Biology subjects

Navaratnam, D. S.

Publications and source records attributed to Navaratnam, D. S..

9 recordsLinked to original sources

bioRxiv2026

SCN- induces prestin contraction through reduced intermonomer angles and a modified transporter like movement of each monomer.

Prestin is the membrane based molecular motor responsible for outer hair cell electromotility that expands and contracts in response to transmembrane voltage. How voltage brings these about is unknown but cryoEM structures in the presence of ligands has led to the extrapolation of structures with the transporter cycle and its expansion contraction coupling. We find prestin to adopt novel increasingly contracted states that are brought about by a reduced intermonomer angles in the obligate dimers and a transmembrane contraction of each monomer at its cytoplasmic half in the presence of SCN-. These changes are associated with a loss in the intermonomer beta sheet at its N terminus. There is a hyperpolarizing shift in Vh along with increased affinity for SCN-. SCN- binding at the internal binding site likely cause the structural changes since they are reversed by SCN- displacement by salicylate.

physiology↗

bioRxiv2026

Conformational changes underlying electromechanical transduction in prestin resemble a transport transition in pendrin

Prestin (SLC26A5), a membrane protein in cochlear outer hair cells, drives electromechanical transduction essential for mammalian hearing. Unlike other SLC26 anion transporters, prestin functions as a voltage-dependent molecular motor, transitioning between contracted and expanded conformations. How this transition relates to the transporter cycle of SLC26 family members remains unclear. Here, multi-microsecond molecular dynamics (MD) simulations starting from a contracted conformation reveal a rapid, spontaneous transition to an expanded state that resembles the inward-facing conformation of the anion exchanger pendrin (SLC26A4 from mouse). An accompanying transmembrane area expansion is localized to the inner membrane leaflet. In line with this observation, reduced unitary sensor charge movement accompanies neutralization of charged residues localized near the inner leaflet. Simulations uncover a previously uncharacterized contracted conformation that resembles outward-facing pendrin; the MD also predicts an extracellular anion-binding site in prestin. A 3.27-[A] resolution cryo-electron microscopy structure of prestin in the presence of thiocyanate anions confirms this site, which appears to be common in the SLC26 family, notably also in pendrin. Based on the simulations, the extracellular site in pendrin is involved in facilitating anion access to the canonical intracellular site in a conformation-dependent manner while in prestin, the intracellular site is not accessible from the extracellular side. Furthermore, like prestin, pendrin exhibits a non-linear capacitance, an indication of voltage-dependent conformational switching. Together, these findings suggest that prestin and pendrin share core structural and functional properties, notably parallels between expansion-contraction states and anion binding sites, though transition speeds and conformation-dependent ion-accessibility may differ.

biophysics↗

bioRxiv2025

Electromotility can be disassociated from gating charge movement in outer hair cells of conditional alpha2 spectrin knockout mice.

Electromotility in mammalian outer hair cells (OHC) is the mechanism underlying cochlear amplification. It is brought about by the piezoelectric-like property of the membrane protein prestin (Slc26a5) that lies in the OHCs lateral plasma membrane. Prestin connects to an underlying cytoskeletal network of circumferential actin filaments that bridge longitudinal spectrin filaments. This network, in turn, lies between the plasma membrane and a closely apposed ER-like tubular array of subsurface cisternae (SSC). Two previous papers examining spectrin knockouts in embryonic hair cells were confined to analyzing the effects on the apical cuticular plate and overlying stereocilia. In this paper, we examine the effects of conditional knockouts of alpha2 spectrin in postnatal OHCs. We find a significant auditory phenotype likely due to the novel disassociation of prestins gating charge movement from OHC electromotility. In addition, OHCs show enlargement in their SSC and plasma membrane-SSC space with preserved cuticular plates and overlying stereocilia, which contrasts with the findings in embryonic knockouts.

physiology↗

bioRxiv2024

Chloride binding and cholesterol effects on high frequency complex nonlinear capacitance (cNLC) in the mouse outer hair cell: experiment and molecular dynamics

