PRRT2-Na+ CHANNELS INTERACTION: PATHOGENETIC BASIS OF PRRT2-ASSOCIATED PAROXYSMAL DISORDERS AND NEW THERAPEUTIC STRATEGIES

Proline-Rich Transmembrane protein 2 (PRRT2) is a neuron-specific protein whose mutations are involved in pleiotropic paroxysmal syndromes including epilepsy, kinesigenic dyskinesia, episodic ataxia and migraine. PRRT2 is a type-2 membrane protein with a transmembrane domain and a long proline-rich N-terminal cytoplasmic region. According to several data, PRRT2 regulates membrane exposure and the biophysical properties of voltage-dependent Na+ channels (Nav) 1.2 and 1.6 that negatively modulate intrinsic excitability. Nav channels form complexes with β-subunits that facilitate the membrane targeting and the activation of the α-subunits. The objective of this thesis is to characterize the molecular and functional PRRT2-Nav interaction clarify: (i) whether PRRT2 and β-subunits interact or compete for common binding sites on the α-subunit, generating Nav complexes with distinct functional properties, (ii) based on its membrane topology, study the structure-function PRRT2 relationships regarding the interaction with Nav, (iii) focus on some point PRRT2 mutations involved in the binding to the Nav directly implicated in PRRT2-related pathologies. Since PRRT2 and β-subunits have opposite effects on Nav channels, it is unclear whether PRRT2 and β-subunits interact or compete for common binding sites on the α-subunit, leading to Nav complexes with different functional features. Using a heterologous expression system, we observed that β-subunits and PRRT2 do not interact with each other acting as independent non-competitive modulators of Nav1.2 channel trafficking and biophysical properties. The data indicate that β4-subunit and PRRT2 form a push-pull system that finely tunes the membrane expression and function of Nav channels and the intrinsic neuronal excitability. In addition, we observed that the unstructured N-terminal cytoplasmic region mimicked full-length PRRT2 by binding to the Nav1.2 more efficiently than the isolated transmembrane domain. Only the C-terminal intramembrane domain was able to modulate Nav properties, maintaining the striking specificity for Nav1.2 vs Nav1.1 channels. These results identify PRRT2 as a multi-domain protein in which the N-terminal cytoplasmic region acts as a binding antenna for Na+ channels, while the transmembrane domain mechanistically regulates channel exposure on the membrane and its biophysical properties. Since the majority of the PRRT2 pathogenic mutations cause the loss of protein expression making in vitro studies difficult or impossible, a restricted number of missense mutations maintains the protein expression and the trafficking to the membrane allowing their characterization. Hence, their expression and function were studied in the same system used for the previous points. Two residues were identified, V286M and A320, that, if mutated, cause Nav binding alteration, and therefore that can be involved in the direct modulation of PRRT2 functions.