Abstract
In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels.
Highlights
In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits
When one or more ligands bind to a multimeric receptor, it exerts a conformational change, resulting either in modulating a conductance for ions or a changed readiness to interact with other proteins
Ligand-gated ion channels provide the unique feature that the final conformational change, the pore opening, can be directly excellently quantified by the patch-clamp technique[2], often by using single-channel analysis and hidden Markovian state models[3]
Summary
The sampling rate was 5 kHz and the filter of the amplifier (4-pole Bessel) was set to 2 kHz. For single-channel measurements, the patch pipettes were pulled from quartz tubing with an outer and inner diameter of 1.0 and 0.5 mm (VITROCOM, New Jersey, USA). The sampling rate was 20 kHz and the filter of the amplifier (4-pole Bessel) was set to 5 kHz. The pipette solution contained (in mM): 150 KCl, 1 EGTA, 5 Hepes (pH 7.4 with KOH) and 200 μ M niflumic acid to block endogenous chloride channels. Some concentration-activation relationships required the sum of a high (H) and a low affinity (L) component: I/I max = A/(1 + (EC50,H/[cGMP])nH) + (1 − A)/(1 + (EC50,L/[cGMP])nL).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
