H+ countertransport and electrogenicity of the sarcoplasmic reticulum Ca2+ pump in reconstituted proteoliposomes

Biophys J. 1993 Apr;64(4):1232-42. doi: 10.1016/S0006-3495(93)81489-9.

Abstract

The Ca2+ transport adenosine triphosphatase of sarcoplasmic reticulum was reconstituted in unilamellar liposomes prepared by reverse-phase evaporation. The size of the resulting proteoliposomes was similar to that of native sarcoplasmic reticulum vesicles, but their protein content was much lower, with a protein/lipid ratio (wt/wt) of 1:40-160, as compared with 1:1 in the native membrane. The proteoliposomes sustained adenosine triphosphate-dependent Ca2+ uptake at rates proportional to the protein content (1-2 mumol Ca2+/mg protein/min), reaching asymptotic levels corresponding to a lumenal calcium concentration of 10-20 mM. The low permeability of the proteoliposomes permitted direct demonstration of Ca2+/H+ countertransport and electrogenicity by parallel measurements in the same experimental system. Countertransport of one H+ per one Ca2+ was demonstrated, and inhibition of the Ca2+ pump by lumenal alkalinization was relieved by the H+ ionophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone. Consistent with the countertransport stoichiometry, net positive charge displacement was produced by Ca2+ transport, as revealed by a rapid oxonol VI absorption rise. The initial rise and the following steady-state level of oxonol absorption were highest when SO4(2-) was the prevalent anion and lowest in the presence of the lipophilic anion SCN-. The influence of anions was attributed to potential driven counterion compensation. The absorption rise was rapidly collapsed by addition of valinomycin in the presence of K+. Experimentation with Ca2+ and H+ ionophores was consistent with a primary role of Ca2+ and H+ in net charge displacement. The estimated value of the steady-state electrical potential observed under optimal conditions was approximately 50 mV and was accounted for by the estimated charge transfer associated with Ca2+ and H+ countertransport under the same conditions.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Animals
  • Biophysical Phenomena
  • Biophysics
  • Calcium / metabolism
  • Calcium-Transporting ATPases / physiology*
  • Coloring Agents
  • Electrochemistry
  • In Vitro Techniques
  • Ion Transport
  • Isoxazoles
  • Membrane Potentials
  • Proteolipids / metabolism*
  • Protons
  • Rabbits
  • Sarcoplasmic Reticulum / metabolism*
  • Spectrophotometry

Substances

  • Coloring Agents
  • Isoxazoles
  • Proteolipids
  • Protons
  • proteoliposomes
  • oxonol VI
  • Adenosine Triphosphate
  • Calcium-Transporting ATPases
  • Calcium