Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, C119
Ionic conductance and block of the outer hair cell ACh receptor
M. G. Evans1, P. Darbon1,2
1. ISTM, Keele University, Keele, United Kingdom. 2. Nociception et Douleur, Universit? Louis Pasteur, Strasbourg, France.
Cochlear outer hair cells (OHCs) play a crucial role in hearing, acting to amplify the vibrations of the basilar membrane induced by sound. They are under efferent inhibitory control from the crossed olivocochlear bundle via a nicotinic ACh receptor (AChR) at the OHC efferent synapse. Recently considerable progress has been made towards understanding exactly what type of AChR is involved. Experiments on neonatal inner hair cells suggest that the receptor contains both α9 and α10 subunits, based on a close functional similarity between the hair cell receptor and the recombinant α9α10 receptor, and the fact that these subunits are found in mammalian hair cells (see 1). We have investigated the AChR current in OHCs in order to provide a basis for its eventual classification, and also to assess its similarity to α9α10. Experiments were done on isolated OHCs from guinea pigs using the whole-cell recording technique under conditions where the coactivating Ca2+-activated K+ currents were abolished, usually by strong internal buffering using BAPTA (10 mM) in Cs+-based internal solutions. Under these conditions, in response to 100 μM ACh, AChR currents activated rapidly (100 ms), reversed at -6 ± 3 mV (mean ± S.E.M. n=4) and showed inward (at negative voltages) and outward rectification, although outward currents were about three times larger than inward currents for an equivalent driving force. Replacement of 70% of the internal Cs+ with N-methyl-D-glucamine (NMG+), produced a shift in the reversal potential to +9 ± 2 mV (n=3), as expected for a non-specific cation channel that was less permeant to NMG+ than to Na+ or Cs+. Current-voltage curves were fitted with a single energy barrier model (2) that indicated a blocking site or energy barrier about 0.3 of the way through the channel from the outside. We assume that this site is normally occupied by Ca2+ or other divalents since increasing external Ca2+ from 1 mM to ≥10 mM at constant Mg2+ blocked the AChR currents. Removal of external Ca2+ at constant Mg2+ resulted in a reduction in current amplitude (50% at +50 mV) and a more symmetrical rectification. An additional finding was that replacement of 78% of the external Na+ with NMG+ resulted in substantially reduced AChR currents at all voltages but especially at negative voltages, which suggested that NMG+ blocked the channel. Internal NMG+ did not block the currents. Our data on the external Ca2+ sensitivity of the AChR are qualitatively similar to published data on α9α10 (3), where Ca2+ both potentiates (maximum at 0.5 mM) and blocks (> 0.5 mM) the receptor current. Also, both receptors show similar bi-directional rectification. At this stage the data would suggest a close similarity between the OHC AChR and α9α10, although further data are needed to test the hypothesis that they are the same.
Where applicable, experiments conform with Society ethical requirements