GABA Type A Receptor Activation in the Allosteric Coagonist Model Framework: Relationship between EC₅₀ and Basal Activity

Abstract

The concerted transition model for multimeric proteins is a simple formulation for analyzing the behavior of transmitter-gated ion channels. We used the model to examine the relationship between the EC for activation of the GABA type A (GABA) receptor by the transmitter GABA and basal activity employing concatemeric ternary GABA receptors expressed in oocytes. Basal activity, reflecting the receptor function in the absence of the transmitter, can be changed either by mutation to increase constitutive activity or by the addition of a second agonist (acting at a different site) to increase background activity. The model predicts that either mechanism for producing a change in basal activity will result in identical effects on the EC We examined receptor activation by GABA while changing the level of basal activity with the allosterically acting anesthetics propofol, pentobarbital, or alfaxalone. We found that the relationship between EC and basal activity was well described by the concerted transition model. Changes in the basal activity by gain-of-function mutations also resulted in predictable changes in the EC Finally, we altered the number of GABA-binding sites by a mutation and again found that the relationship could be well described by the model. Overall, the results support the idea that interactions between the transmitter GABA and the allosteric agonists propofol, pentobarbital, or alfaxalone can be understood as reflecting additive and independent free energy changes, without assuming any specific interactions.

Analysis of GABAA Receptor Activation by Combinations of Agonists Acting at the Same or Distinct Binding Sites

“…The receptors comprised concatemeric b2-a1-g2L (bag) and b2-a1 (ba) constructs. The design and properties of the receptors have been described previously (Bracamontes and Steinbach, 2009;Bracamontes et al, 2011;Akk et al, 2018). Receptors formed of bag and ba constructs without further mutations are referred to as wild-type concatemeric receptors.…”

“…Note that in (B) two inactive states (YRX and Y 2 RX) are hidden and in (C) YRX is hidden. probability of a constitutively active receptor (P open,const ) 5 0.00011] (Akk et al, 2018); therefore, the holding current in the absence of agonists was considered to have a value of P open 5 0. The current level corresponding to P open 5 1 was estimated by exposing the receptors to the combination of saturating GABA plus 100 mM pentobarbital (Ziemba and Forman, 2016).…”

“…The parameter L is a measure of background activity. For wild-type concatemeric receptors in the absence of additional agonists, L, calculated as (1 2 P open,const )/P open,const , was held at 8000 (Akk et al, 2018). To analyze the steroid concentration-response data recorded in the wildtype concatemeric receptor in the presence of a low concentration of GABA, L was constrained to (1 2 P open,GABA )/P open,GABA .…”

“…This process is exemplified by coapplication of an allosteric agonist with an orthosteric agonist, e.g., coapplication of propofol with GABA. The peak response to the combination of GABA 1 propofol can be accurately predicted based on energetic additivity of the effects of each individual agent (Ruesch et al, 2012;Akk et al, 2018;Shin et al, 2018). Coapplication of two allosteric agonists that interact with distinct sites would be mechanistically similar, resulting in additivity of free energies provided by each agonist toward stabilization of the open state (Shin et al, 2017).…”

“…Here, we have analyzed activation of the concatemeric a1b2g2L GABA A receptor by combinations of orthosteric and/or allosteric agents, using the coagonist concerted transition model (Monod et al, 1965;Forman, 2012;Akk et al, 2018). In this model (Fig.…”