, 1989, Hudspeth and Gillespie, 1994, Ricci and Fettiplace, 1997 and Ricci et al., 1998). BTK inhibitors Here, we used either apical perfusion or a local pipette to apply a 20 μM Ca2+ solution onto the hair bundle of rat OHCs and observed an increase in the peak current amplitude due to removal of a Ca2+ block of the channel (Figure 7A;

Pan et al., 2012 and Ricci and Fettiplace, 1998). We consistently found a large change in the resting open probability, as previously described (Figures 7A and 7B) (Beurg et al., 2008 and Beurg et al., 2010). In contrast to previous data from low-frequency hair cells (Ricci and Fettiplace, 1997 and Ricci et al., 1998), the shift observed in resting open probability was unaffected by intracellular Ca2+ buffering. With

no correction for baseline changes, our current-displacement plots underestimate the actual baseline shift, but nonetheless, reveal a large leftward shift (Figure 7B). Additionally, unlike in low-frequency cells, lowering external Ca2+ did not slow the adaptation time constants in OHCs; rather, the proportion of the slower time constant was increased (Figure 7C). Due to the difficulty of pulling the hair bundle with our stiff probe, we used the permeable blocker dihydrostreptomycin (DHS) to provide a better estimate of resting open probability by blocking the MET current in lowered external Ca2+ (Figures 7D and 7E). These results confirmed that large shifts in the resting open probability were independent of internal Ca2+ buffering. Taken together, these data suggest VEGFR inhibitor that external Ca2+ regulation of MET resting open probability is independent of adaptation and intracellular DNA ligase Ca2+ levels and is mediated by an external Ca2+ site. Long-standing theories, largely based on data obtained from turtle, frog, and mammalian vestibular hair cells, posit that Ca2+ entry through MET channels is required for adaptation (Corey and Hudspeth, 1983b, Crawford et al., 1991, Eatock et al., 1987, Peng et al.,

2011 and Ricci et al., 1998). Subsequent experiments identified two components of adaptation (Vollrath and Eatock, 2003 and Wu et al., 1999), each driven by Ca2+ entry, a fast component, where multiple mechanisms have been proposed (Bozovic and Hudspeth, 2003, Cheung and Corey, 2006, Choe et al., 1998, Crawford et al., 1991 and Stauffer et al., 2005) and a slower (motor) component, controlled by myosin isozymes (Gillespie and Cyr, 2004). Initial work from mammalian auditory hair cells suggested adaptation was faster but largely similar to that reported in other hair cell types, and mechanisms of mammalian auditory adaptation have remained largely unexplored (Kennedy et al., 2003). Our data challenge these views of adaptation by demonstrating that Ca2+ entry does not drive adaptation in mammalian auditory hair cells and that motor adaptation as described in other hair cell types has at best a limited role.