We are currently focused on identifying how and when hair cells acquire the remarkable ability of mechanosensitivity and sensory signaling. In particular, we want to know which molecules are involved in the signal transduction cascade and when they are acquired during normal development. To address these questions we have taken several approaches. We have examined the normal development of hair cells from the wild-type mouse inner ear to define the temporal pattern of functional acquisition of signaling components (Géléoc & Holt, 2003; Géléoc et al. 2004). To identify candidate molecules that contribute to essential hair cell functions we have identified temporal correlations between the physiological expression patterns and the expression pattern of hair cells genes using quantitative RT-PCR. To test the hypotheses generated based on these correlations we examine hair cells of mice that carry naturally occurring mutations, as well as transgenic animals including, targeted gene deletions and target gene replacements with mutant genes. In addition, we have pioneered the use of adenoviral vectors to drive expression of dominant-negative constructs to suppress the function of endogenous hair cell proteins; overexpression of wild-type genes to rescue mutant phenotypes; expression of GFP tagged constructs to facilitate protein localization; and suppression of endogenous gene expression using siRNAs. To assay for changes in function we image FM1-43 uptake, an indicator of functional mechanotransduction, and use the whole-cell, tight-seal recording technique in voltage-clamp mode to record transduction currents or voltage-dependent currents. We use a fast piezoelectric bimorph with a submillisecond rise-time to evoke hair bundle deflections. In current-clamp mode we record membrane potential to examine the functional consequences of altered gene and protein expression. Using these approaches we have identified the physiological consequences of mutations in two structural proteins that are required for integrity of the sensory hair bundle, protocadherin 15 (PCDH15) and the very large G-protein coupled receptor1 (VLGR1). In the case of PCDH15 (Senften et al. 2006), we found that a naturally occurring mutation, the av3j allele, causes a loss of mechanosensitivity in vestibular and auditory hair cells of early postnatal mice. Localization of the protein and its binding to myosin 7a suggest it may be a component of the extracellular linkages that help maintain the hair bundle in a rigid, upright configuration. Generation of a mouse that carried a targeted deletion of the 7th transmembrane domain of VLGR1 resulted in a lack of FM1-43 uptake and lack of mechanotransduction currents in auditory but not vestibular hair cells (McGee et al. 2006). Immunolocalization of VLGR1 to the base of auditory hair cells suggests it may be component of the ankle link, a linkage required for the normal development and function of hair cells. To investigate the role of myosin molecules we have used a chemical-genetic strategy and generated a mouse that expresses a mutation in the ATP binding pocket of myosin 1c. The mutation, known as Y61G sensitizes the motor protein to inhibition by an ADP analog, NMB-ADP. We have found that acute application of NMB-ADP disrupts both fast and slow adaptation in vestibular hair cells of mutant but not wild-type mice (Stauffer et al. 2005). Since fast adaptation has been implicated in auditory amplification, we suggest that myosin 1c may be a component of the elusive cochlear amplifier. To examine the function of voltage-dependent conductances localized to the basolateral membrane, we have generated modified adenoviral vectors and infected hair cells of organotypic cultures. One class of potassium channel, known as KCNQ4, is highly expressed in both auditory and vestibular hair cells and causes a dominant, progressive hearing loss when mutated. To identify the K+ currents carried by KCNQ4 and the function of those currents we generated a vector that expressed GFP as a marker and the dominant-negative form of KCNQ4. We found that the mutant KCNQ4 suppressed the endogenous K+ currents carried by wild-type KCNQ4 in both auditory and vestibular hair cells. In current-clamp mode we found that the cells that expressed the mutant channels had depolarized resting potentials and had larger receptor potentials relative to wild-type controls. Since the conductance is active at rest, we conclude that KCNQ4 functions to maintain hyperpolarized resting potentials and attenuate the hair cell receptor potential. Our presentation will highlight some of these recent findings and the approaches we have taken to understand the hair cell transduction cascade from its origin in the hair bundle to its transmission at the afferent synapse.