CHARGED LOCAL-ANESTHETICS BLOCK IONIC-CONDUCTION IN THE SHEEP CARDIACSARCOPLASMIC-RETICULUM CALCIUM-RELEASE CHANNEL

We have examined the effect of the charged local anesthetics QX314, QX 222, and Procaine on monovalent cation conduction in the Ca2+ release channel of the sheep cardiac sarcoplasmic reticulum. All three blocker s,only affect cation conductance when present at the cytoplasmic face of the channel. OX222 and Procaine act as voltage-dependent blockers. With 500 Hz filtering, this is manifest as a relatively smooth reducti on in single-channel current amplitude most prominent at positive hold ing potentials. Quantitative analysis gives an effective valence of ap proximately 0.9 for both ions and Kb(0)s of 9.2 and 15.8 mM for QX222 and Procaine, respectively. Analysis of the concentration dependence o f block suggests that QX222 is binding to a single site with a K(m) of 491 muM at a holding potential of 60 mV. The use of amplitude distrib ution analysis, with the data filtered at 1 to 2 kHz, reveals that the voltage and concentration dependence of QX222 block occurs largely be cause of changes in the blocker on rate. The addition of QX314 has a d ifferent effect, leading to the production of a substate with an ampli tude of approximately one-third that of the control. The substate's oc currence is dependent on holding potential and QX314 concentration. Qu antitative analysis reveals that the effect is highly voltage dependen t, with a valence of approximately 1.5 caused by approximately equal c hanges in the on and off rates. Kinetic analysis of the concentration dependence of the substate occurrence reveals positive cooperativity w ith at least two QX314s binding to the conduction pathway, and this is largely accounted for by changes in the on rate. A paradoxical increa se in the off rate at high positive holding potentials and with increa sing QX314 concentration at 80 mV suggests the existence of a further QX314-dependent reaction that is both voltage and concentration depend ent. The substate block is interpreted physically as a form of partial occlusion in the vestibule of the conduction pathway giving a reducti on in single-channel current by electrostatic means.