Proceedings of The Physiological Society
University of Bristol (2005) J Physiol 567P, PC209
Fast and slow responses to acetylcholine in outer hair cells
Darbon, Pascal; Wright, Daniel J; Evans, Michael G;
1. MacKay Institute of Communication and Neuroscience, Keele University, Newcastle-under-Lyme, United Kingdom.
Outer hair cells (OHCs) of the mammalian cochlea have a nicotinic acetylcholine receptor (AChR) comprising α9 and α10 subunits and forming a cation channel with a high calcium permeability (see Lioudyno et al. 2004). Acetylcholine produces inhibition of OHCs as calcium influx through this receptor gates a SK-type calcium activated potassium current. Experiments on isolated OHCs showed that the response was rapid, activating in 200 ms (Evans, 1996). We have observed a slow current in OHCs that appears to flow through the same AChR. Guinea pigs were humanely killed and OHCs isolated from cochlear turns 2 and 3 as described previously (Evans, 1996). Whole-cell voltage-clamp recordings were made from OHCs at room temperature. ACh (100μM) was pressure-applied from a pipette positioned about 20 μm from the cell base. The pipette (internal) solutions were KCl/5 mM EGTA (see Evans, 1996) or CsCl/10 mM BAPTA, the latter being used to monitor the AChR current in isolation. With the CsCl/BAPTA solution, I-V plots of the AChR current had a reversal potential of -4 mV and prominent rectification, particularly in the outward direction. The current activated in 150 ms. With the KCl/EGTA solution, the ACh-evoked current usually had an N-shaped I-V relation as expected for a calcium activated potassium current dependent on calcium influx. Occasionally ACh application produced a much slower current, activating over about 1 s after a 0.5 s delay, either on its own or in addition to the faster potassium current. This current was similar to the AChR current in terms of reversal potential and block by α-bungarotoxin (0.4 μM), but was larger (up to -1 nA at -60 mV) and inwardly rectifying. It was still present in low external calcium (0-0.4 mM) and was more commonly observed under these conditions (in ~50% of recordings compared to ~5% in controls). Application of ACh during exposure (4 min) to external thapsigargin (1-5 μM) or 2,5-di-(t-butyl)hydroquinone (BHQ, 50 μM) did not produce the slow current, suggesting that calcium stores are not involved. This current appears to be a slower kinetic version of the fast AChR current.
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