Dhasakumar S. Navaratnam MD, PhD
Associate Professor of Neurology and of Neurobiology
Hearing and Balance Organs
We work on the molecular basis of a number of physiological phenomena related to the hearing and balance organs. Phenomenon of electrical resonance. 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). Inherent to this view is that the two proteins are physically proximate.
We had previously erroneously believed that the range in BK channel currents was brought about by alternative splicing. We now hypothesize that this variation in current is brought about by association with other proteins. We have isolated several binding partners using the yeast two hybrid technique and are in the process of evaluating their ability to alter BK kinetics and bring about channel clustering and co-localization. The role of Prestin (in Collaboration with Dr. Joseph Santos-Sacchi). Prestin is a recently described protein in outer hair cells that is responsible for the sharp tuning seen in the hearing organ of mammals. It is critical for normal hearing. Knocking out of this protein results in the loss of hearing in mice.
Extensive Research Description
Phenomenon of electrical resonance
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 systematic variation in the frequency of such an oscillation in hair cells that occurs across the tonotopic axis is brought about primarily by a variation in the kinetic properties of the Potassium current. Previously we had shown that some of this variation in kinetic properties of this current was brought about by alternative splicing of this BK potassium channel. However, a large part of this variation in 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. Towards this end we have made yeast two hybrid libraries from the cochlea organ and are screening these libraries for binding proteins using the purported intracellular domains of the BK channel as “bait”. In addition we have also demonstrated by fluorescence immunohistochemistry that the calcium channel and the potassium channel are in fact in close physical approximation in these hair cells.
There is considerable theoretical reasons to support this observation. We have preliminary data that these two proteins are physically linked to one another. We are presently attempting to duplicate these results using a more sensitive method of detection (tandem mass spec at the Keck facility). We are also seeking to identify proteins that might serve as scaffolding linking both these channels. The role of Prestin (in Collaboration with Dr Joseph Santos Saatchi, Prof in Otolaryngology see www.yaleearlab.org) Prestin is a recently described protein in outer hair cells that is responsible for the sharp tuning seen in the hearing organ of mammals.
It is critical for normal hearing. Knocking out of this protein results in the loss of hearing in mice. Prestin is a “motor” protein that gives rise to electromotility in outer hair cells. Its an unusual phenomenon in that motility in outer hair cells can be rapid, upto 20KHz, which is orders of magnitude faster than conventional motor proteins like myosin. We are attempting to determine the functional domains within this protein by mutagenesis. We have made over a dozen mutants of this protein and are presently evaluating how it affects the motor function of this protein.
Regeneration in the auditory epithelium
Hearing loss in the elderly population affects upto 60% of this age group. This hearing loss is largely brought about by a loss in hair cells in the auditory epithelium. In previous experiments we had demonstrated that increasing levels of the second messenger cyclic AMP results in a cascade of events giving rise to cell division and regeneration in hair cells.
These results have since been duplicated in mice. We are attempting to identify the molecular events that underlie such regeneration and we have undertaken a subtractive hybridization experiment to determine the identity of mRNA transcripts that are upregulated with increasing cAMP. We have identified several molecules.
One such molecule is Id 2 a protein that has a binding domain but lacks the activation domain of BHLH transcription factors. Id2 is thought to repress other BHLH transcription factors by preventing their dimerization. To determine the effects of Id2 on regeneration we have used antisense oligos to suppress the expression of this protein in cochleas treated with forskolin (which increases cAMP levels).
Our preliminary data suggests that suppressing Id2 results in a reduced number of cells entering the cell cycle (measured by incorporation of BrDU). We are presently attempting to further corroborate this finding and will undertake experiments to determine the binding partners of Id2.
Cholinergic synaptic function
Acetylcholinesterase in cholinergic synapses removes acetylcholine. Acetylcholine, the neurotransmitter in cholinergic neurons, is rapidly removed from cholinergic synapses by hydrolysis by the enzyme acetylcholinesterase.
Failure to remove acetylcholine in a timely fashion gives rise to a sustained depolarization of the postsynaptic membrane and a failure to respond to further neurotransmitter release. Thus the timely removal of acetylcholine brought about by the localization of acetylcholinesterase in these synapses is critical for normal neurotransmission to occur. We have identified two proteins that likely serve to localize the protein to these neuronal synapses. One of these proteins is related to the bacterial Cu transporter CUTA1.
The other is a novel protein which however has a proline rich domain that has been shown to be important in interactions between acetylcholinesterase and the colq protein that localizes the enzyme to the synapse in the neuromuscular junction. We have made several mutants of the latter protein and are presently attempting to relate the ability of these mutants to alter the localization of acetylcholineterase to synaptic membranes.