Research Program

Electrical tuning is a phenomenon by which certain vertebrates discriminate between different frequencies of sound. Electrical resonance results when the inherent oscillation in the membrane potential of hair cells corresponds to sound of a particular frequency. This gives rise to a resonance and amplification of signal with consequent transmitter release from these cells. The inherent oscillation in membrane potential in a hair cell is brought about by an inward calcium current and an outward potassium current (calcium dependent). The molecular identity of these currents has been established with the Ca current carried by a L type DHP sensitive Ca channel, and the K current carried by the large conductance Ca activated K channel (BK). The systematic variation in the frequency of membrane potential oscillation in hair cells that occurs along the tonotopic axis is brought about primarily by a variation in the kinetic properties of the BK current. BK channels are made up of the Slo protein and a variable number of associated proteins. Previously, we had shown that some of the variation in kinetic properties along the tonotopic axis was brought about by alternative splicing of the Slo gene. However, a large part of this variation in BK kinetics cannot be explained by alternative splicing alone. Our present hypothesis is that this variation in current is brought about by association with other proteins and systematic changes in phosphorylation that intersects with alternative splicing. For instance, gene expression differences along the tonotopic axis strongly suggest that PKA is more active in low frequency hair cells while PKC is more active in high frequency hair cells. Moreover, we have determined novel interactions between CDK5 and Slo and demonstrated that CDK5 is expressed in increasing amounts in high frequency hair cells.

We have also demonstrated by fluorescence immunohistochemistry that the BK channel and the calcium channel are, in fact, in close physical approximation in these hair cells. There are considerable theoretical reasons to support this observation. We have determined that while localization to the basolateral surface of hair cells is determined by signals within the protein, a number of BK channel associated proteins also play a critical role in its surface expression. Additionally, we determine that phosphorylation both directly (CDK5) and indirectly (PKC) affects BK channel expression on the surface of hair cells.

Prestin is the molecular motor responsible for outer hair cell motility, that is, in turn, responsible for ochlear amplification responsible for the exquisite hearing sensitivity in mammals. Prestin is unusual in that it resides in the membrane and can alternate between a contacted and expanded state at very fast rates (up to 100,000 in bats for instance). My lab is focused on the structure-function relationship of the protein and on specific cell biological aspects of the protein. We have determined that prestin has intrinsic charge that is responsible for its voltage sensitivity. Unlike classic voltage sensitive ion channels, however, residues conferring voltage sensitivity are distributed throughout the transmembrane regions of the protein. Moreover, we have determined that prestin can transport anions. This latter finding was somewhat controversial and has been substantiated by recent papers from John Ashmore’s group, and Schänzler and Fahlke in Germany. More recently, we are investigating its chloride binding site (prestin is modulated by intracellular chloride). Our current working hypothesis is that prestin, in lieu of its anion transporter activity and intracellular chloride sensitivity, is able to expand and contract on a cycle-by-cycle basis upon “activation” by chloride conferring its ability to act as a motor. This idea proposed by Dr Santos-Sacchi gets around a key limitation of the prestin hypothesis that requires voltage sensitivity to operate on a cycle by cycle basis which is limiting above approximately 2000Hz owing to the membrane time constant.

A goal in our lab is to identify the molecular events that prevent the adult mammalian cochlea and vestibular epithelium from regenerating new hair cells. We have previously concentrated on the bird auditory epithelium since it shows mitotic quiescence, but is able to mount a robust proliferative response to hair cell damage. We reasoned that understanding the molecular events that lead to the conversion of supporting cells from mitotic quiescence to proliferation would give us insights into the mammal. However, we have been discouraged by this strategy. We have gained a lot of insights into the molecular events that underlies regeneration, but have not been able to convert these insights into the mammal. For instance, we determined that microRNA 181 was responsible for a large number of gene expression changes in the regenerating chick auditory epithelium. Transfection of microRNA181 results in a proliferative response in the bird auditory epithelium. Such an effect is not observed in mammalian utricles.

Our present efforts are now directed at understanding the molecular basis of how the vestibular epithelium in mammals undergoes an age associated loss in mitotic ability.