Membrane cholesterol strongly influences confined diffusion of prestin.
ABSTRACT: Prestin is the membrane motor protein that drives outer hair cell (OHC) electromotility, a process that is essential for mammalian hearing. Prestin function is sensitive to membrane cholesterol levels, and numerous studies have suggested that prestin localizes in cholesterol-rich membrane microdomains. Previously, fluorescence recovery after photobleaching experiments were performed in HEK cells expressing prestin-GFP after cholesterol manipulations, and revealed evidence of transient confinement. To further characterize this apparent confined diffusion of prestin, we conjugated prestin to a photostable fluorophore (tetramethylrhodamine) and performed single-molecule fluorescence microscopy. Using single-particle tracking, we determined the microscopic diffusion coefficient from the full time course of the mean-squared deviation. Our results indicate that prestin undergoes diffusion in confinement regions, and that depletion of membrane cholesterol increases confinement size and decreases confinement strength. By interpreting the data in terms of a mathematical model of hop-diffusion, we quantified these cholesterol-induced changes in membrane organization. A complementary analysis of the distribution of squared displacements confirmed that cholesterol depletion reduces prestin confinement. These findings support the hypothesis that prestin function is intimately linked to membrane organization, and further promote a regulatory role for cholesterol in OHC and auditory function.
Project description:Glycosylation is a common post-translational modification of proteins and is implicated in a variety of cellular functions including protein folding, degradation, sorting and trafficking, and membrane protein recycling. The membrane protein prestin is an essential component of the membrane-based motor driving electromotility changes (electromotility) in the outer hair cell (OHC), a central process in auditory transduction. Prestin was earlier identified to possess two N-glycosylation sites (N163, N166) that, when mutated, marginally affect prestin nonlinear capacitance (NLC) function in cultured cells. Here, we show that the double mutant prestin(NN163/166AA) is not glycosylated and shows the expected NLC properties in the untreated and cholesterol-depleted HEK 293 cell model. In addition, unlike WT prestin that readily forms oligomers, prestin(NN163/166AA) is enriched as monomers and more mobile in the plasma membrane, suggesting that oligomerization of prestin is dependent on glycosylation but is not essential for the generation of NLC in HEK 293 cells. However, in the presence of increased membrane cholesterol, unlike the hyperpolarizing shift in NLC seen with WT prestin, cells expressing prestin(NN163/166AA) exhibit a linear capacitance function. In an attempt to explain this finding, we discovered that both WT prestin and prestin(NN163/166AA) participate in cholesterol-dependent cellular trafficking. In contrast to WT prestin, prestin(NN163/166AA) shows a significant cholesterol-dependent decrease in cell-surface expression, which may explain the loss of NLC function. Based on our observations, we conclude that glycosylation regulates self-association and cellular trafficking of prestin(NN163/166AA). These observations are the first to implicate a regulatory role for cellular trafficking and sorting in prestin function. We speculate that the cholesterol regulation of prestin occurs through localization to and internalization from membrane microdomains by clathrin- and caveolin-dependent mechanisms.
Project description:Full expression of electromotility, generation of non-linear capacitance (NLC), and high-acuity mammalian hearing require prestin function in the lateral wall of cochlear outer hair cells (OHCs). Estimates of the number of prestin molecules in the OHC membrane vary, and a consensus has not emerged about the correlation between prestin expression and prestin-associated charge movement in the OHC. Using an inducible prestin-expressing cell line, we demonstrate that the charge density, but not the voltage at peak capacitance, directly correlates with the amount of prestin in the plasma membrane. This correlation is evident in studies involving a controlled increase of prestin expression with time after induction and inducer dose-response. Conversely, membrane prestin levels and charge density gradually decline together following the reduction of prestin levels from a steady state by removal of the inducer. Thus, charge density directly correlates with the level of membrane prestin expression, whereas changing membrane levels of prestin have no effect on the voltage at peak capacitance in this inducible prestin-expressing cell line.
Project description:Prestin is the motor protein responsible for cochlear outer hair cell (OHC) somatic electromotility. Eliminating this abundant basolateral membrane protein not only causes loss of frequency selectivity and hearing sensitivity, but also leads to OHC death. A membrane-based yeast two-hybrid approach was used to screen an OHC-enriched cDNA (complementary Deoxyribonucleic Acid) library in order to identify prestin-associated proteins. Several proteins were recognized as potential prestin partners, including vesicle-associated membrane protein associated protein A (VAPA or VAP-33). VAPA is an integral membrane protein that plays an important role in membrane trafficking, endoplasmic reticulum homeostasis, and the stress-signaling system. The connection between VAPA and prestin was confirmed through co-immunoprecipitation experiments. This new finding prompted the investigation of the interaction between VAPA and prestin in outer hair cells. By comparing VAPA expression between wild-type OHCs and OHCs derived from prestin-knockout mice, we found that VAPA is expressed in OHCs and the quantity of VAPA expressed is related to the presence of prestin. In other words, less VAPA protein is found in OHCs lacking prestin. Thus, prestin appears to modify the expression of VAPA protein in OHCs. Intriguingly, more prestin protein appears at the plasma membrane when VAPA is co-expressed with prestin. These data suggest that VAPA could be involved in prestin's transportation inside OHCs and may facilitate the targeting of this abundant OHC protein to the plasma membrane.
