Role of the NMDAR-GluN2B subunits in the function of supramolecular NMDAR-BK channel complexes.

Abstract

Large conductance calcium- and voltage-activated potassium channels (BK) are widely expressed across many tissues, contributing to many physiological functions. Membrane depolarization and relatively high (micromolar) intracellular concentrations of Ca2+ are required for their activation. Such concentrations are reached in the vicinity of Ca2+-permeant ion channels, to which BK proteins are closely located in various tissues, including the brain. N-methyl-D-aspartate receptors (NMDAR) are sodium (Na+)- and Ca2+-conducting glutamate-activated ion channels that are functionally coupled to BK channels in the olfactory bulb and the dentate gyrus, contributing to decreased neuronal intrinsic excitability. Recently, our group demonstrated that postsynaptic NMDAR-BK complexes at basal dendrites of somatosensory cortex layer 5 pyramidal neurons regulate synaptic transmission and long-term plasticity. However, the specific contribution of different NMDAR subunits (GluN1 and GluN2A/GluN2B) to the NMDAR-BK interaction remains elusive. Defects in this association may be of pathological relevance in the context of EIEE27 syndrome (epileptic encephalopathy, early infantile, 27), which has been related to mutations in the GluN2B-encoding gene (GRIN2B). In this work I aimed to understand the role of GluN2B subunits in the function of NMDAR-BK complexes and, more specifically, if GluN2B-related human mutations modify NMDAR-BK coupling. Function of NMDAR-BK complexes containing different EIEE27-related GluN2B mutations was examined using electrophysiology in heterologous expression systems. Proximity between channel proteins within complexes was assessed using in situ proximity ligation assay (PLA). Membrane expression efficiency was studied employing TIRF microscopy (Total Internal Reflection Fluorescence). Our results reveal some disease-related GluN2B mutations reduce the NMDAR-BK interaction, either by altered interactions with BK channels and/or by functional uncoupling between the channels within the NMDAR-BK complexes.