HCN (hyperpolarization-activated cyclic-nucleotide sensitive cation nonselective) channels are activated by voltage and modulated by the direct binding of cAMP to their cytoplasmic C-terminal region named CNBD (cyclic nucleotide binding domain) (Zagotta et al., 2003). HCN channels are further regulated by TRIP8b, a brain-specific auxiliary subunit which controls channel trafficking and gating. In particular, TRIP8b interacts with the HCN channel CNBD and antagonizes the facilitatory effect of cAMP on channel opening (Hu et al., 2013 ). Recently, the cryo-EM structure of human HCN1 was solved in both the cAMP-free and cAMP-bound states (Lee and Mackinnon, 2017). A comparison of the two structures shows that, in the full-length protein, cAMP binding induces all the same changes previously highlighted in the isolated CNBD fragment using NMR (Saponaro et al., 2014), plus the unexpected folding of two additional helices at the C-terminus of the CNBD. These helices were not included in any of the previous experiments conducted using the isolated C-linker/CNBD fragment. To understand whether these two dynamic helices play a role in regulating both cAMP and TRIP8b binding, we have compared, using an in vitro binding assay, two CNBD constructs with or without the newly discovered helices. Surprisingly, their presence increases the affinity for cAMP without affecting TRIP8b binding. This result assigns to the new C-terminal helices of the HCN channel CNBD a specific role in controlling cyclic nucleotide affinity.
Assigning Function to the D and E Helices of HCN CNBD / A. Saponaro, A. Porro, C. Donadoni, B. Santoro, G. Thiel, A. Moroni. - In: BIOPHYSICAL JOURNAL. - ISSN 1542-0086. - 114:3 (Suppl. 1)(2018 Feb 19), pp. 1514-Pos Board B423.303a-1514-Pos Board B423.303a. ((Intervento presentato al 62. convegno Annual Biophysical Meeting : February, 17th - 21st tenutosi a San Francisco (CA, USA) nel 2018.
|Titolo:||Assigning Function to the D and E Helices of HCN CNBD|
SAPONARO, ANDREA COSIMO (Primo)
PORRO, ALESSANDRO (Secondo)
MORONI, ANNA (Ultimo)
|Parole Chiave:||HCN channels, CNBD, cAMP|
|Settore Scientifico Disciplinare:||Settore BIO/09 - Fisiologia|
Settore BIO/10 - Biochimica
|Data di pubblicazione:||19-feb-2018|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1016/j.bpj.2017.11.1721|
|Appare nelle tipologie:||01 - Articolo su periodico|