SPERMINE MODULATION OF SPECIFIC [H-3] GABAPENTIN BINDING TO THE DETERGENT-SOLUBILIZED PORCINE CEREBRAL-CORTEX ALPHA(2)DELTA CALCIUM-CHANNELSUBUNIT

1 Recent studies have identified the [H-3]-gabapentin-binding protein, purified from porcine cerebral cortical membranes, as the alpha(2) de lta subunit of voltage-sensitive calcium channels (Gee et al., 1996). The present study investigates the influence of the polyamine spermine on specific [H-3]-gabapentin binding to detergent-solubilized porcine cerebral cortical membranes. 2 Spermine, spermidine, 1,10 diaminodeca ne, Mg2+ and Zn2+, all divalent cations, displaced [H-3]gabapentin bin ding to detergent-solubilized membranes in a concentration-dependent m anner with a maximal inhibition of 65-75%. Radioligand binding studies showed that spermine did not directly interact with the [H-3]-gabapen tin-binding site. Spermine inhibited [H-3]-gabapentin binding by inter acting with a polyamine-sensitive allosteric site on the membrane prot ein. The steep concentration-dependence of spermine inhibition of [H-3 ]-gabapentin binding may suggest multi-site co-operativity. 3 Prolonge d dialysis of cerebral cortical membranes and Tween 20-solubilized mem branes resulted in a >2.0 fold increase in [H-3]-gabapentin binding. T he increase in binding was due to the removal of a heat stable, low mo lecular weight (<12,000Da) endogenous molecule which influences [H-3]- gabapentin binding competitively. 4 Dialysis of detergent-solubilized cerebral cortical membranes also resulted in a decrease in the maximum inhibition of [H-3]-gabapentin binding by spermine. Since the rates o f the increase in [H-3]gabapentin binding and the loss of the ability of spermine to inhibit [H-3]-gabapentin binding on dialysis were diffe rent it was inferred that a second endogenous ligand was removed durin g dialysis. 5 During initial steps of purification of the [H-3]-gabape ntin-binding protein there was a decrease in the maximum inhibition of [H-3]-gabapentin binding by spermine. The loss of the second endogeno us molecule during initial purification would reasonably explain the r eduction in inhibition of binding by spermine. However, spermine stimu lation of [H-3]-gabapentin binding to material that eluted from the ge l-filtration column later in the purification scheme does not appear t o be due to removal of a dialysable endogenous factor or to the dissoc iation of other calcium channel subunit(s). 6 Adding back dialysate, b efore or after boiling, to detergent solubilized membranes resulted in a dose-dependent restoration of the inhibition of [H-3]-gabapentin bi nding and of the maximal inhibition [H-3]-gabapentin binding by spermi ne. This result is consistent with the re-addition of two endogenous h eat stable ligands. 7 The finding that [H-3]-gabapentin binding to the pure alpha(2) delta subunit was stimulated by spermine indicates that the alpha(2) delta subunit of voltage-sensitive calcium channels bear s a modulatory spermine site. Such a spermine site has not been identi fied before. Spermine stimulation of [H-3]-gabapentin binding to the p urified protein was reversed to inhibition after adding back dialysate . Thus the inhibitory spermine effect in membranes is also probably du e to one or more modulatory sites on the alpha(2) delta subunit.