A Self-restricted CD38-connexin 43 Cross-talk Affects NAD+ and Cyclic ADP-ribose Metabolism and Regulates Intracellular Calcium in 3T3 Fibroblasts

Abstract

Connexin 43 (Cx43) hexameric hemichannels, recently demonstrated to mediate NAD+ transport, functionally interact in the plasma membrane of several cells with the ectoenzyme CD38 that converts NAD+ to the universal calcium mobilizer cyclic ADP-ribose (cADPR). Here we demonstrate that functional uncoupling between CD38 and Cx43 in CD38-transfected 3T3 murine fibroblasts is paralleled by decreased [Ca2+]i levels as a result of reduced intracellular conversion of NAD+ to cADPR. A sharp inverse correlation emerged between [Ca2+]i levels and NAD+ transport (measured as influx into cells and as efflux therefrom), both in the CD38+ cells (high [Ca2+] i, low transport) and in the CD38-fibroblasts (low [Ca 2+]i, high transport). These differences were correlated with distinctive extents of Cx43 phosphorylation in the two cell populations, a lower phosphorylation with high NAD+ transport (CD38-cells) and vice versa (CD38+ cells). Conversion of NAD+-permeable Cx43 to the phosphorylated, NAD+-impermeable form occurs via Ca 2+-stimulated protein kinase C (PKC). Thus, a self-regulatory loop emerged in CD38+ fibroblasts whereby high [Ca2+] i restricts further Ca2+ mobilization by cADPR via PKC-mediated disruption of the Cx43-CD38 cross-talk. This mechanism may avoid: (i) leakage of NAD+ from cells; (ii) depletion of intracellular NAD+ by CD38; (iii) overproduction of intracellular cADPR resulting in potentially cytotoxic [Ca2+]i.