Reduced Activation of the Synaptic-Type GABAA Receptor Following Prolonged Exposure to Low Concentrations of Agonists: Relationship between Tonic Activity and Desensitization

Abstract

Synaptic GABA_A receptors are alternately exposed to short pulses of a high, millimolar concentration of GABA and prolonged periods of low, micromolar concentration of the transmitter. Prior work has indicated that exposure to micromolar concentrations of GABA can both activate the postsynaptic receptors generating sustained low-amplitude current and desensitize the receptors, thereby reducing the peak amplitude of subsequent synaptic response. However, the precise relationship between tonic activation and reduction of peak response is not known. Here, we have measured the effect of prolonged exposure to GABA or the combination of GABA and the neurosteroid allopregnanolone, which was intended to desensitize a fraction of receptors, on a subsequent response to a high concentration of agonist in human α1β3γ2L receptors expressed in Xenopus oocytes. We show that the reduction in the peak amplitude of the post-exposure test response correlates with the open probability of the preceding desensitizing response. Curve fitting of the inhibitory relationship yielded an IC₅₀ of 12.5 µM and a Hill coefficient of -1.61. The activation and desensitization data were mechanistically analyzed in the framework of a three-state Resting-Active-Desensitized model. Using the estimated affinity, efficacy, and desensitization parameters, we calculated the amount of desensitization that would accumulate during a long (2-minute) application of GABA or GABA plus allopregnanolone. The results indicate that accumulation of desensitization depends on the level of activity rather than agonist or potentiator concentration per se. We estimate that in the presence of 1 µM GABA, approximately 5% of α1β3γ2L receptors are functionally eliminated because of desensitization. SIGNIFICANCE STATEMENT: We present an analytical approach to quantify and predict the loss of activatable GABA_A receptors due to desensitization in the presence of transmitter and the steroid allopregnanolone. The findings indicate that the peak amplitude of the synaptic response is influenced by ambient GABA and that changes in ambient concentrations of the transmitter and other GABAergic agents can modify tonically and phasically activated synaptic receptors in opposite directions.

Fig. 1.

Prolonged exposure to GABA reduces the number of activatable receptors. The current traces show responses to brief applications of 1 mM GABA before (“test application #1”) and after (“test application #2”) a 2-minute desensitizing application of 1 µM (A) or 300 µM GABA (B). (C) shows the GABA concentration dependence of the ratio of amplitudes of test responses (test application #2/test application #1). The data show mean ± S.D. from five to seven cells per concentration. Each cell was exposed to a single concentration of GABA. The curve was fitted with the Hill equation. The IC₅₀ of the curve is 12.5 µM, the Hill coefficient is −1.61, and the high concentration asymptote is 0.30.

Fig. 2.

A three-state Resting-Active-Desensitized model. The receptor can occupy a resting (R), active (A), or desensitized (D) state. The receptor has two binding sites for ligand X. The parameter L (= R/A) describes the equilibrium between the resting and active states. The parameter Q (= A/D) describes the equilibrium between the active and desensitized states. K_X, K_Xc_X, and K_Xc_Xd_X are the equilibrium dissociation constants for X in the resting, active, and desensitized receptor, respectively.

Fig. 3.

Calculated probability distributions. (A) shows the probabilities of being in the resting (P_R,s.s.), active (P_A,peak and P_A,s.s.), or desensitized states (P_D,s.s.) at various concentrations of agonist X. The modeling was done using the values of L of 8000 and Q of 0.2. The equilibrium dissociation constant of X in the resting state (K_X) was assigned a value of 10 µM, c_X was 0.005, and d_X was 1. P_A,s.s. and P_D,s.s. were calculated using eqs. 1 and 4, respectively. P_R,s.s. was calculated as 1 − P_A,s.s. − P_D,s.s.. P_A,peak was calculated by assigning an arbitrary high value (10⁷) to Q (ratio of active to desensitized receptors) in eq. 1, effectively reducing the RAD model to a two-state Resting-Active model (Germann et al., 2019a). (B) shows the sum of the fractions of active and resting (i.e., activatable) receptors at different values of P_A,s.s. (steady-state probability of being in the active state) with the value of Q constrained to 1000, 1, or 0.2. The maximal values for P_A,s.s. in the presence of saturating agonist are calculated as 1/(1 + 1/(Qd^N) + Lc^N) and equal 0.83, 0.46, and 0.16 at these values of Q, whereas the corresponding values for the probability that a receptor is either resting or active are 0.99, 0.54, and 0.19.

Fig. 4.

Activation and desensitization of the α1β3γ2L GABA_A receptor. (A) shows sample traces in the presence of 0.3, 3, 30, or 300 µM GABA. The amplitudes of peak and steady-state currents were converted to units of open probability (P_A) by normalizing the current levels to the peak response to 1 mM GABA + 50 µM propofol in the same cell. (B) shows the peak and steady-state open probabilities as a function of GABA concentration. The data points show mean ± S.D. from five to six cells per concentration. The curves were fitted with eq. 5 (peak data) or eq. 1 (steady-state data), yielding a K_GABA of 39 µM, a c_GABA of 0.0027, and a Q of 0.24. The values of L and N_GABA were held at 8000 and 2, respectively.

Fig. 5.

Desensitization of the α1β3γ2L GABA_A receptor in the presence of GABA or GABA + 3α5αP. Panel A shows the fraction of desensitized receptors (P_D,s.s.) after different levels of desensitizing activity (P_A,s.s.) elicited by GABA or the combination of GABA + 3α5αP. The data points give mean ± S.D. from five to seven cells per experimental condition. For GABA, the symbols show data obtained in the presence of 1, 2, 5, 10, 30, 100, and 300 µM GABA (order from low to high P_A,s.s.). For GABA + 3α5αP, the symbols show data obtained in the presence of 1 µM GABA + 0.1 µM 3α5αP, 2 µM GABA + 0.5 µM 3α5αP, 3 µM GABA + 1 µM 3α5αP, 5 µM GABA + 1 µM 3α5αP, 20 µM GABA + 1 µM 3α5αP, and 100 µM GABA + 1 µM 3α5αP (order from low to high P_A,s.s.). The solid line is not fit to the data; it was calculated using eq. 4 and the activation and desensitization parameters established for the α1β3γ2L receptor (K_GABA = 39 µM, c_GABA = 0.0027, N_GABA = 2, L = 8000, Q = 0.24) with no free parameters. In the absence of agonist, P_A,s.s. is calculated as 1/(1 + 1/Q + L). In the presence of saturating agonist, P_A,s.s. is calculated as 1/(1 + 1/(Qd^N) + Lc^N). (B) compares the fraction of desensitized receptors after different levels of desensitizing activity in the α1β3γ2L (black lines) and α4β2δ receptor (blue lines). The activation and desensitization parameters for the α4β2δ receptor are: K_GABA = 15.7 nM, c_GABA = 0.45, N_GABA = 2, Q = 0.78, and L = 6.7 (Pierce et al., 2019). The regions in the plot indicated with thick solid lines give the levels of P_A,s.s. and the associated P_D,s.s. in the presence of putative ambient GABA (0.1–1 µM).