The cell adhesion molecule coxsackie virus and adenovirus receptor (CAR) modulates intracellular Ca2+ concentration and Cl- conductance in cultivated mouse cortical neurons (2009)

How cell adhesion proteins of the Ig superfamily modulate intracellular signaling cascades and thereby influence neuronal communication is not well understood. In contrast to other CAMs, the coxsackie virus and adenovirus receptor (CAR) is strongly expressed in the embryonic and early postnatal brain. Initially it is found on all neural cells but becomes concentrated in axon- and dendrite-rich areas, suggesting that it might play a role in the development of neuronal circuits. Here, I investigated the function of CAR by patch-clamp recordings using CAR-deficient neurons or reagents binding to CAR (fiber knob Ad2, anti-CAR antibodies, recombinant extracellular CAR domains). Initially I studied passive membrane properties of cultivated neurons. Unexpectedly, I observed an increased Rm in CAR-deficient neurons. In parallel, a decrease of Rm was measured in wild type neurons in the presence of the fiber knob Ad2 – a trimeric protein binding to CAR that disrupts cell- cell contacts. Most likely CAR-mediated changes in membrane resistance (Rm) are the result of an altered Cl- conductance across the membrane, which is impaired in CAR-deficient neurons and enhanced in Ad2 treated neurons. This conclusion is based on several electrophysiological recordings including the comparison of the Cl- conductance between CAR-deficient and WT neurons, the Cl- ion substitution in the recording solution, the pharmacological block of the Cl- current as well as shifting the reversal potential of Cl-. In addition, investigations on the conductance of other ions such as Na+ and K+ across the membrane as well as investigations on gap junctions or on Na+/K+-ATPase, HCN or voltage-gated Na+, K+, Ca2+ channels revealed no evidence for their implication in the deficits observed in CAR KO neurons. Furthermore, it could be shown that Ad2 is able to increase the [Ca2+]i, by releasing Ca2+ from intracellular Ca2+ stores. An Ad2-mediated increase in the [Ca2+]i leads, most probably, to activation of the Ca2+-activated Cl- channels (CaCCs). Several characteristical properties for the CaCCs could be observed including outward rectification, voltage- and Ca2+-dependency and specific halide selectivity for these channels. This enhanced Cl- conductance in Ad2 treated neurons results in a significantly lower Rm. The absence of CAR and the Ad2-mediated changes in the membrane conductance influence also neuronal network activity. CAR-deficient neurons showed a significantly higher frequency of action potentials (APs) compared to WT cells, whereas Ad2 treated neurons revealed a significantly lower frequency of APs than control cells. Furthermore, investigation of neuronal network formation and synaptic activity in CAR-deficient neurons revealed an imbalance in the excitatory and inhibitory input ratio, suggesting a function of CAR in the formation of neuronal networks. The data presented in this work lead to the conclusions that CAR plays an important role during the development of neurons by modulating the [Ca2+]i, the membrane conductance as well as synaptic activity and action potential generation.