformation to a 4-fold symmetrical gating ring as suggested by previous functional and biochemical data.
Chimeric HCN Channels for Studying cAMP-Induced Conformational Changes in the C-Linker / B. Introini, A. Saponaro, A. Bonicci, O. Rauh, F. Cantini, L. Banci, G. Thiel, A. Moroni. - In: BIOPHYSICAL JOURNAL. - ISSN 1542-0086. - 116:3 (Suppl. 1)(2019 Mar 15), pp. 1487-Plat.301A-1487-Plat.301A. ((Intervento presentato al 63. convegno Biophysical Society Annual Meeting nel 2019.
|Titolo:||Chimeric HCN Channels for Studying cAMP-Induced Conformational Changes in the C-Linker|
INTROINI, BIANCA (Primo)
SAPONARO, ANDREA COSIMO (Secondo)
MORONI, ANNA (Ultimo)
|Parole Chiave:||HCN channels, Clinker, cAMP, EPR, synthetic biology|
|Settore Scientifico Disciplinare:||Settore BIO/09 - Fisiologia|
Settore BIO/10 - Biochimica
|Data di pubblicazione:||15-mar-2019|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1016/j.bpj.2018.11.1631|
|Appare nelle tipologie:||01 - Articolo su periodico|