Programme

Block of the divalent anion channel in the SR of rabbit skeletal muscle by disulfonic stilbene derivatives

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 (P_i) and 60pS for SO₄^2-). 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 P_i transport across the SR.

SR vesicles containing the anion channels were isolated from rabbit skeletal muscle that was removed from dead rabbits. Transport of P_i across the SR vesicle membrane was inferred from the P_i 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 KP_i, 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 P_i facilitated calcium uptake by rabbit SR vesicles were used to assay the degree of P_i 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 P_i transport. Thus it appears that while this channel conducts P_i, it is not the major pathway for P_i 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.