Number of extracellular-transmembrane interfaces required for activation of homomeric Cys-loop receptors.

ANDERSEN N; CORRADI J; BARTOS M; SINE SM; BOUZAT C

Baltimore, Maryland

Congreso; 55th Annual Meeting Biophysical Society; 2011

Biophysical Society

Each subunit in a homo-pentameric Cys-loop receptor contains a specialized transduction zone located at the extracellular-transmembrane interface that links the ligand binding domain to the ion conductive channel. To determine the contribution of each transduction zone to stability of the open channel, we constructed a subunit with both a disabled transduction zone and a reporter mutation that alters unitary conductance, and co-expressed mutant and normal subunits. The resulting receptors show single channel current amplitudes that are quantized according to the number of reporter mutations per receptor, allowing correlation of mean open time with the number of intact transduction zones. We find that each transduction zone contributes an equal increment to the stability of the open channel. However by combining subunits with either disabled agonist binding sites or transduction zones, we find that although each binding site is formed by a pair of subunits, detectable channel opening requires an intact transduction zone in both subunits. By manipulating the numbers and locations of transduction zones and binding sites, we find that a transduction zone in a subunit at an inactive binding site can still stabilize the open channel. The findings show that although the agonist binding sites and transduction zones contribute allosterically to open channel stability, their stoichiometry and positioning requirements are distinct.