Abstract
Mutations in PRoline Rich Transmembrane protein 2 (PRRT2) cause pleiotropic syndromes including benign infantile epilepsy, paroxysmal kinesigenic dyskinesia, episodic ataxia, that share the paroxysmal character of the clinical manifestations. PRRT2 is a neuronal protein that plays multiple roles in the regulation of neuronal development, excitability, and neurotransmitter release. To better understand the physiopathology of these clinical phenotypes, we investigated PRRT2 interactome in mouse brain by a pulldown-based proteomic approach and identified α1 and α3 Na+/K+ ATPase (NKA) pumps as major PRRT2-binding proteins. We confirmed PRRT2 and NKA interaction by biochemical approaches and showed their colocalization at neuronal plasma membrane. The acute or constitutive inactivation of PRRT2 had a functional impact on NKA. While PRRT2-deficiency did not modify NKA expression and surface exposure, it caused an increased clustering of α3-NKA on the plasma membrane. Electrophysiological recordings showed that PRRT2-deficiency in primary neurons impaired NKA function during neuronal stimulation without affecting pump activity under resting conditions. Both phenotypes were fully normalized by re-expression of PRRT2 in PRRT2-deficient neurons. In addition, the NKA-dependent afterhyperpolarization that follows high-frequency firing was also reduced in PRRT2-silenced neurons. Taken together, these results demonstrate that PRRT2 is a physiological modulator of NKA function and suggest that an impaired NKA activity contributes to the hyperexcitability phenotype caused by PRRT2 deficiency.
Highlights
Introduction Mutations in thePRRT2 gene cause a variety of paroxysmal disorders including paroxysmal kinesigenic dyskinesia (PKD), benign infantile epilepsy, episodic ataxia, and migraine that can be present alone or in combination
We show that PRRT2 interacts with both the α1 and the α3 subunits of Na+/K+ ATPase (NKA) and that PRRT2 deficiency affects α3-NKA clustering on the plasma membrane and impairs NKA function during neuronal stimulation, a phenotype that was rescued by PRRT2 expression in PRRT2-lacking neurons
We focused on a region of the Coomassie-stained gels spanning between 95 and 130 kDa, where we discriminated few distinct bands in the PRRT2-HA lane that were virtually absent in the bacterial alkaline phosphatase (BAP)-HA lane (Supplementary Fig. 1A, insets)
Summary
Introduction Mutations in thePRRT2 gene cause a variety of paroxysmal disorders including paroxysmal kinesigenic dyskinesia (PKD), benign infantile epilepsy, episodic ataxia, and migraine that can be present alone or in combination. Most patients (>80%) carry the same frameshift mutation (c.649dupC; p.Arg217Profs*8) that generates a precocious stop codon followed by degradation of the mRNA or the protein, leading to haploinsufficiency[1,2,3,4]. PRRT2 is a neuronal type-2 transmembrane protein that is a major determinant of network stability[5,6,7]. PRRT2 knock out (KO) mice are viable and fertile, but show hyperkinetic movements, motor paroxysms, and an increased seizure propensity that are very reminiscent of the human PRRT2-linked pathologies[11]. Relevant is the involvement of the cerebellum, where PRRT2 deficiency in granule cells recapitulates the behavioral phenotype of PRRT2 KO mice[11,12]
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