Prestin (SLC26a5) function evolved to enhance auditory sensitivity and frequency selectivity by providing mechanical feedback via outer hair cells (OHC) into the organ of Corti. Its effectiveness is governed by the voltage-dependent kinetics of the proteins charge movements, namely, nonlinear capacitance (NLC). We study the frequency response of NLC in the mouse OHC, a species with ultrasonic hearing. We find that the characteristic frequency cut-off (Fis) for the mouse in near 27 kHz. Single point mutations within the chloride binding pocket of prestin (e.g., S396E, S398E) lack the proteins usual anion susceptibility. In agreement, we now show absence of anion binding in these mutants through molecular dynamics (MD) simulations. NLC Fis in the S396E knock-in mouse is unaltered, indicating that high frequency activity is not governed by chloride, but more likely by viscoelastic loads within the membrane. We also show that the allosteric action of chloride does not underlie piezoelectric-like behavior in prestin, since tension sensitivity of S396E NLC is comparable to that of WT. Because prestin structures of all species studied to-date are essentially indistinguishable, with analogous chloride binding pockets, auditory requirements of individual species for cochlear amplification likely evolved to enhance prestin performance by modifying, not its protein-anion interaction, but instead external mechanical loads on the protein. SignificancePrestin is believed to provide cochlear amplification in mammals that possess a wide range of frequency sensitivities. Previously, chloride anions have been shown to control prestin kinetics at frequencies below 10 kHz. However, now we find that chloride binding is not influential for prestin kinetics in the very high range of the mouse. We suggest that such high frequency prestin performance is governed by impinging mechanical loads within the membrane, and not interactions with anions.

biophysics↗

bioRxiv2022

Ultrasonic measures of prestin (SLC26a5) charge movements in membrane patches.

Charged moieties in the outer hair cell (OHC) molecular motor protein, prestin, are driven by transmembrane voltage to ultimately provide for cochlear amplification. The speed of voltage-dependent conformational switching underlies its ability to influence micromechanics of the cell and the organ of Corti. Corresponding voltage-sensor charge movements in prestin, classically assessed as a voltage-dependent, nonlinear membrane capacitance (NLC), have been used to gauge its frequency response. Using megahertz sampling of prestin charge movements, we extend interrogations of prestin performance into the ultrasonic range (up to 120 kHz) and find response magnitude larger than previously reported. We also confirm kinetic model predictions of prestin by directly observing its cut-off frequency under voltage-clamp as the intersection frequency (Fis) of the real and imaginary components of complex NLC (cNLC), showing values near 19 kHz. At higher frequencies, the imaginary component roll-off exactly tracks that of Abs(cNLC). The frequency response of prestin displacement current noise determined from the Nyquist relation aligns with this cut-off. On the other hand, previous measures of stationary thermal-driven noise of prestin indicated that the cut-off was several fold greater than that of NLC, in violation of the fluctuation-dissipation theorem. We have attempted to confirm this apparent paradox, but find that low frequency (<10kHz), voltage-dependent 1/f noise, likely due to intrinsic prestin conductance, can limit the accessible bandwidth for stationary noise analysis. Nevertheless, within those bandwidths, frequency response comparisons of stationary measures and Nyquist relation measures are consistent. We conclude that voltage stimulation accurately assesses the spectral limits of prestin activity. SignificanceUsing megahertz sampling, we extend measures of prestin charge movement into the ultrasonic range and find that the frequency roll-off is less than previously reported. Nevertheless, analysis of complex nonlinear capacitance confirms low-pass behavior, with a characteristic cut-off frequency near 19 kHz. The frequency response of prestin noise garnered by the admittance-based Nyquist relation confirms this cut-off frequency. In conflict with previous results, however, we find a similar low-pass frequency response using direct measures of prestin noise in the absence of voltage stimulation. Our data indicate that voltage perturbation provides an accurate assessment of prestin performance.

