NECAB1 and NECAB2 are Prevalent Calcium-Binding Proteins of CB1/CCK-Positive GABAergic Interneurons.

Vivien Miczán,Benjámin Barti,Judit R Glavinics,Krisztina Kelemen,Zsófia I László,István Katona,Máté Kisfali,Kata Kenesei

doi:10.1093/cercor/bhaa326

Abstract

The molecular repertoire of the “Ca2+-signaling toolkit” supports the specific kinetic requirements of Ca2+-dependent processes in different neuronal types. A well-known example is the unique expression pattern of calcium-binding proteins, such as parvalbumin, calbindin, and calretinin. These cytosolic Ca2+-buffers control presynaptic and somatodendritic processes in a cell-type-specific manner and have been used as neurochemical markers of GABAergic interneuron types for decades. Surprisingly, to date no typifying calcium-binding proteins have been found in CB1 cannabinoid receptor/cholecystokinin (CB1/CCK)-positive interneurons that represent a large population of GABAergic cells in cortical circuits. Because CB1/CCK-positive interneurons display disparate presynaptic and somatodendritic Ca2+-transients compared with other interneurons, we tested the hypothesis that they express alternative calcium-binding proteins. By in silico data mining in mouse single-cell RNA-seq databases, we identified high expression of Necab1 and Necab2 genes encoding N-terminal EF-hand calcium-binding proteins 1 and 2, respectively, in CB1/CCK-positive interneurons. Fluorescent in situ hybridization and immunostaining revealed cell-type-specific distribution of NECAB1 and NECAB2 throughout the isocortex, hippocampal formation, and basolateral amygdala complex. Combination of patch-clamp electrophysiology, confocal, and STORM super-resolution microscopy uncovered subcellular nanoscale differences indicating functional division of labor between the two calcium-binding proteins. These findings highlight NECAB1 and NECAB2 as predominant calcium-binding proteins in CB1/CCK-positive interneurons.

Highlights

IntroductionTo subserve pleiotropic physiological functions, Ca2+signaling dynamics must be tightly controlled in a spatially and temporally restricted manner (Berridge et al 2000)
Ionized calcium (Ca2+) is the most versatile intracellular messenger
In order to detect candidate calcium-binding proteins that may shape specific Ca2+-signaling dynamics and serve as neurochemical markers of cannabinoid receptor/cholecystokinin (CB1/CCK)-positive interneurons, we first exploited the original datasets provided by the Karolinska Institute that were obtained from cells in the CA1 subfield of the hippocampus and in the somatosensory cortex of mice (Zeisel et al 2015)

Summary

Introduction

To subserve pleiotropic physiological functions, Ca2+signaling dynamics must be tightly controlled in a spatially and temporally restricted manner (Berridge et al 2000). A myriad of proteins, the so-called “Ca2+-signaling toolkit,” were evolved to mediate and regulate Ca2+-entry, cytosolic free Ca2+-levels, and Ca2+-extrusion/uptake. Each of the 249 EF-hand Ca2+-binding proteins encoded in the mouse genome show characteristic distribution patterns in the brain (Girard et al 2015). The molecular and anatomical diversity together with the highly different Ca2+-binding kinetics of these proteins indicate that cell-typespecific regulation of the spatio-temporal properties of Ca2+signaling is essential for specific computational functions in brain circuits. The cellular complexity in the brain represents a major challenge and our knowledge about how the specific molecular components of the “Ca2+-signaling toolkit” determine distinct physiological functions has remained rather limited in most cell types

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