Block of the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel by tetra-alkyl ammonium cations

Abstract

The purified ryanodine receptor channel of the sheep cardiac muscle sarcoplasmic reticulum (SR) membrane functions as a calcium-activated cation-selective channel under voltage-clamp conditions following reconstitution into planar phospholipid bilayers. We have investigated the effects of the tetra-alkyl ammonium (TAA) cations, (CnH2n+1)4N+ and the trimethyl ammonium cations, ethyltrimethyl ammonium and propyltrimethyl ammonium, on potassium conductance through the receptor channel. Small TAA cations (n = 1-3) and the trimethyl ammonium derivatives act as asymmetric, voltage-dependent blockers of potassium current. Quantitative analysis of the voltage dependence of block indicates that the conduction pathway of the sheep cardiac SR ryanodine receptor channel contains two distinct sites for the interaction of these small organic cations. Sites are located at approximately 50% for tetramethyl ammonium (TMA+) and 90% for tetraethyl ammonium (TEA+) and tetrapropyl ammonium (TPrA+) of the voltage drop across the channel from the cytosolic face of the protein. The chemical substitution of an ethyl or propyl group for one of the methyl groups in TMA+ increases the voltage dependence of block to a level similar to that of TEA+ and TPrA+. The zero-voltage dissociation constant (Kb(0)) falls with the increasing number of methyl and methylene groups for those blockers acting 90% of the way across the voltage drop. This is interpreted as suggesting a hydrophobic binding site at this point in the conduction pathway. The degree of block increases as the concentration of small TAA cations is raised. The concentration dependence of tetraethyl ammonium block indicates that the cation interacts with a single site within the conduction pathway with a Km of 9.8 +/- 1.7 mM (mean +/- SD) at 40 mV. Larger TAA cations (n = 4-5) do not induce voltage-dependent block of potassium current of the form seen with the smaller TAA cations. These data support the contention that the sheep cardiac SR ryanodine receptor channel may be occupied by at most one ion at a time and suggest that a large proportion of the voltage drop falls over a relatively wide region of the conduction pathway.