Connexin-43 hemichannels mediate cyclic ADP-ribose generation and its Ca2+-mobilizing activity by NAD+/cyclic ADP-ribose transport.
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 Fcγ receptor (FcγR) 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 FcγR stimulation. FcγR stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between FcγR 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.
Departments of Microbiology and Biochemistry, Chonbuk National University Medical School and Hospital, Jeonju 561-182, Korea.
Eun-Kyung
EK
Song
So-Young
SY
Rah
Young-Rae
YR
Lee
Chae-Hwa
CH
Yoo
Yu-Ri
YR
Kim
Ji-Hyun
JH
Yeom
Kwang-Hyun
KH
Park
Jong-Suk
JS
Kim
Uh-Hyun
UH
Kim
Myung-Kwan
MK
Han
D051997:ADP-ribosyl Cyclase 1 / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D036563:Cyclic ADP-Ribose / Q000235:genetics / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009243:NAD / Q000235:genetics / Q000378:metabolism*; D017452:Receptors, IgG / Q000235:genetics / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000378:metabolism
D018031:Connexin 43; C500216:GJA1 protein, human; C543076:GJA1 protein, mouse; D008562:Membrane Glycoproteins; D017452:Receptors, IgG; D019837:Ryanodine Receptor Calcium Release Channel; D009243:NAD; D036563:Cyclic ADP-Ribose; D017868:Cyclic AMP-Dependent Protein Kinases; C498154:CD38 protein, human; C498155:Cd38 protein, mouse; D051997:ADP-ribosyl Cyclase 1; D002118:Calcium