Reconstitution of a passive Ca(2+)-transport pathway from the basolateral plasma membrane of rat parotid gland acinar cells.
We have previously reported that rat parotid gland basolateral plasma membrane vesicles (BLMV) have a relatively high affinity Ca2+ transport pathway and an unsaturable Ca2+ flux component (Lockwich et al., 1994. J. Membrane Biol. 141:289-296). In this study, we have solubilized BLMV with octylglucoside (1.5%) and have reconstituted the solubilized proteins into proteoliposomes (PrL) composed of E. coli bulk phospholipids, by using a detergent dilution method. PrL exhibited 3-5-fold higher 45Ca2+ influx than control liposomes (without protein). Ca2+ uptake into PrL was dependent on the [protein] in PrL and steady state [Ca2+] in PrL was in equilibrium with external [Ca2+]. These data demonstrate that a passive, protein-mediated Ca2+ transport has been reconstituted from BLMV into PrL. 45Ca2+ influx into liposomes did not saturate with increasing [Ca2+] in the assay medium. In contrast, PrL displayed saturable 45Ca2+ influx and exhibited a single Ca2+ flux component with an apparent KCa = 242 +/- 50.9 microM and Vmax = 13.5 +/- 1.14 nmoles Ca2+/mg protein/ minute. The KCa of Ca(2+)-transport in PrL was similar to that of the high affinity Ca2+ influx component in BLMV while the Vmax was about 4-fold higher. The unsaturable Ca2+ flux component was not detected in PrL. 45Ca2+ influx in PrL was inhibited by divalent cations in the order of efficacy, Zn2+ > Mn2+ > Co2+ = Ni2+, and appeared to be more sensitive to lower concentrations of Zn2+ than in BLMV. Consistent with our observations with BLMV, the carboxyl group reagent N,N'-dicyclohexylcarbodiimide (DCCD) inhibited the reconstituted Ca2+ transport in PrL. Importantly, in both BLMV and PrL, DCCD induced a 40-50% decrease in Vmax of Ca2+ transport without an alteration in KCa. These data strongly suggest that the high affinity, passive Ca2+ transport pathway present in BLMV has been functionally reconstituted into PrL. We suggest that this approach provides a useful experimental system towards isolation of the protein(s) involved in mediating Ca2+ influx in the rat parotid gland basolateral plasma membrane.
Lockwich
T
T
National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20292, USA.
Chauthaiwale
J
J
Ambudkar
S V
SV
Ambudkar
I S
IS
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005960:Glucosides / Q000494:pharmacology; D008081:Liposomes / Q000378:metabolism; D008297:Male; D010306:Parotid Gland / Q000166:cytology*; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D003902:Detergents; D005960:Glucosides; D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes; C018619:octyl-beta-D-glucoside; D004024:Dicyclohexylcarbodiimide; D002118:Calcium