STUDY OF THE INTERACTION BETWEEN HCN CHANNELS AND THEIR REGULATORY SUBUNITS TRIP8B AND MINT PROTEINS

Ion channels are membrane proteins that control crucial physiological and pathological processes in all organisms. Channels are often composed by different subunits, forming macromolecular protein complexes. They include α subunits, which are the pore-forming channel subunits, and they also include β subunits, which associate to the α subunits and can regulate channel function, surface expression and trafficking (Yu et al. 2005). Hyperpolarization-activated cyclic nucleotide gated channels (HCN1-4) are the molecular determinants of the so-called pacemaker current If/h current which controls cell excitability both in the brain and in the heart (Robinson & S. a Siegelbaum 2003). HCN channels are dually activated by membrane hyperpolarization and cAMP binding to their cyclic nucleotide binding domain (CNBD); moreover, several regulatory β subunits have been reported to regulate HCN channels (Robinson & S. a Siegelbaum 2003), among which TRIP8b. The cytoplasmatic tetratricopeptide repeat containing Rab8b interacting protein (TRIP8b) is an auxiliary β subunit of neuronal HCN isoforms, governing ion channel trafficking and gating. The interaction between HCN and TRIP8b proteins is already well explained in molecular details and takes place at two distinct sites: an upstream binding site where the CNBD of HCN channels interacts with an 80 aa domain in the conserved central core of TRIP8b (called miniTRIP8b); and a downstream site where the C-terminal SNL (Ser-Asn-Leu) tripeptide of the channel interacts with the tetratricopeptide repeat domain of TRIP8b (Santoro et al. 2011). In my thesis, I identified some key residues on the CNBD important for complex formation, and finally I was able to obtain an HCN double mutant N547D/A548C unable to bind TRIP8b at the upstream binding site. This will prevent the regulation of channel activity by its β subunit without affecting the regulation of channel trafficking. This mutant represents an important tool that can be used in vivo to investigate the physiological importance of TRIP8b regulation of HCN channels in certain brain regions, such as the hippocampus. Recently HCN channels were found to interact with Mint proteins. Less is known about this new interaction. Mints are a class of adaptor proteins which contain protein-protein interaction domains through which they mediate the assembly of functional multiprotein complexes (Rogelj et al. 2006). The study of Mint proteins has become central in Alzheimer’s disease. Indeed, Mint proteins are known to directly bind the cytoplasmic tail of APP. This binding is important to regulate APP trafficking and notably also its cleavage, thus β-amyloid peptide (Aβ) production (Rogelj et al. 2006). Mints were recently reported to interact with HCN channels (Saito et al. 2012; Kimura et al. 2004) but neither the nature of the binding (direct vs. indirect) nor the interaction site between the two partners is so far known. In my thesis I have performed a preliminary characterization of this new interaction by a combination of in vitro and in vivo experimental approaches. In particular I have investigated the functional effects of the interaction between Mint proteins and HCN channels and found that Mint expression causes a decrease in HCN channel current, due to their redistribution from the plasma membrane to intracellular compartments. In addition I was able to identify a specific region located C-terminal to the CNBD of the channel which is important for the interaction.