cAMP and voltage modulate rat auditory mechanotransduction by decreasing the stiffness of gating springs

Hair cells of the auditory and vestibular systems transform mechanical input into electrical potentials through the mechanoelectrical transduction process (MET). Deflection of the mechanosensory hair bundle increases tension in the gating springs that open MET channels. Regulation of MET channel sensitivity contributes to the auditory system’s precision, wide dynamic range and, potentially, protection from overexcitation. Modulating the stiffness of the gating spring modulates the sensitivity of the MET process. Here, we investigated the role of cyclic adenosine monophosphate (cAMP) in rat outer hair cell MET and found that cAMP up-regulation lowers the sensitivity of the channel in a manner consistent with decreasing gating spring stiffness. Direct measurements of the mechanical properties of the hair bundle confirmed a decrease in gating spring stiffness with cAMP up-regulation. In parallel, we found that prolonged depolarization mirrored the effects of cAMP. Finally, a limited number of experiments implicate that cAMP activates the exchange protein directly activated by cAMP to mediate the changes in MET sensitivity. These results reveal that cAMP signaling modulates gating spring stiffness to affect auditory sensitivity.

Hair cells of the auditory and vestibular systems convert mechanical input from sound and head movement into an electrical current through a process termed mechanoelectrical transduction (MET). At the apical surface of each hair cell is a specialized mechanosensitive organelle called the hair bundle, composed of a collection of actin-filled stereocilia arranged in graded height. The tips of the shorter rows house MET channels (1), nonspecific cation channels hypothesized to be comprised of TMC1/2, TMIE, and a number of other accessory proteins (2–4). MET channels are gated by a tension force imparted by a gating spring, which is part of or connected with the filamentous tip links that span adjacent rows of stereocilia (5–9). Positive stimuli directed along the sensitive axis of the hair bundle (10) result in displacement toward the tallest row and increase tension in the gating spring, increasing the open probability of the MET channel.Mechanisms that regulate the sensitivity of the MET channel are hypothesized to contribute to the wide dynamic range and frequency selectivity of the auditory system, with MET adaptation being the most widely studied (11–14). Based on the gating spring theory, changes in gating spring stiffness alter the setpoint and the sensitivity of the MET channel (5, 6). The second messenger cyclic adenosine monophosphate (cAMP) has been proposed to modulate the sensitivity of the MET channel in turtle and bullfrog hair cells. In turtle auditory hair cells, cAMP shifts the operating point of the MET channel after a period of minutes, extending the dynamic range of the channel (15). In bullfrog hair cells, pharmacological up-regulation of cAMP reduced the frequency of spontaneous hair bundle oscillations (16). Furthermore, in humans, a loss of function mutation in adenylyl cyclase 1 (ADCY1, an enzyme that catalyzes cAMP production) is associated with hearing loss (17) and zebrafish carrying an equivalent mutation (adcy1b morphants) exhibited significantly reduced fn1-43 dye uptake in lateral line hair cells, indicating dysfunctional MET. These studies indicate a potential role for cAMP in modulating MET channel sensitivity in mammals.In this study, we determine the mechanism of cAMP modulation of the MET channel in a mammalian cochlear hair cell model. We first characterized the cAMP effect in rat outer hair cells (OHCs). Second, we describe a long depolarization manipulation (LDM), which we found mirrored the effects of cAMP up-regulation. Third, by measuring the mechanical changes of the hair bundle, we confirmed that cAMP and long depolarization-induced effects on channel sensitivity are due to a reduction in gating spring stiffness. Finally, we find that cAMP targets the exchange protein directly activated by cAMP (EPAC) rather than protein kinase A (PKA) for modulating MET sensitivity. This cAMP regulation of gating spring stiffness provides a cell signaling means for controlling the sensitivity of MET channels.