Connexin-43 Hemichannels Mediate Cyclic ADP-ribose Generation and Its Ca2+-mobilizing Activity by NAD+/Cyclic ADP-ribose Transport (original) (raw)

2011, Journal of Biological Chemistry

Background: The ADP-ribosyl cyclase CD38 produces cyclic ADP-ribose from NAD ϩ in the extracellular space. Cyclic ADP-ribose induces intracellular Ca 2ϩ mobilization. Results: We demonstrate that connexin 43 hemichannels import cyclic ADP-ribose to the intracellular target ryanodine receptor. Conclusion: Connexin 43 hemichannels mediate the extracellular production and Ca 2ϩ-mobilizing action of cyclic ADP-ribose. Significance: We show that connexin 43 hemichannels resolve the topological hindrance between CD38 and ryanodine receptor. The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD ؉. cADPR increases intracellular Ca 2؉ through the intracellular ryanodine receptor/Ca 2؉ release channel (RyR). It has been known that intracellular NAD ؉ approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. However, it is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR although CD38 itself or nucleoside transporter has been proposed to import cADPR. Moreover, it has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD ؉. Here we demonstrate that Cx43 hemichannels, but not CD38, import cADPR to increase intracellular calcium through RyR. We also demonstrate that physiological stimulation such as Fc␥ receptor (Fc␥R) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD ؉ export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. Protein kinase A (PKA) activation also induced calcium mobilization in the same way as Fc␥R stimulation. Fc␥R stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between Fc␥R stimulation and NAD ؉ /cADPR transport. Cx43 knockdown blocked extracellular cADPR import and extracellular cADPR-induced calcium mobilization in J774 cells. Cx43 overexpression in Cx43-negative cells conferred extracellular cADPR-induced calcium mobilization by the mediation of cADPR import. Our data suggest that Cx43 has a dual function exporting NAD ؉ and importing cADPR into the cell to activate intracellular calcium mobilization. Cyclic ADP-ribose (cADPR) 3 is produced from NAD ϩ by the ADP-ribosyl cyclase CD38, a type II transmembrane glycoprotein first identified as a lymphocyte differentiation antigen. cADPR directly binds to types II and III ryanodine receptors (RyRs) to induce Ca 2ϩ release from RyR containing Ca 2ϩ stores located in the sarcoplasmic and endoplasmic reticulum in a variety of cells (1-3). In a limited number of cell systems, cADPR binds to FKBP12.6 and mediates responsiveness of RyR toward cADPR (4-5). The cADPR-mediated increase in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i) controls various biological processes including egg fertilization (6-7), cell activation, and proliferation (8-9), muscle contraction (10), hormone secretion (11-12), and immune responses (13). The catalytic site of CD38 is located outside of the cell, but the substrate NAD ϩ is inside. Moreover, all cADPR targets are located inside and not in direct contact with extracellular cADPR. Therefore, the CD38/cADPR/Ca 2ϩ signaling system has two topological questions, the way to export NAD ϩ to CD38 and the way to import cADPR to its intracellular targets. These topological problems have been described as the topological paradox (14). Many efforts have been done to solve the topological paradox. It has been reported that approach of intracellular NAD ϩ to ecto-CD38 could be carried out by connexin 43 (Cx43) hemichannels, a component of gap junctions (15), and that approach of cADPR to its intracellular target