The slow component of adaptation was slower in Tmc1Δ/Δ;Tmc2+/Δ cells than either Tmc1+/Δ;Tmc2Δ/Δ or Tmc1Bth/Δ;Tmc2Δ/Δ cells ( Figure 3E). The extent of adaptation also varied among the three genotypes ( Figure 3F). The differences in fast adaptation time constants may be a consequence of different calcium permeabilities ( Figure 2F) or they may Raf inhibitor reflect inherent differences in the adaptation properties of TMC proteins. Wu et al. (1999) modeled adaptation in auditory hair cells and suggested that fast

adaptation required a calcium binding site in close proximity to the channel pore. Thus, it is plausible that amino acid sequence differences between TMC1 and TMC2 may contribute to the different fast adaptation properties reported here. Because slow adaption

is thought to involve myosin motors ( Holt et al., 2002) at a remote location ( Wu et al., 1999), minor changes in calcium entry in cells this website bathed in 50 μM calcium may have little impact on the local calcium concentration at the slow adaptation site due to diffusion and the activity of calcium pumps and buffers. Another core property of an ion channel is its single-channel conductance. To examine contributions of TMC proteins to the properties of single transduction channels we designed a stimulation and recording paradigm. Inner hair cell bundles consist of an array of loosely organized stereocilia, with few lateral connections and the tallest row towering above the rest. To exploit this morphology, we engineered Phosphatidylinositol diacylglycerol-lyase stimulus pipettes that tapered to a few hundred nanometers in diameter at their distal tips which we used to deflect single stereocilia (Figure S4). We recorded the response of single stereocilium deflections in whole-cell mode at a holding potential of −84 mV. At this potential, voltage-dependent sodium and calcium channels were deactivated and we substituted Cs+ for K+ in the recording pipette to block residual potassium currents. The cells were bathed in an endolymph concentration of calcium, 50 μM, which had the dual effect of enhancing transduction current

amplitudes relative to standard extracellular calcium (i.e., minimizing calcium block) and prolonging channel open times by reducing calcium-dependent adaptation. We began with a characterization of Tmc1Δ/Δ;Tmc2Δ/Δ cells which lacked single-channel currents entirely and thus permitted evaluation of the recording paradigm in a quiescent background ( Figure 4A). We observed no voltage-dependent or ligand-gated ion channel activity. Under these conditions the root mean square (RMS) noise was 2.2 pA, which we reasoned would allow us to resolve currents with amplitudes greater than 4.4 pA. When we used this technique to record from Tmc1Δ/Δ;Tmc2+/Δ inner hair cells, we observed prominent single-channel currents ( Figure 4B) in response to small stereocilium deflections. The single-channel events were blocked by application of 0.