Functional Relationships between Agonist Binding Sites and Coupling Regions of Homomeric Cys-Loop Receptors

Abstract

Each subunit in a homo-pentameric Cys-loop receptor contains a specialized coupling region positioned between the agonist binding domain and the ion conductive channel. To determine the contribution of each coupling region to the stability of the open channel, we constructed a receptor subunit (α7-5HT3A) with both a disabled coupling region and a reporter mutation that alters unitary conductance, and co-expressed normal and mutant subunits. The resulting receptors show single channel current amplitudes that are quantized according to the number of reporter mutations per receptor, allowing correlation of the number of intact coupling regions with mean open time. We find that each coupling region contributes an equal increment to the stability of the open channel. However by altering the numbers and locations of active coupling regions and binding sites, we find that a coupling region in a subunit flanked by inactive binding sites can still stabilize the open channel. We also determine minimal requirements for channel opening regardless of stability, and find that channel opening can occur in a receptor with one active coupling region flanked by functional binding sites, or with one active binding site flanked by functional coupling regions. The overall findings show that whereas the agonist binding sites contribute inter-dependently and asymmetrically to open channel stability, the coupling regions contribute independently and symmetrically.

Neurotransmitter GABA Activates Muscle but Notα7 Nicotinic Receptors

“…We evaluated whether GABA is able to activate human a7 and the high conductance form of the chimeric a7-5HT 3 A receptor, which is a good model for studies of a7 because it shows high surface expression and channel activity is easily detected (Rayes et al, 2005;Andersen et al, 2011). In cells transfected with a7 (Fig.…”

“…where I 0 and I ' are peak and the steady state current values, respectively, and t d is the decay time constant (Andersen et al, 2011). EC 50 and Hill coefficient values were calculated by nonlinear regression analysis using the Hill equation:…”

Stoichiometry for drug potentiation of a pentameric ion channel

“…To determine subunit stoichiometry, we used an electrical fingerprinting strategy (17,18) in which mutations that alter the single-channel conductance without affecting PNU potentiation (Fig. S2) are introduced into one of the two kinds of subunits.…”

“…We used a single-channel electrical fingerprinting strategy (17,18) to determine the subunit stoichiometry of α7 receptors in which the number of wild-type and PNU-resistant subunits varied. This approach allowed us to simultaneously measure PNU potentiation and the number of intact PNU sites in individual α7 receptors in real-time.…”

γ-Aminobutyric Acid Type A (GABAA) Receptor α Subunits Play a Direct Role in Synaptic Versus Extrasynaptic Targeting

“…The ultrafast drug application system (ALA Inc., Long Island, NY) consists of solution reservoirs, manual switching valves, a solenoid-driven four-way pinch valve, and two tubes (inner diameter 500 m) oriented at 50°for rapid solution exchange (43,44). One tube contains normal bath solution and the other contains 10 mM GABA to maximally activate GABA A -Rs.…”