biophysics↗

bioRxiv2021

Single particle cryo-EM structure of the outer hair cell motor protein prestin

The mammalian outer hair cell (OHC) protein prestin (Slc26a5), a member of the solute carrier 26 (Slc26) family of membrane proteins, differs from other members of the family owing to its unique piezoelectric-like property that drives OHC electromotility. OHCs require prestin for cochlear amplification, a process that enhances mammalian hearing. Despite substantial biophysical characterization, the mechanistic basis for the prestins electro-mechanical behavior is not fully understood. To gain insight into such behavior, we have used cryo-electron microscopy at subnanometer resolution (overall resolution of 4.0 [A]) to investigate the three-dimensional structure of prestin from gerbil (Meriones unguiculatus). Our studies show that prestin dimerizes with a 3D architecture strikingly similar to the dimeric conformation observed in the Slc26a9 anion transporter in an inside open/intermediate state, which we infer, based on patch-clamp recordings, to reflect the contracted state of prestin. The structure shows two well-separated transmembrane (TM) subunits and two cytoplasmic sulfate transporter and anti-sigma factor antagonist (STAS) domains forming a swapped dimer. The dimerization interface is defined by interactions between the domain-swapped STAS dimer and the transmembrane domains of the opposing half unit, further strengthened by an antiparallel beta-strand at its N terminus. The structure also shows that each one of its two transmembrane subunits consists of 14 transmembrane segments organized in two inverted 7-segment repeats with a topology that was first observed in the structure of bacterial symporter UraA (Lu F, et al., Nature 472, 2011). Finally, the solved anion binding site structural features of prestin are quite similar to that of SLC26a9 and other family members. Despite this similarity, we find that SLC26a9 lacks the characteristic displacement currents (or NonLinear Capacitance(NLC)) found with prestin, and we show that mutation of prestins Cl- binding site removes salicylate competition with anions in the face of normal NLC, thus refuting the yet accepted extrinsic voltage sensor hypothesis and any associated transport-like requirements for voltage-driven electromotility.

neuroscience↗

bioRxiv2021

Outer Hair Cell Function is Normal in βV Spectrin Knockout Mice

Reports have proposed a putative role for {beta}V spectrin in outer hair cells (OHCs) of the cochlea. In an ongoing investigation of the role of the cytoskeleton in electromotility, we tested mice with a targeted exon deletion of {beta}V spectrin (Spnb5), and unexpectedly find that Spnb5(-/-) animals auditory thresholds are unaffected. Similarly, these mice have normal OHC electromechanical activity (otoacoustic emissions) and non-linear capacitance. In contrast, magnitudes of auditory brainstem response (ABR) wave 1-amplitudes are significantly reduced. Evidence of a synaptopathy was absent with normal hair cell CtBP2 counts. In Spnb5(-/-) mice, the number of afferent and efferent nerve fibers is decreased. Consistent with this data, Spnb5 mRNA is present in Type I and II spiral ganglion neurons, but undetectable in OHCs. Together, these data establish that {beta}V spectrin is important for hearing, affecting neuronal structure and function. Significantly, these data exclude {beta}V spectrin as functionally important to OHCs as has been previously suggested.

neuroscience↗

bioRxiv2020

Efferent feedback enforces bilateral coupling of spontaneous activity in the developing auditory system

In the developing auditory system, spontaneous activity generated in the cochleae propagates into the central nervous system to promote circuit formation before hearing onset. Effects of the evolving peripheral firing pattern on spontaneous activity in the central auditory system are not well understood. Here, we describe the wide-spread bilateral coupling of spontaneous activity that coincides with the period of transient efferent modulation of inner hair cells from the medial olivochlear (MOC) system. Knocking out the 9/10 nicotinic acetylcholine receptor, a requisite part of the efferent cholinergic pathway, abolishes these bilateral correlations. Pharmacological and chemogenetic experiments confirm that the MOC system is necessary and sufficient to produce the bilateral coupling. Moreover, auditory sensitivity at hearing onset is reduced in the absence of pre-hearing efferent modulation. Together, our results demonstrate how ascending and descending pathways collectively shape spontaneous activity patterns in the auditory system and reveal the essential role of the MOC efferent system in linking otherwise independent streams of bilateral spontaneous activity during the prehearing period.

neuroscience↗

bioRxiv2019

Calcium induced calcium release in proximity to hair cell BK channels revealed by PKA activation

Large conductance calcium-activated potassium (BK) channels play a critical role in electrical resonance, a mechanism of frequency selectivity in chicken hair cells. We determine that BK currents are dependent on inward flow of Ca2+, and intracellular buffering of Ca2+. Entry of Ca2+ is further amplified locally by Ca2+ induced Ca2+ release (CICR) in close proximity to plasma membrane BK channels. Ca2+ imaging reveals peripheral clusters of high concentrations of Ca2+ that are suprathreshold to that needed to activate BK channels. PKA activation increases BK currents likely by recruiting more BK channels due to spatial spread of high Ca2+ concentrations in turn from increasing CICR. STORM imaging confirms the presence of nanodomains with ryanodine and IP3 receptors in close proximity to the Slo subunit of BK channels. Together, these data require a rethinking of how electrical resonance is brought about and suggest effects of CICR in synaptic release. Both genders were included in this study.

physiology↗