ELIC Channels Activate Slowly in Response to Agonist Concentration Jumps

Abstract

Prokaryotic members of the Cys-loop superfamily of ligand-gated channels, such as GLIC and ELIC, have given us X-ray structures of different functional states of the channel. Zimmermann & Dutzler (PLOS Biology, 9, e1001101, 2011) have recently shown that ELIC is activated by a range of primary amines, including GABA, making this channel an attractive model for investigating agonist mechanisms. As a first characterisation of ELIC channel kinetics, we have used the technique of rapid theta-tube application to elicit macroscopic agonist currents. Outside-out patches from HEK293 cells expressing ELIC were held at −100 mV and agonists were applied by concentration jumps, 400-550 ms in duration, with exchange times faster than 0.4 ms (20-80%, measured with diluted extracellular solution after patch rupture). We tested propylamine (10-50 mM), cysteamine (10-50 mM) and GABA. For propylamine and cysteamine, the speed with which the current on-relaxation developed reached a clear maximum at 50 mM. At this concentration the time constant for the current risetime was 5.4 +/- 0.75 ms (n = 6) and 7.3 +/- 0.82 ms (n = 9) for cysteamine and propylamine, respectively. Thus, the most striking property of agonist ELIC responses was their slow time course, when compared with mammalian Cys-loop channels, such as the ACh muscle nicotinic or the glycine channel. In similar experiments with glycine channels, the response risetime was effectively too fast to be characterised by this technique, as its time constant reached sub-millisecond values at 3 mM glycine.