1.Alcohol/Benzodiazepine Withdrawal Syndrome and its Treatment by Modulating Desensitization/Resensitization Kinetics of GABA_AR-Mediated Currents

Alcohol and benzodiazepine are useful to mitigate anxiety through enhancing GABA_AR-mediated inhibition. However, alcohol and benzodiazepine are known as abused drugs. The alcohol or benzodiazepine withdrawal syndrome appears following a reduction on alcohol or benzodiazepine use after a period of excessive use ^[1][2][3][4]. The alcohol or benzodiazepine withdrawal symptoms typically include anxiety, sweating, hand tremor and sleep disturbance. The underlying mechanisms involve neuronal adaptations, which are revealed as decreased GABAergic responses ^[5] and enhancement of NMDA responses ^[6][7][8][9]. Although the exact mechanism for the reduced responsiveness of GABA_ARs remains uncertain, changes in surface GABA_AR protein level and subunit composition, changes in turnover, recycling, and production rates, degree of phosphorylation and decreased coupling mechanisms between GABA and alcohol/benzodiazepine sites are thought to be involved in the reduced responsiveness ^{[10][11][12][13]}. It has recently been demonstrated that the benzodiazepine diazepam caused downregulation of GABAergic inhibition through phospholipase C (PLCδ)/Ca²⁺/calcineurin signaling pathway ^[14]. The study showed that overexpression of PRIP-1 suppressed diazepam-dependent activation of PLCδ and diazepam-dependent downregulation of GABA_ARs in HEK293 cells ^[14], indicating that PRIP-1 acts as an inhibitor by outcompeting the PLCδ binding to GABA_ARs. Because intracellular Ca²⁺ and calcineurin activity are increased in PRIP-DKO mice ^[15], these findings suggest that the diazepam-induced long-term downregulation of GABAergic inhibition is mediated by PLCδ/Ca²⁺/calcineurin signaling pathway. Nevertheless, it is also true that calcineurin causes resensitization of GABA_AR-mediated currents by facilitating their desensitization ^[15][16]. Given the apparently contradictory behaviors of GABA_AR-mediated currents by calcineurin activation, the two different behaviors of GABA_AR-mediated currents may depend on whether calcineurin activation occurs before or after activation of GABA_ARs.

As for the treatment of the alcohol/benzodiazepine withdrawal syndrome, propofol and barbiturate which enhance GABA_AR-mediated inhibition are useful. Indeed, it was demonstrated that propofol and barbiturates (pentobarbital and phenobarbital) were effective for the treatment of alcohol withdrawal syndrome ^[17][18], and barbiturate (pentobarbital) was effective for the treatment of benzodiazepine withdrawal syndrome ^[19]. However, it remains unclear how propofol and barbiturate ameliorate reduced GABA responsiveness in patients with alcohol/benzodiazepine withdrawal syndrome. Although the concentrations of propofol and barbiturates that generated the hump-like current are very high ^[20][21][22] compared to the dose used for treatment of the withdrawal syndrome ^[17][18], the generation of hump-like GABA_AR currents itself may suggest the occurrence of resensitization of GABA_AR-mediated currents. Indeed, the desensitization and deactivation of GABA_AR-mediated currents are facilitated and slowed, respectively, by propofol/barbiturate at much lower concentrations ^[21][22]. Then, propofol and barbiturate may improve the reduced GABA responsiveness through the resensitization of GABA_AR-mediated currents. Therefore, the regulatory mechanisms of desensitization/resensitization of GABA_AR-mediated currents are important to better understand the benzodiazepine/alcohol withdrawal syndrome and to develop the treatment method.

2. Drugs that Cause Desensitization and Resensitization of GABA_AR-Mediated Currents

The drugs that cause desensitization and resensitization of GABA_AR-mediated currents are summarized in Table 1. Hump-like GABA_AR currents after a strong desensitization were seen at the offset of propofol application at a high concentration (600 μM) in hippocampal pyramidal neurons ^[22], etomidate application at a high concentration (1 mM) in rat spinal dorsal horn neurons ^[23], pentobarbital application at high concentrations (1–3 mM) in frog sensory neurons ^[21][22], rat hippocampal neurons ^[24] and recombinant GABA_ARs ^{[25][26][27][28][29][30]} or phenobarbital application at a high concentration (10 mM) in rat hippocampal neurons ^[24], although these were not seen at the offset of GABA application. It is believed that the generation of hump-like currents may be caused by removal of the blockade by anesthetic agents as partial antagonists ^[25], although their mechanisms remain unclear and the involvement of desensitization is not necessarily denied. To better understand the actions of these anesthetic drugs, the molecular and regulatory mechanisms of desensitization/resensitization of GABA_AR-mediated currents might be important.

