Single-channel kinetic analysis of alpha7-5HT3 receptors

CECILIA BEATRIZ BOUZAT; D RAYES; G SPITZMAUL; S SINE

Long Beach

Congreso; 49th Annual Meeting Biophysical Society; 2005

Nicotinic acetylcholine receptors are members of the Cys-loop superfamily of neurotransmitter receptors, and assemble into either homo- or hetero-pentamers. Homo-pentameric receptors such as alpha7 are widespread throughout the nervous system, and are thought to most closely resemble the ancestral receptor of the superfamily. However, despite the fundamental importance of alpha7, single channel kinetic analysis has been limited because it does not express on the surface of mammalian cells. This limitation was overcome by generating a chimeric receptor containing alpha7 sequence in the extracellular domain and 5HT3 sequence in the pore domain, but the single channel conductance of the chimera was too low for detection. To increase conductance of the chimera, we introduced the mutations R432Q, R436D, R440A in the M3-M4 linker of 5HT3 (Bouzat, et al., 2004, Nature, 430, 896-900), enabling kinetic analyses of the 6 pA single channel events elicited by ACh (membrane potential -70 mV). Both open and closed time histograms are described as the sum of multiple exponential components, but these do not change over a wide range of ACh concentrations (50 µM to 20 mM), and channel events do not activate in clusters of closely-spaced openings flanked by prolonged silent periods. Similar results are seen with the agonists nicotine and choline. Fast application of ACh to cell-free outside-out patches elicits macroscopic currents with a near-instantaneous rise and a decay of 10 ms, indicating fast activation followed by desensitization comparable to the mean duration of the longest component of channel openings. The absence of clusters of channel events and the invariant multi-component dwell time histograms are explained by similar rates of channel closing and desensitization, indicating the overall response is governed jointly by activation and desensitization processes.