The Journal of Membrane Biology

, Volume 187, Issue 3, pp 185–201 | Cite as

Reincorporated Plasma Membrane Ca2+-ATPase can Mediate B-Type Ca2+ Channels Observed in Native Membrane of Human Red Blood Cells

  • C. Pinet
  • S. Antoine
  • A.G. Filoteo
  • J.T. Penniston
  • A. Coulombe

Recently, we reported indirect evidence that plasma membrane Ca2+-ATPase (PMCA) can mediate B-type Ca2+ channels of cardiac myocytes. In the present study, in order to bring more direct evidence, purified PMCA from human red blood cells (RBC) was reconstituted into giant azolectin liposomes amenable to the patch-clamp technique. Purified RBC PMCA was used because it is available pure in larger quantity than cardiac PMCA. The presence of B-type Ca2+ channels was first investigated in native membranes of human RBC. They were detected and share the characteristics of cardiac myocytes. They spontaneously appeared in scarce short bursts of activity, they were activated by chlorpromazine (CPZ) with an EC50 of 149 mmole/l or 1 mmole/l vanadate, and then switched off by 10 mmole/l eosin or dose-dependently blocked by 1–5 mmole/l ATP. Independent of membrane potential, the channel gating exhibited complex patterns of many conductance levels, with three most often observed conductance levels of 22, 47 and 80 pS. The activation by vanadate suggests that these channels could play a role in the influx of extracellular Ca2+ involved in the vanadate-induced Gardos effect. In PMCA-reconstituted proteoliposomes, nearly half of the ATPase activity was retained and clear "channel-like" openings of Ba2+- or Ca2+-conducting channels were detected. Channel activity could be spontaneously present, lasting the patch lifetime or, when previously quiescent, activity could be induced by application of 50 mmole/l CPZ only in presence of 25 U/ml calmodulin (CaM), or by application of 1 mmole/l vanadate alone. Eosin (10 mmole/l) and ATP (5 mmole/l) significantly reduced spontaneous activity. Channel gating characteristics were similar to those of RBC, with main conductance levels of 21, 40 and 72 pS. The lack of direct activation by CPZ alone might be attributed to a purification-induced modification or absence of unidentified regulatory component(s) of PMCA. Despite a few differences in results between RBC and reincorporated PMCA, most probably attributable to the decrease in ATPase activity following the procedure of reincorporation, the present experimental conditions appear to reveal a channel-mode of the PMCA that shares many similarities with the B-type Ca2+ channel.

Keywords

Vanadate Eosin ATPase Activity Chlorpromazine Cardiac Myocytes 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Springer-Verlag New York Inc. 2002

Authors and Affiliations

  • C. Pinet
    • 1
  • S. Antoine
    • 1
  • A.G. Filoteo
    • 2
  • J.T. Penniston
    • 2
  • A. Coulombe
    • 1
  1. 1.Laboratoire de Physiologie Cardio-vasculaire et Thymique (CNRS UMR 8078), Hôpital Marie Lannelongue, 133 Avenue de la Résistance, F-92350 Le Plessis Robinson, FranceFR
  2. 2.Department of Biochemistry and Molecular Biology, Mayo Foundation, Rochester, Minnesota 55905, USAUS

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