Connexin 43 hemi channels mediate Ca2+-regulated transmembrane NAD+ fluxes in intact cells1
- SANTINA BRUZZONE*,
- LUCREZIA GUIDA*,
- ELENA ZOCCHI*,
- LUISA FRANCO† and
- ANTONIO DE FLORA*,2
- *Department of Experimental Medicine, Section of Biochemistry, University of Genova, 16132 Genova, Italy; and
- †Biocrystallography Center-CNR, University Federico II, 80134 Naples, Italy
- ↵Correspondence: 2Correspondence: Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV, 1, 16132 Genova, Italy. E-mail: toninodf{at}unige.it
SPECIFIC AIMS
Several mammalian cell types have been recently shown to express a NAD+ transport system on the plasma membrane. The fundamental and pleiotropic roles played by NAD+ in physiological processes (ranging from redox reactions and metabolism, signaling mechanisms, and DNA repair) prompted us to characterize the NAD+ transporter of NIH 3T3 fibroblasts, since this murine cell line had previously been shown to exhibit gradient-directed transmembrane fluxes of NAD+, both influx of externally added NAD+ and release of intracellular NAD+ into the medium.
PRINCIPAL FINDINGS
1. The NAD+ transporter from 3T3 cells can be reconstituted into unilamellar proteoliposomes
Total membrane proteins from 3T3 fibroblasts were reconstituted into unilamellar proteoliposomes, which were then tested for NAD+ influx using either 32[P]-NAD+ or unlabeled dinucleotide. This process was dependent on time, protein concentration, and pH, with maximum influx being observed at pH 8.3. Influx of NAD+ was almost completely inhibited by NADP+, NADPH, NADH, nicotinamide, ADP, ATP, and FAD. GSSG and thiol reagents abolished NAD+ transport completely.
2. Calcium inhibits NAD+ transport across the plasma membrane of intact 3T3 fibroblasts
Although NAD+ transport in reconstituted proteoliposomes was unaffected by Ca2+, extracellular and intracellular Ca2+ were found to inhibit influx and efflux of NAD+ in intact 3T3 cells. Chelation of extracellular Ca2+ (5 mM EDTA) increased ∼ threefold NAD+ transport (both influx and release) over values observed in Ca2+-containing medium (DME) (Fig. 1a⇓ ). Increasing intracellular Ca2+ by means of 20 μM A23187 ionophore blocked NAD+ fluxes completely (Fig. 1a⇓ ). Moreover, extracellular Ca2+ inhibited NAD+ influx in a concentration-dependent fashion (Fig. 1b⇓ ).
Inhibition by Ca2+ of NAD+ transport in intact 3T3 fibroblasts. a) Chelation of extracellular Ca2+ (5 mM EDTA) and intracellular Ca2+ loading (20 μM A23187) affect influx (white bars, n=5) and efflux …
