Clinical Significance of Desensitization and Resensitization of GABAAR-Mediated Currents
Propofol or barbiturate application at low concentrations increases desensitization and slows deactivation of GABA-induced current and propofol/barbiturate at high concentrations directly elicits after-responses upon their washout in hippocampal or sensory neurons. It is postulated that the generation of such after-responses is caused by removal of the blockade by anesthetic agents as partial antagonists. However, the increased desensitization was invariably followed by slowdown of deactivation of GABA-induced current, and the after-response may arise as a consequence of extreme slowdown of deactivation following strong desensitization. It is thus possible that propofol and barbiturate can facilitate resensitization of GABA responses. Propofol and barbiturate are useful to treat the alcohol/benzodiazepine withdrawal syndrome. Considering that the slowdown of deactivation following desensitization and the after-response induced by propofol or barbiturate application, the regulatory mechanisms of desensitization/resensitization of GABAAR-mediated currents might be important for understanding the treatment of the alcohol/benzodiazepine withdrawal syndrome.
Alcohol/benzodiazepine withdrawal syndrome and its treatment by modulating desensitization/resensitization kinetics of GABAAR-mediated currents
Alcohol and benzodiazepine are useful to mitigate anxiety through enhancing GABAAR-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 . 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  and enhancement of NMDA responses . Although the exact mechanism for the reduced responsiveness of GABAARs remains uncertain, changes in surface GABAAR 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 . It has recently been demonstrated that the benzodiazepine diazepam caused downregulation of GABAergic inhibition through phospholipase C (PLCδ)/Ca2+/calcineurin signaling pathway . The study showed that overexpression of PRIP-1 suppressed diazepam-dependent activation of PLCδ and diazepam-dependent downregulation of GABAARs in HEK293 cells , indicating that PRIP-1 acts as an inhibitor by outcompeting the PLCδ binding to GABAARs. Because intracellular Ca2+ and calcineurin activity are increased in PRIP-DKO mice , 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 GABAAR-mediated currents by facilitating their desensitization . Given the apparently contradictory behaviors of GABAAR-mediated currents by calcineurin activation, the two different behaviors of GABAAR-mediated currents may depend on whether calcineurin activation occurs before or after activation of GABAARs.
As for the treatment of the alcohol/benzodiazepine withdrawal syndrome, propofol and barbiturate which enhance GABAAR-mediated inhibition are useful. Indeed, it was demonstrated that propofol and barbiturates (pentobarbital and phenobarbital) were effective for the treatment of alcohol withdrawal syndrome , and barbiturate (pentobarbital) was effective for the treatment of benzodiazepine withdrawal syndrome . 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  compared to the dose used for treatment of the withdrawal syndrome , the generation of hump-like GABAAR currents itself may suggest the occurrence of resensitization of GABAAR-mediated currents. Indeed, the desensitization and deactivation of GABAAR-mediated currents are facilitated and slowed, respectively, by propofol/barbiturate at much lower concentrations . Then, propofol and barbiturate may improve the reduced GABA responsiveness through the resensitization of GABAAR-mediated currents. Therefore, the regulatory mechanisms of desensitization/resensitization of GABAAR-mediated currents are important to better understand the benzodiazepine/alcohol withdrawal syndrome and to develop the treatment method.
Drugs that cause desensitization and resensitization of GABAAR-mediated currents
The drugs that cause desensitization and resensitization of GABAAR-mediated currents are summarized in Table 1. Hump-like GABAAR currents after a strong desensitization were seen at the offset of propofol application at a high concentration (600 μM) in hippocampal pyramidal neurons , etomidate application at a high concentration (1 mM) in rat spinal dorsal horn neurons , pentobarbital application at high concentrations (1–3 mM) in frog sensory neurons , rat hippocampal neurons  and recombinant GABAARs  or phenobarbital application at a high concentration (10 mM) in rat hippocampal neurons , 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 , 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 GABAAR-mediated currents might be important.
Table 1. Drugs that modulate GABA responses and directly activate GABAARs at higher concentrations.
A possible kinetic mechanism for resensitization of GABAA currents
In layer II/III pyramidal cells of the mouse barrel cortex, the deletion of PRIP-1/2 enhanced the desensitization of GABAAR-mediated currents but paradoxically induced a hump-like tail-current at the offset of the GABA puff , and also resulted in the prolongation of the decay phase of eIPSCs . 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  (Figure 1). It was examined whether the possible increase in the fast desensitization rate (d2) and the possible decrease in the unbinding rate (koff) 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, GABAAR-mediated currents were induced without a hump-like tail-current in response to 2-sec GABA puff at 50 µM . In contrast, in the simulated PRIP-DKO pyramidal cell, GABAAR-mediated currents displayed a prominent desensitization and was followed by a prominent hump-like tail-current . Thus, a slowdown of koff 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 koff 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 koff 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  revealed that a prolongation of eIPSCs/eIPSPs in PRIP-DKO pyramidal cells results from resensitization of GABAAR-mediated currents, which is brought about by an acceleration of d2 through the modulation of its [GABA] dependency together with a slowdown of koff.
Figure 1. A kinetic model for a hump-like tail-current. (A) A kinetic model of GABAARs 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 GABAAR-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  and .
Based on the experimental and simulation studies, the regulatory mechanisms of GABAARs are schematically depicted (Figure 2).
Figure 2. Close, open (resensitized) and desensitized states of GABAARs. When GABA binds to GABAARs, the receptors open the pore, and consequently increasing the permeability of the ion pore to Cl-. In response to a prolonged application of GABA, GABAARs are desensitized (d) by increased calcineurin activity due to potentiated Ca2+-induced Ca2+ release (CICR) followed by store-operated Ca2+ entry (SOCE) . GABAARs are resensitized through the de-desensitization (r) at the offset of the GABA puff. PRIP outcompetes the PLCδ in binding to GABAAR β subunits . d: desensitization, r: resensitization, RYR: ryanodine receptor, SOCC: store-operated Ca2+ channel, IP3R: inositol trisphosphate receptor.
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