Project description:Prestin is a membrane protein in the outer hair cell (OHC) that has been shown to be essential for electromotility. OHCs from prestin-null mice do not express prestin, do not have a nonlinear capacitance (the electrical signature of electromotility), and are smaller in size than wild-type OHCs. We sought to determine whether prestin-null OHCs can be transduced to incorporate functional prestin protein in a normal fashion. A recombinant helper-dependent adenovirus expressing prestin and green fluorescent protein (HDAd-prestin-GFP) was created and tested in human embryonic kidney cells (HEK cells). Transduced HEK cells demonstrated membrane expression of prestin and nonlinear capacitance. HDAd-prestin-GFP was then applied to cochlear sensory epithelium explants harvested from wild-type and prestin-null mice at postnatal days 2-3, the age at which native prestin is just beginning to become functional in wild-type mice. At postnatal days 4-5, we investigated transduced OHCs for (1) their prestin expression pattern as revealed by immunofluorescence; (2) their cell surface area as measured by linear capacitance; and (3) their prestin function as indicated by nonlinear capacitance. HDAd-prestin-GFP efficiently transduced OHCs of both genotypes and prestin protein localized to the plasma membrane. Whole-cell voltage clamp studies revealed a nonlinear capacitance in transduced wild-type and prestin-null OHCs, but not in non-transduced cells of either genotype. Prestin transduction did not increase the linear capacitance (cell surface area) for either genotype. In peak nonlinear capacitance, voltage at peak nonlinear capacitance, charge density of the nonlinear capacitance, and shape of the voltage-capacitance curves, the transduced cells of the two genotypes resembled each other and previously reported data from adult wild-type mouse OHCs. Thus, prestin introduced into prestin-deficient OHCs segregates normally to the cell membrane and generates a normal nonlinear capacitance, indicative of normal prestin function.
Project description:Outer hair cells (OHC) act as amplifiers and their function is modified by medial olivocochlear (MOC) efferents. The unique OHC motor protein, prestin, provides the molecular basis for somatic electromotility, which is required for sensitivity and frequency selectivity, the hallmarks of mammalian hearing. Prestin proteins are the major component of the lateral membrane of mature OHCs, which separates apical and basal domains. To investigate the contribution of prestin to this unique arrangement, we compared the distribution of membrane proteins in OHCs of wildtype (WT) and prestin-knockout (KO) mice. In WT, the apical protein PMCA2 was exclusively localized to the hair bundles, while it was also found at the lateral membrane in KOs. Similarly, a basal protein KCNQ4 did not coalesce at the base of OHCs but was widely dispersed in mice lacking prestin. Since the expression levels of PMCA2 and KCNQ4 remained unchanged in KOs, the data indicate that prestin is required for the normal distribution of apical and basal membrane proteins in OHCs. Since OHC synapses predominate in the basal subnuclear region, we also examined the synaptic architecture in prestin-KO mice. Although neurite densities were not affected, MOC efferent terminals in prestin-KO mice were no longer constrained to the basal pole as in WT. This trend was evident as early as at postnatal day 12. Furthermore, terminals were often enlarged and frequently appeared as singlets when compared to the multiple clusters of individual terminals in WT. This abnormality in MOC synaptic morphology in prestin-KO mice is similar to defects in mice lacking MOC pathway proteins such as ?9/?10 nicotinic acetylcholine receptors and BK channels, indicating a role for prestin in the proper establishment of MOC synapses. To investigate the contribution of prestin's electromotility, we also examined OHCs from a mouse model that expresses non-functional prestin (499-prestin). We found no changes in PMCA2 localization and MOC synaptic morphology in OHCs from 499-prestin mice. Taken together, these results indicate that prestin, independent of its motile function, plays an important structural role in membrane compartmentalization, which is required for the formation of normal efferent-OHC synapses in mature OHCs.
Project description:Cholesterol affects diverse biological processes, in many cases by modulating the function of integral membrane proteins. We observed that alterations of cochlear cholesterol modulate hearing in mice. Mammalian hearing is powered by outer hair cell (OHC) electromotility, a membrane-based motor mechanism that resides in the OHC lateral wall. We show that membrane cholesterol decreases during maturation of OHCs. To study the effects of cholesterol on hearing at the molecular level, we altered cholesterol levels in the OHC wall, which contains the membrane protein prestin. We show a dynamic and reversible relationship between membrane cholesterol levels and voltage dependence of prestin-associated charge movement in both OHCs and prestin-transfected HEK 293 cells. Cholesterol levels also modulate the distribution of prestin within plasma membrane microdomains and affect prestin self-association in HEK 293 cells. These findings indicate that alterations in membrane cholesterol affect prestin function and functionally tune the outer hair cell.
