Joseph Santos-Sacchi - US grants

The organ of Corti is composed of a variety of cell types including sensory, supporting and neural elements. Taken together, these cells comprise a functionally intricate and cohesive electrical unit that initiates the analysis of acoustic information within our environment. This electrical unit is extremely complex and nearly anatomically inaccessible, making an analysis of the whole quite a challenge. Fortunately, during the last several years the in vitro approach, including the isolated cochlea and cell preparations, has aided in the elucidation of cell function; the strategy is to understand the cells first on an individual basis, and finally to integrate this knowledge into a complete understanding of the organ of Corti. The overall aim of this project is to analyze the membrane properties of the outer hair cell (OHC), one of the major players in auditory function, principally using variations on the whole cell voltage clamp technique. Three areas of investigation are proposed. Specifically, we intend to 1) study in detail the mechanical sensitivity of the OHC lateral membrane as it relates to chloride flux, 2) study the effects of chloride and anions on the OHC motor activity, and 3) study mechanical coupling among OHCs in an explant organ of Corti preparation. The results that we will obtain will lead to a deeper understanding of inner ear function and aid in understanding auditory pathologies, which may result from OHC insult and homeostatic imbalance, including sensorineural hearing loss and tinnitus.

The overall aim of this project is to analyze the membrane properties of several isolated cell types from the inner ear using the whole cell voltage clamp technique. The cell types (outer hair cells [OHCs], Type II spiral ganglion cells, and Deiter cells) were chosen because they all functionally relate (directly or indirectly to the OHC system. This system has recently been shown to be very important for normal cochlear function. Several properties of OHCs will be studied, including their inward Ca+2 currents, the mechanism of gadolinium induced block of voltage induced OHC motility, and the frequency response of OHC motility. These evaluations will be made by measuring the voltage dependence of these currents and motility blocking effects, by comparing the motility characteristics to those of stretch receptors, and by measuring OHC mechanical response magnitude induced by AC stimuli. The ionic conductances of Type II spiral ganglion cells and Deiter cells will be analyzed to determine their roles in the OHC system. Specifically, Deiter cells, which are coupled to other supporting cells via gap junctions, will be evaluated via voltage clamp to determine whether they possess mechanisms (e.g., inwardly directed K+ currents) which might aid in the ionic homeostasis of the organ of Corti. Type II spiral ganglion cells will be evaluated in similar fashion to determine whether they possess the ionic conductances necessary for the transfer of acoustic information to the central nervous system. Since no electrophysiological information is available on this cell type, this is an important issue. Understanding these phenomena will shed light upon the role that OHCs play in fine frequency tuning within the organ of Corti via mechanical feedback mechanisms. They will also help elucidate the effects of pathologies of the outer hair cell system, including ionic imbalances (e.g., Meniere's disease), noise induced hearing loss, and tinnitus.

The organ of Corti is composed of a variety of cell types including sensory, supporting and neural elements. Taken together, these cells comprise a functionally intricate and cohesive electrical unit that initiates the analysis of acoustic information within our environment. This electrical unit is extremely complex and nearly anatomically inaccessible, making an analysis of the whole quite a challenge. Fortunately, during the last several years the in vitro approach, including the isolated cochlea and cell preparations, has aided in the elucidation of cell function; the strategy is to understand the cells first on an individual basis, and finally to integrate this knowledge into a complete understanding of the organ of Corti. The overall aim of this project is to analyze the membrane properties of the outer hair cell (OHC), one of the major players in auditory function, principally using variations on the whole cell voltage clamp technique. Three areas of investigation are proposed. Specifically, we intend to 1) analyze the mobility OHC sensor/motors in the lateral plasma membrane, 2) study in detail and make modifications to the electrical correlate of OHC motility, its nonlinear capacitance and analyze what such modifications will do to the high frequency mechanical activity of the OHC, and 3) study the mechanical coupling among OHCs that we have just discovered. These results will lead to a deeper understanding of inner ear function and aid in understanding auditory pathologies which may result from OHC insult and homeostatic imbalance, including sensorineural hearing loss and tinnitus.

DESCRIPTION (provided by applicant): With the identification of prestin as the elusive lateral membrane motor protein of the outer hair cell (OHC), we are faced with the possibility of understanding how this single molecule can affect the mammal's exquisite sense of hearing. To that end, we have focused our interest on determining what protein structures may give rise to the motor's known biophysical attributes, including its temperature, tension, and voltage dependence. We hypothesize that these molecular activities arise and/or are influenced by interactions of prestin's intracellular C and N termini with other intracellular proteins and anions, and possibly by multimeric interactions, as well. We propose to target a focused set of aims, including 1) determine the contribution of prestin's C and N termini to prestin's signature biophysical attributes, 2) determine prestin's trafficking route in a prestin cell line that we have developed, and 3) determine what structures are different between prestin (slc26a5) and its closet family member slc26a6 that account for prestin's nonlinear capacitance. In order to reach these goals, we will employ a host of electrophysiological, molecular biological and biochemical methods. We believe that the information that we obtain through these studies will aid in understanding how the OHC enables us to hear so well and in turn how we might combat pathologies of the OHC that afflict millions.

Project Summary In this proposal we seek to develop single molecule FRET in prestin. The method will allow us to assess the function of the protein using optical measures. Single molecule FRET has been developed in other proteins to aid in the understanding of their function. Presently, we do not have a functional assay of prestin with a single molecule resolution. The rationale for the experiments is based on structural data interpolated from the structure of several members of the extended anion transporter family to which prestin belongs. Available structural data together with modeling data suggest that members of this family have 14 transmembrane domains with an inverted repeat 7+7 structure to each monomer. The functional units of these proteins appear to be dimers. Each monomer has a gate and core domain. We model prestin to behave like these transporters using an elevator mechanism to move from contracted to expanded states. An analogous movement in these family members from inside open to outside open conformations result in a relative motion between two parts of the same monomer. Thus, there is a movement of the gate domain (Tm domains 5-8 and 12-14) relative to the stable core domain (Tm domains 1-4 and 9-11) containing the Tm anion binding site. Based on these models we will insert several cysteine residues in the gate domain that will allow us to label a single dimer with two different fluorophores with FRET activity. We have already generated a cysteine free protein that shows reduced but measurable gating charge movement (NLC), confirming its functionality. The distance between the fluorophores is modelled to vary by 30A0 that would give rise to a significant change in FRET efficiency. Establishing a single molecule FRET assay will allow us to measure how physiological parameters affect the function of the protein.

With the identification of prestin, an anion transporter (SLC26) family member, as the elusive lateral membrane motor protein of the outer hair cell (OHC), we are faced with the possibility of understanding how this single molecule can effect the mammal?s exquisite sense of hearing. We propose to target a focused set of aims, including 1) determining the role of a leakage conductance in prestin, distinct from its transporter pathway, in hearing and defining its structural basis; 2) determine the role of anion binding residues, and the influence of mechanical load in governing prestin?s frequency dependence; simultaneous measures of sensor charge movement (NLC), electromotility and evoked-forces are planned and 3) determine the structural components of pillars that link prestin to the underlying cytoskeleton, and confirming the importance of such links in hearing. In order to reach these goals, we will employ a host of genetic, electrophysiological, molecular biological and biochemical methods. We believe that the information that we obtain through these studies will aid in understanding how the OHC enables us to hear so well and in turn how we might combat pathologies of the OHC that afflict millions.