Table 1. Drugs that modulate GABA responses and directly activate GABA_ARs at higher concentrations.

3. A possible Kinetic Mechanism for Resensitization of GABA_A Currents

In layer II/III pyramidal cells of the mouse barrel cortex, the deletion of PRIP-1/2 enhanced the desensitization of GABA_AR-mediated currents but paradoxically induced a hump-like tail-current at the offset of the GABA puff ^[15], and also resulted in the prolongation of the decay phase of eIPSCs ^[31]. To understand the kinetic mechanisms underlying the generation of the hump-like tail-currents and the prolongation of eIPSCs, these currents were simulated using a model previously proposed ^[32] (Figure 1). It was examined whether the possible increase in the fast desensitization rate (d₂) and the possible decrease in the unbinding rate (k_off) can lead to the generation of the hump-like tail-current at the offset of the GABA puff. In the simulated wild-type pyramidal cell, GABA_AR-mediated currents were induced without a hump-like tail-current in response to 2-sec GABA puff at 50 µM ^[15]. In contrast, in the simulated PRIP-DKO pyramidal cell, GABA_AR-mediated currents displayed a prominent desensitization and was followed by a prominent hump-like tail-current ^[15]. Thus, a slowdown of k_off and an acceleration of d₂ resulted in a generation of a hump-like tail-current. Following a sharp decrease in [GABA] at the offset of GABA puff, a sharp decrease in d₂ to a level smaller than the fast de-desensitization (i.e. resensitization) rate constant (r₂) occurred to subsequently induce a hump-like tail-current. It can be concluded that a higher calcineurin activity in PRIP-DKO layer III pyramidal cells might have caused a slowdown of k_off and an acceleration of d₂ through the modulation of its GABA concentration dependency, leading to a generation of hump-like tail-currents in PRIP-DKO pyramidal cells. It was also investigated whether the increase in d₂ and the decrease in k_off can also lead to the prolongation of eIPSCs. Simulated IPSCs in PRIP-DKO and the wild-type pyramidal cells that have the half-durations similar to those obtained in the real experiments ^[15] revealed that a prolongation of eIPSCs/eIPSPs in PRIP-DKO pyramidal cells results from resensitization of GABA_AR-mediated currents, which is brought about by an acceleration of d₂ through the modulation of its [GABA] dependency together with a slowdown of k_off.

Figure 1. A kinetic model for a hump-like tail-current. (A) A kinetic model of GABA_ARs representing mono- and double-liganded states, each providing access to open and desensitized states. (B and C) Top; Presumed [GABA] changes created by puff application of GABA with a rectangular pressure pulse through a puff pipette containing 200 μM GABA in the extracellular medium was assumed to be diluted 4 times, and the onset and offset of the puff application were assumed to be attenuated with a time constant raging between 0.1 and 0.3 sec. Bottom; superimposed traces of the simulated GABA_AR-mediated currents under the condition that the attenuation time constant is 0.3 and 0.1 sec (solid and interrupted traces, respectively) in simulated wild-type (B) and PRIP-DKO (C) pyramidal cells. (D) Superimposed traces of a simulated wild-type and PRIP-DKO eIPSC induced by a GABA transient shown on an expanded time scale (inset) with a small maximum conductance. Adopted from ^[15] and ^[31]. 

Based on the experimental and simulation studies, the regulatory mechanisms of GABA_ARs are schematically depicted (Figure 2).

Figure 2. Close, open (resensitized) and desensitized states of GABA_ARs. When GABA binds to GABA_ARs, the receptors open the pore, and consequently increasing the permeability of the ion pore to Cl^-. In response to a prolonged application of GABA, GABA_ARs are desensitized (d) by increased calcineurin activity due to potentiated Ca²⁺-induced Ca²⁺ release (CICR) followed by store-operated Ca²⁺ entry (SOCE) ^[15]. GABA_ARs are resensitized through the de-desensitization (r) at the offset of the GABA puff. PRIP outcompetes the PLCδ in binding to GABA_AR β subunits ^[14]. d: desensitization, r: resensitization, RYR: ryanodine receptor, SOCC: store-operated Ca²⁺ channel, IP₃R: inositol trisphosphate receptor.