Previous studies have identified two types of anion channels in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle (Kourie et al., 1996). One of these anion channels has an appreciable anion conductance (saturating at 20pS for phosphate (Pi) and 60pS for SO42-). It was proposed that these channels could be responsible for movement of phosphate across the SR membrane (Laver et al., 2001). To further investigate this hypothesis we have searched for specific inhibitors of the divalent anion channel with the rationale that these inhibitors would prevent Pi transport across the SR.
SR vesicles containing the anion channels were isolated from rabbit skeletal muscle that was removed from dead rabbits. Transport of Pi across the SR vesicle membrane was inferred from the Pi assisted component of Ca2+ uptake by the SERCa pump. Ca2+ uptake was measured from the optical absorbance of antipyrylazo III (Dulhunty et al., 1999). Ca2+ uptake experiments were carried out using solutions containing 100 mM KCl or KPi, 4mM MgCl2, 1mM ATP, 5μM ruthenium red, 0.5 mM antipyrylazo III, 5 mM TES (pH 7). Anion channels were incorporated into lipid bilayers using standard techniques (Laver et al., 2001). Cytoplasmic solutions contained 260 mM Mg2+ (250 mM MgSO4 and 10 mM MgCl2), 1 mM CaCl2 10 mM TES at pH 7.4. Luminal solutions contained 60 mM Mg2+ (50 mM MgSO4 and 10 mM MgCl2), 10 mM TES, pH 7.4.
Lipid bilayer studies showed that the divalent anion channels were inhibited by the disulfonated stilbene derivatives, Diisothiocyanostilbene-2',2'-di-sulfonic acid (DIDS), 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS), 4,4'-dibenzamidostilbene-2,2'-disulfonic acid (DBDS) and by suramin. Reversible block by these compounds inhibited these channels with high affinity from the cytoplasmic side (∼0.1-1μM) and low affinity from the luminal side (0.1 - 1 mM). The voltage-dependent kinetics of drug binding and dissociation indicated that these compounds can dissociate from the channel to either side of the membrane (i.e. they are permeant blockers). DIDS also produces non-reversible inhibition of the channel.
Measurements of Pi facilitated calcium uptake by rabbit SR vesicles were used to assay the degree of Pi transport across the SR membrane. The presence of DBDS at concentrations sufficient to block the divalent anion channel in lipid bilayers (∼1-10μM) had no effect on Pi transport. Thus it appears that while this channel conducts Pi, it is not the major pathway for Pi in the SR membrane.
Dulhunty, A.F., Laver, D.R., Gallant, E.M., Casarotto, M.G., Pace, S.M. & Curtis, S. (1999) Biophysical Journal, 77:189-203
Kourie, J.I., Laver, D.R., Junankar, P.R., Gage. P.W. & Dulhunty, A.F. (1996) Biophysical Journal, 70:202-221.
Laver, D.R., Lenz, G.K.E., Dulhunty, A.F. (2001) Journal of Physiology, 535:715-728.