Context dependent benzodiazepine modulation of GABA(A) receptor opening frequency

Abstract

The anxiolytic, hypnotic, and anti-convulsant properties of benzodiazepines (BDZs) require modulation of distinct GABA(A) receptor alpha-subtypes. BDZ modulation of GABA(A) receptors is often described in terms of increased opening frequency, and contrasted with the increased open durations occurring with barbiturate modulation. Several studies spanning single channel, rapid kinetic, and whole cell techniques have suggested that BDZs effect this observed change in frequency through increased affinity for GABA. BDZ-sensitive αβγ isoforms exist at extrasynaptic as well as synaptic locations, where they encounter markedly different concentration and time-course of GABA exposure. Interestingly, this affinity-based mechanism (specifically, decreasing the GABA unbinding rate) is only predicted to increase opening frequency under conditions that allow the unbinding and rebinding cycles typical of prolonged exposure to low GABA concentrations, which are more likely to occur at extrasynaptic GABA(A) receptors. In contrast, when rebinding is less likely, such as may occur in certain synaptic conditions, the number, but not the frequency, of channel openings increases in response to BDZ modulation. In conclusion, not only can multiple kinetic mechanisms alter channel opening frequency, but a single mechanism - increased affinity - impacts opening frequency differently under different contexts of GABA(A) receptor activation.

Keywords: Mechanism; affinity.; extra-synaptic; ion channel; synaptic.

Fig. (1)

GABA_A receptor model and macroscopic currents. (A). 4- state kinetic model containing a single resting unbound closed state (C_u), a bound closed state (C_b),and one open (O) and one desensitized (D) state. Rate constants have units of s-1, except kon, which is in units of M^-1s^-1, as follows: k_on = 5 x 10₆; k_off = 1000; k_open = 800; k_close = 500; k_desens = 800; k_resens = 100. (B). Experimental traces from α1β3γ2L receptors in a single whole cell patch clamp experiment. Only the terminal portion of each trace is shown (a 400 ms concentration jump in 1 mM GABA preceded the washout). Solid line, deactivation while cell remains adherent to the recording dish; note the slow time course reflecting GABA rebinding due to imprecise washout. Dashed line, deactivation while cell remains adherent, but had been jumped from GABA into bicuculline (1 µM), instead of control wash; note the sharper deactivation. Gray line, deactivation after cell was lifted from the recording dish, improving solution exchange (control wash); note the similarity with the bicuculline wash, as lifting the cell or exposing the intact cell to bicuculline similarly prevented GABA rebinding. (C). Increasing GABA affinity by decreasing k_off prolongs deactivation without altering the current shape during the GABA pulse (peak amplitude, desensitization). The asterisk indicates baseline parameters; slower deactivation occurred when k_off was decreased to 200 and 50 s^-1. Adapted from reference [5] with permission.

Fig. (2)

Simulated single channels under extrasynaptic conditions. (A). Single channels were simulated such that unbound closed (C_u), bound closed (C_b and D), and bound open (O) states are distinguished via different conductance levels (levels 1, 2 and 3, respectively) (QUB software, www.qub.buffalo.edu). Notice the increased time spent in bound states when k_off is decreased (lower panel). Repeated 2 second exposures to 1 μM GABA were used to generate the data summarized in panels B and C. The Markov states corresponding to the conductance levels (1-3) are also shown on the right of traces in panel A and B. (B). Overall opening frequency increased as affinity was increased (via decreasing k_off). (C). When frequency analysis is restricted to times when GABA is bound, it is unchanged by decreasing k_off. Adapted from reference [5] with permission.

Fig. (3)

Simulated single channels under synaptic conditions. (A). Simulated single channel response to a 1 ms GABA pulse (1 mM), using the same model as in Figure 2. The Markov states corresponding to the conductance levels (1-3) are also shown on the right of the trace. (B). The number of openings recorded after the brief synaptic GABA pulse increased as affinity increased (by decreasing k_off). (C). The frequency of openings was, however, unchanged across the same range of k_off. Adapted from reference [5] with permission.

Fig. (4)

Macroscopic desensitization sets the limit of k_off-mediated slowing of deactivation. Simulated macroscopic currents (solid lines) evoked by 1 ms pulses of 1 mM GABA, across a range of k_off values (200, 100, 30, 10, and 0). k_off = 200 is indicated by the solid circle, while k_off = 0 is indicated by the open circle. A typical BZDinduced prolongation is represented by the koff value of 100 (adjacent to koff = 200, indicated by the solid circle). These are shown in comparison with a 200 ms pulse of 1 mM GABA (gray line). Notice that the progressive slowing of deactivation following the brief pulses approaches a limit set by the macroscopic shape of the current during prolonged exposure.