Modulation by bicuculline and penicillin of the block by t-butyl-bicyclo-phosphorothionate (TBPS) of GABA(A)-receptor mediated Cl(-)-current responses in rat striatal neurones

Abstract

1. T-butyl-bicyclo-phosphorothionate (TBPS) is a prototypical representative of the cage-convulsants which act through a use-dependent block of the GABA(A)-receptor-ionophore complex. Using current recordings from cultured neurones of rat striatum the manner was investigated in which two antagonists, bicuculline and penicillin, presumably acting at the agonist binding site and in the ionic channel, respectively, modify the rate of block by TBPS. 2. Penicillin (5 or 10 mM) did not slow the rate of block by TBPS, but produced a significant enhancement of block rate, which, however, was inversely related to the degree of antagonism by penicillin of the GABA-induced current. 3. Bicuculline (10 microM) reduced the rate of block by TBPS. However, this effect was 3 fold weaker than its GABA-antagonistic action. The slowing of block rate and the current antagonism exhibited a biphasic, positive-negative relationship. Co-application of bicuculline (100 microM) in a concentration that produced nearly complete antagonism and TBPS (10 microM) resulted in a marked ( approximately 40%) reduction of subsequent GABA response amplitudes compatible with a direct, bicuculline-induced conformational change in the receptor required for the binding of and block by TBPS. 4. The lack of protection afforded by the channel blocker penicillin as well as the lack of correlation between bicuculline antagonism of the Cl(-)-current and its efficiency in protecting against TBPS block is evidence against an open channel blocking mechanism for TBPS. TBPS does, therefore, not appear to gain access to its binding site via the open pore but through alternative routes regulated from the agonist binding site.

Figure 1

Effect of penicillin on amplitude and kinetics of GABA-responses. (A) Control response to a 20 ms pressure application from a pipette containing GABA at 100 μM. (B) Response obtained immediately following perfusion of the cell with a solution containing penicillin (5 mM). Note the slower rising phase and the early rapid and late slow decay phases, as well as the enhanced current noise characteristic of simple open channel block (see text). Inset b: The superimposition of the current integrals indicates that despite the slow decay, the total open probability time (i.e. P_o^*t) of the Cl⁻-channels during the response is reduced by >50% by the action of penicillin.

Figure 2

Effect of penicillin on the rate of block of GABA-responses by TBPS. Graph of the current amplitude vs sequential number of test GABA-applications (delivered 1/5 s). Upon application of penicillin (5 mM, open bar) response amplitude is immediately reduced to <50% of control. Application of TBPS (10 μM, closed bar) produces a rapid exponential block with an e-fold reduction in amplitude after 1.66 applications of GABA. Upon washout of TBPS, a slower, exponential recovery to control values is observed. After washout of penicillin and recovery of control amplitudes, TBPS is applied again inducing again a rapid decline in current amplitudes with an e-fold reduction in amplitude after 2.08 GABA-applications, i.e. it is slower than that observed in the presence of penicillin. Insets a and b show superimposed original current traces from the respective portions of the experiment: four traces recorded before TBPS-application are shown together with those during development of the block to the steady state. Data from the same experiment as in Figure 1.

Figure 3

Summary of results of six experiments with penicillin and TBPS. The graph shows relative change in block rate plotted against the change in current integral (i.e. total channel open time) produced by penicillin (5 or 10 mM). Note that all values for change in block rate are >100%, indicating that penicillin invariably produced an enhancement of block. However, this graph suggests a negative correlation (solid line, correlation coefficient: 0.93) with the total reduction in charge transfer produced by penicillin (see text).

Figure 4

Effect of bicuculline on block rate of GABA-responses by TBPS. The graph shows the response amplitude plotted against the application number. Application of bicuculline (10 μM, open bar) produces a strong reduction in inward current responses. Addition of TBPS at 3 μM (closed bar) shows a slow, exponential block with an e-fold reduction of amplitude occurring after 8.19 GABA-pulses and washout of TBPS produces little recovery. Upon washout of bicuculline, TBPS is applied again at 3 μM and produces a faster block with e-fold amplitude reduction after 4.44 pulses.

Figure 5

Summary of eight experiments with bicuculline (10 μM) and TBPS. The graph shows the relationship between change in the blocking rate in TBPS and the decrease in current integral obtained with 10 μM bicuculline. Note that there is a biphasic relationship. The solid line is a quadratic fit. The protective effect of bicuculline increases up to an 80% decrease in current integral but declines thereafter (see text).

Figure 6

Effect of co-application of TBPS with a near-maximal concentration of bicuculline. (A) Response amplitude is plotted against number of GABA-applications. Application of 100 μM bicuculline (open bar) produces a near complete suppression of the GABA response. This effect is readily reversible upon washout. Co-application of TBPS (10 μM) with bicuculline (closed bar) produces immediate total block of the response. Upon washout of both drugs, the response is found to be suppressed without a clear tendency for recovery. (B) Superimposed original traces coresponding to A. Similar results were obtained in four other cells.