Detection and trapping of intermediate states priming nicotinic receptor channel opening

Abstract

In the course of synaptic transmission in the brain and periphery, acetylcholine receptors (AChRs) rapidly transduce a chemical signal into an electrical impulse. The speed of transduction is facilitated by rapid ACh association and dissociation, suggesting a binding site relatively non-selective for small cations. Selective transduction has been thought to originate from the ability of ACh, over that of other organic cations, to trigger the subsequent channel-opening step. However, transitions to and from the open state were shown to be similar for agonists with widely different efficacies. By studying mutant AChRs, we show here that the ultimate closed-to-open transition is agonist-independent and preceded by two primed closed states; the first primed state elicits brief openings, whereas the second elicits long-lived openings. Long-lived openings and the associated primed state are detected in the absence and presence of an agonist, and exhibit the same kinetic signatures under both conditions. By covalently locking the agonist-binding sites in the bound conformation, we find that each site initiates a priming step. Thus, a change in binding-site conformation primes the AChR for channel opening in a process that enables selective activation by ACh while maximizing the speed and efficiency of the biological response.

Fig. 1

Agonist-independent channel gating. a, Spontaneous single channel currents through AChR containing Leu-to-Ser mutations at position 9′ of the second transmembrane domain (L9’S) of the β and δ subunits. Cell-attached configuration; membrane potential, -70 mV; bandwidth, 10 kHz; channel openings are upward deflections. Dwell time histograms are shown with probability density functions obtained by fitting the scheme, right, to the dwell times. Rate constants are from Table S1. b, Same as panel a but with 10 nM ACh. c, Plot of fitted channel opening and closing rate constants against ACh concentration (Table S1).

Fig. 2

Covalent priming of the AChR. a, Torpedo AChR (PDB code: 2bg9); α-subunit green, δ-subunit orange. Boxed region, magnified right, indicates Cys substitutions at positions α192 and δ121. b, upper trace: spontaneous currents through AChR containing L9’S mutations in the β and δ subunits and Cys substitutions at both ACh binding sites. Lower trace: Spontaneous currents from the patch above following application of H₂O₂. c, upper trace: spontaneous currents through AChR with L9’S mutations and Cys substitutions at both ACh binding sites treated with H₂O₂. Lower trace: Spontaneous currents from the patch above following application of DTT. In panels b and c, dwell time histograms are fitted by sums of exponentials. Results are summarized in Table S2.

Fig. 3

Mutating residues linking binding and pore domains increases or decreases priming. a, Upper trace: spontaneous single channel currents through AChR containing L9’S mutations in the β and δ subunits and αY190F. Lower trace: ACh-evoked single channel currents from a different patch containing the same mutant AChR. b, Spontaneous currents through AChR containing L9’S mutations in the β and δ subunits and αP272A. c, Spontaneous currents through AChR containing L9’S mutations in the β and δ subunits and εP121L. Dwell time histograms are shown with fitted probability density functions (Table S1).

Fig. 4

Primed model of AChR activation. Agonist binding, priming and channel gating steps are indicated (inset). C, C’ and C” symbolize closed states, while O’ and O” symbolize open states. For the wild type AChR in the absence of ACh, the C’ and C” states are negligible, indicating the first step in the activation process generates AC, from which there are three possible paths toward A₂C”. Fitting the path, bind-bind-prime-prime, did not give well defined rate constants, possibly due to an inability to distinguish interconnected A₂C’ and A₂C” states. Fitting the path, bind-prime-prime-bind, also did not give well defined rate constants, possibly because the second binding step would be to a primed site presumed to have reduced accessibility to small molecules. The remaining path in red was fitted to agonist-dependent dwell times from the wild type AChR, yielding the rate constants in Table S4.