[Frontiers in Bioscience 12, 4641-4660, May 1, 2007]

[Frontiers in Bioscience 12, 4641-4660, May 1, 2007]

Ca²⁺/H⁺ exchange via the plasma membrane Ca²⁺ ATPase in skeletal muscle

J. DeSantiago, D. Batlle, M. Khilnani, S. Dedhia, J. Kulczyk, R. Duque, J. Ruiz, C. Pena-Rasgado, Hector Rasgado-Flores

1 Dept. Physiology and Biophysics, Rosalind Franklin University of Medicine and Science, The Chicago Medical School, N. Chicago, IL 60064, ²Dept. Medicine, Division of Nephrology and Hypertension, Northwestern University. The Feinberg School of Medicine, Chicago, IL 60611

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and Methods: 3.1. Experimental set-up; 3.2. External (superfusion) solutions; 3.2. Internal (perfusion) solutions; 3.3. Measurement of pHi; 3.4. Measurement of the intracellular pH buffering power; 3.5. Measurement of tracer efflux; 3.6. Determination of the stoichiometry of Ca²⁺/H⁺ exchange; 3.7. Voltage-clamping of Barnacle muscle cells; 3.8. Reagents; 3.9. Statistics
4. Results: 4.1. Effect of changes in external H⁺ (pH_o) on Ca²⁺efflux; 4.2. Effect of removal of intracellular ATP on Ca²⁺efflux; 4.3. Effect of plasma membrane Ca²⁺ATPase inhibitors on Ca²⁺efflux induced by external acidification; 4.4. Activation of Ho-dependent Ca²⁺ efflux by (Ca²⁺)_i; 4.5. Requirement of Ca²⁺/H⁺ exchange for the function of the PMCA; 4.6. Stoichiometry of Ca²⁺/H⁺ exchange; 4.7. Voltage-dependence of PMCA-mediated Ca²⁺/H⁺ exchange
5. Discussion: 5.1. Evidence that Ca²⁺efflux via Ca²⁺/H⁺ is mediated by the PMCA; 5.2. Dependence on H_o for activity of the Ca²⁺pump; 5.3. Stoiochioometry, coupling ratio and electrogenicity of the sarcolemmal Ca²⁺/H⁺ exchange; 5.4. Thermodynamic considerations; 5.5. Voltage-dependence of PMCA-mediated Ca²⁺/H⁺ exchange; 5.6. Physiological inmplications of mediation of Ca²⁺/H⁺ exchange via the PMCA
6. Acknowledgments
7. References

1. ABSTRACT

The aims of this work were to determine: 1) whether Ca²⁺exit via the plasmalemmal Ca²⁺ATPase (PMCA) is coupled to H⁺entry via a Ca²⁺/H⁺ exchange; 2) whether operation of PMCA has an absolute requirement on external H⁺ (H_o); and 3) the stoichiometry and voltage-dependence of the Ca²⁺/H⁺ exchange. Barnacle muscle cells were used because of the ease with which they can be internally-perfused (e.g., with ⁴⁵Ca), voltage-clamped and impaled with a pH electrode. Thus, the simultaneous measurement of plasmalemmal Ca²⁺and H⁺ fluxes can be measured. The effects of H_o, intracellular ATP, PMCA blockers, and membrane potential (V_M) were studied on PMCA-mediated Ca²⁺/H⁺ exchange. The results indicate that: i) Ca²⁺efflux is promoted by external acidification, is accompanied by a membrane depolarization, and by an intracellular acidification greater than the one resulting from H_o "leak" and PMCA-mediated ATP hydrolysis; ii) H_o-dependent Ca²⁺efflux is inhibited by PMCA blockers and by ATP depletion and is accelerated by membrane depolarization (~3 fold by 20 mV depolarization); iii) the coupling ratio of the Ca²⁺/H⁺ exchange depends on H_o: at an extracellular pH (pH_o)=6.5, the ratio is 1Ca²⁺:~3H⁺; at pH_o=8.2, Ca²⁺efflux rate is 3 times slower and the ratio is 1Ca²⁺:<1H⁺.