NECAB1 and NECAB2 are Prevalent Calcium-Binding Proteins of CB(1)/CCK-Positive GABAergic Interneurons

The molecular repertoire of the “Ca(2+)-signaling toolkit” supports the specific kinetic requirements of Ca(2+)-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 Ca(2+)-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 CB(1) cannabinoid receptor/cholecystokinin (CB(1)/CCK)-positive interneurons that represent a large population of GABAergic cells in cortical circuits. Because CB(1)/CCK-positive interneurons display disparate presynaptic and somatodendritic Ca(2+)-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 CB(1)/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 CB(1)/CCK-positive interneurons.