1.Alcohol/bBenzodiazepine wWithdrawal sSyndrome and its tTreatment by mModulating dDesensitization/rResensitization kKinetics of GABA_AR-mMediated cCurrents

Alcohol and benzodiazepine are useful to mitigate anxiety through enhancing GABA

_A

R-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

_A

Rs remains uncertain, changes in surface GABA

_A

R 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)/Ca

²⁺

/calcineurin signaling pathway

^[14]

. The study showed that overexpression of PRIP-1 suppressed diazepam-dependent activation of PLCδ and diazepam-dependent downregulation of GABA

_A

Rs in HEK293 cells

^[14]

, indicating that PRIP-1 acts as an inhibitor by outcompeting the PLCδ binding to GABA

_A

Rs. Because intracellular Ca

²⁺

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/Ca

²⁺

/calcineurin signaling pathway. Nevertheless, it is also true that calcineurin causes resensitization of GABA

_A

R-mediated currents by facilitating their desensitization

^[15][16]

. Given the apparently contradictory behaviors of GABA

_A

R-mediated currents by calcineurin activation, the two different behaviors of GABA

_A

R-mediated currents may depend on whether calcineurin activation occurs before or after activation of GABA

_A

Rs.

As for the treatment of the alcohol/benzodiazepine withdrawal syndrome, propofol and barbiturate which enhance GABA

_A

R-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

_A

R currents itself may suggest the occurrence of resensitization of GABA

_A

R-mediated currents. Indeed, the desensitization and deactivation of GABA

_A

R-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

_A

R-mediated currents. Therefore, the regulatory mechanisms of desensitization/resensitization of GABA

_A

R-mediated currents are important to better understand the benzodiazepine/alcohol withdrawal syndrome and to develop the treatment method.

2. Drugs that cause dCause Desensitization and rResensitization of GABA_AR-mMediated cCurrents

The drugs that cause desensitization and resensitization of GABA

_A

R-mediated currents are summarized in Table 1. Hump-like GABA

_A

R 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

_A

Rs

^{[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

_A

R-mediated currents might be important.

Table 1. Drugs that modulate GABA responses and directly activate GABA

_A

Rs at higher concentrations.

3. A possible kKinetic mMechanism for rResensitization of GABA_A cCurrents

In layer II/III pyramidal cells of the mouse barrel cortex, the deletion of PRIP-1/2 enhanced the desensitization of GABA

_A

R-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

_A

R-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

_A

R-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

_A

R-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

_A

Rs 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

_A

R-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

_A

Rs are schematically depicted (Figure 2).

Figure 2.

Close, open (resensitized) and desensitized states of GABA

_A

Rs. When GABA binds to GABA

_A

Rs, 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

_A

Rs are desensitized (

d

) by increased calcineurin activity due to potentiated Ca

²⁺

-induced Ca

²⁺

release (CICR) followed by store-operated Ca

²⁺

entry (SOCE)

^[15]

. GABA

_A

Rs are resensitized through the de-desensitization (

r

) at the offset of the GABA puff. PRIP outcompetes the PLCδ in binding to GABA

_A

R β subunits

^[14]

.

d

: desensitization,

r

: resensitization, RYR: ryanodine receptor, SOCC: store-operated Ca

²⁺

channel, IP

₃R: inositol trisphosphate receptor.

R: inositol trisphosphate receptor.