Project description:The hair cells of the vertebrate inner ear posses active mechanical processes to amplify their inputs. The stereocilia bundle of various vertebrate animals can produce active movements. Though standard stereocilia-based mechanisms to promote amplification persist in mammals, an additional radically different mechanism evolved: the so-called somatic electromotility which refers to the elongation/contraction of the outer hair cells' (OHC) cylindrical cell body in response to membrane voltage changes. Somatic electromotility in OHCs, as the basis for cochlear amplification, is a mammalian novelty and it is largely dependent upon the properties of the unique motor protein prestin. We review recent literature which has demonstrated that although the gene encoding prestin is present in all vertebrate species, mammalian prestin has been under positive selective pressure to acquire motor properties, probably rendering it fit to serve somatic motility in outer hair cells. Moreover, we discuss data which indicates that a modified α10 nicotinic cholinergic receptor subunit has co-evolved in mammals, most likely to give the auditory feedback system the capability to control somatic electromotility.
Project description:Niemann-Pick Type C1 (NPC1) disease is a fatal neurovisceral disorder caused by dysfunction of NPC1 protein, which plays a role in intracellular cholesterol trafficking. The cholesterol-chelating agent, 2-hydroxypropyl-?-cyclodextrin (HP?CD), is currently undergoing clinical trials for treatment of this disease. Though promising in alleviating neurological symptoms, HP?CD causes irreversible hearing loss in NPC1 patients and outer hair cell (OHC) death in animal models. We recently found that HP?CD-induced OHC death can be significantly alleviated in a mouse model lacking prestin, an OHC-specific motor protein required for the high sensitivity and sharp frequency selectivity of mammalian hearing. Since cholesterol status is known to influence prestin's electromotility, we examined how prestin contributes to HP?CD-induced OHC death in the disease context using the NPC1 knockout (KO) mouse model (NPC1-KO). We found normal expression and localization of prestin in NPC1-KO OHCs. Whole-cell patch-clamp recordings revealed a significant depolarization of the voltage-operating point of prestin in NPC1-KO mice, suggesting reduced levels of cholesterol in the lateral membrane of OHCs that lack NPC1. OHC loss and elevated thresholds were found for high frequency regions in NPC1-KO mice, whose OHCs retained their sensitivity to HP?CD. To investigate whether prestin's electromotile function contributes to HP?CD-induced OHC death, the prestin inhibitor salicylate was co-administered with HP?CD to WT and NPC1-KO mice. Neither oral nor intraperitoneal administration of salicylate mitigated HP?CD-induced OHC loss. To further determine the contribution of prestin's electromotile function, a mouse model expressing a virtually nonelectromotile prestin protein (499-prestin) was subjected to HP?CD treatment. 499-prestin knockin mice showed no resistance to HP?CD-induced OHC loss. As 499-prestin maintains its ability to bind cholesterol, our data imply that HP?CD-induced OHC death is ascribed to the structural role of prestin in maintaining the OHC's lateral membrane, rather than its motor function.
Project description:The motor protein prestin is a member of the SLC26 family of anion antiporters and is essential to the electromotility of cochlear outer hair cells and for hearing. The only direct inhibitor of electromotility and the associated charge transfer is salicylate, possibly through direct interaction with an anion-binding site on prestin. In a screen to identify other inhibitors of prestin activity, we explored the effect of the non-steroid anti-inflammatory drug diflunisal, which is a derivative of salicylate. We recorded prestin activity by whole-cell patch clamping HEK cells transiently expressing prestin and mouse outer hair cells. We monitored the impact of diflunisal on the prestin-dependent non-linear capacitance and electromotility. We found that diflunisal triggers two prestin-associated effects: a chloride independent increase in the surface area and the specific capacitance of the membrane, and a chloride dependent inhibition of the charge transfer and the electromotility in outer hair cells. We conclude that diflunisal affects the cell membrane organization and inhibits prestin-associated charge transfer and electromotility at physiological chloride concentrations. The inhibitory effects on hair cell function are noteworthy given the proposed use of diflunisal to treat neurodegenerative diseases.
Project description:The voltage-dependent movement, or electromotility, of cochlear outer hair cells contributes to cochlear amplification in mammalian hearing. Outer hair-cell electromotility involves a membrane-based motor in which the membrane protein prestin plays a central role. We have investigated the contribution of prestin to the mechanics and electromechanical force (EMF) generation of the membrane using membrane tethers formed from human embryonic kidney (HEK) cells. Several measures of membrane tether mechanics are greater in tethers pulled from HEK cells transfected with prestin when compared to control untransfected HEK cells. A single point mutation of alanine to tryptophan (A100W) in prestin eliminates prestin-associated charge movement and diminishes EMF but does not alter passive membrane mechanics. These results suggest that prestin-associated charge transfer is necessary for maximal EMF generation by the membrane.