Kv3.3 Channels Bind Hax-1 and Arp2/3 to Assemble a Stable Local Actin Network that Regulates Channel Gating.

Mutations in the Kv3.3 potassium channel (KCNC3) cause cerebellar neurodegeneration and impair auditory processing. The cytoplasmic C terminus of Kv3.3 contains a proline-rich domain conserved in proteins that activate actin nucleation through Arp2/3. We found that Kv3.3 recruits Arp2/3 to the plasma membrane, resulting in formation of a relatively stable cortical actin filament network resistant to cytochalasin D that inhibits fast barbed end actin assembly. These Kv3.3-associated actin structures are required to prevent very rapid N-type channel inactivation during short depolarizations of the plasma membrane. The effects of Kv3.3 on the actin cytoskeleton are mediated by the binding of the cytoplasmic C terminus of Kv3.3 to Hax-1, an anti-apoptotic protein that regulates actin nucleation through Arp2/3. A human Kv3.3 mutation within a conserved proline-rich domain produces channels that bind Hax-1 but are impaired in recruiting Arp2/3 to the plasma membrane, resulting in growth cones with deficient actin veils in stem cell-derived neurons.

Megahertz Sampling of Prestin (SLC26a5) Voltage-Sensor Charge Movements in Outer Hair Cell Membranes Reveals Ultrasonic Activity that May Support Electromotility and Cochlear Amplification.

Charged moieties in the outer hair cell (OHC) membrane motor protein, prestin, are driven by transmembrane voltage to power OHC electromotility (eM) and cochlear amplification (CA), an enhancement of mammalian hearing. Consequently, the speed of prestin's conformational switching constrains its dynamic influence on 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, but have been validly measured only out to 30 kHz. Thus, controversy exists concerning the effectiveness of eM in supporting CA at ultrasonic frequencies where some mammals can hear. Using megahertz sampling of guinea pig (either sex) prestin charge movements, we extend interrogations of NLC into the ultrasonic range (up to 120 kHz) and find an order of magnitude larger response at 80 kHz than previously predicted, indicating that an influence of eM at ultrasonic frequencies is likely, in line with recent in vivo results (Levic et al., 2022). Given wider bandwidth interrogations, we also validate kinetic model predictions of prestin by directly observing its characteristic cut-off frequency under voltage-clamp as the intersection frequency (Fis), near 19 kHz, of the real and imaginary components of complex NLC (cNLC). The frequency response of prestin displacement current noise determined from either the Nyquist relation or stationary measures aligns with this cut-off. We conclude that voltage stimulation accurately assesses the spectral limits of prestin activity, and that voltage-dependent conformational switching is physiologically significant in the ultrasonic range.SIGNIFICANCE STATEMENT The motor protein prestin powers outer hair cell (OHC) electromotility (eM) and cochlear amplification (CA), an enhancement of high-frequency mammalian hearing. The ability of prestin to work at very high frequencies depends on its membrane voltage-driven conformation switching. Using megahertz sampling, we extend measures of prestin charge movement into the ultrasonic range and find response magnitude at 80 kHz an order of magnitude larger than previously estimated, despite confirmation of previous low pass characteristic frequency cut-offs. The frequency response of prestin noise garnered by the admittance-based Nyquist relation or stationary noise measures confirms this characteristic cut-off frequency. Our data indicate that voltage perturbation provides accurate assessment of prestin performance indicating that it can support cochlear amplification into a higher frequency range than previously thought.

Cost-Effectiveness of CT, CTA, MRI, and Specialized MRI for Evaluation of Patients Presenting to the Emergency Department With Dizziness.

BACKGROUND. Underlying stroke is often misdiagnosed in patients presenting with dizziness. Although such patients are usually ineligible for acute stroke treatment, accurate diagnosis may still improve outcomes through selection of patients for secondary prevention measures. OBJECTIVE. The purpose of our study was to investigate the cost-effectiveness of differing neuroimaging approaches in the evaluation of patients presenting to the emergency department (ED) with dizziness who are not candidates for acute intervention. METHODS. A Markov decision-analytic model was constructed from a health care system perspective for the evaluation of a patient presenting to the ED with dizziness. Four diagnostic strategies were compared: noncontrast head CT, head and neck CTA, conventional brain MRI, and specialized brain MRI (including multiplanar high-resolution DWI). Differing long-term costs and outcomes related to stroke detection and secondary prevention measures were compared. Cost-effectiveness was calculated in terms of lifetime expenditures in 2022 U.S. dollars for each quality-adjusted life year (QALY); deterministic and probabilistic sensitivity analyses were performed. RESULTS. Specialized MRI resulted in the highest QALYs and was the most cost-effective strategy with US$13,477 greater cost and 0.48 greater QALYs compared with noncontrast head CT. Conventional MRI had the next-highest health benefit, although was dominated by extension with incremental cost of US$6757 and 0.25 QALY; CTA was also dominated by extension, with incremental cost of US$3952 for 0.13 QALY. Non-contrast CT alone had the lowest utility among the four imaging choices. In the deterministic sensitivity analyses, specialized MRI remained the most cost-effective strategy. Conventional MRI was more cost-effective than CTA across a wide range of model parameters, with incremental cost-effectiveness remaining less than US$30,000/QALY. Probabilistic sensitivity analysis yielded similar results as found in the base-case analysis, with specialized MRI being more cost-effective than conventional MRI, which in turn was more cost-effective than CTA. CONCLUSION. The use of MRI in patients presenting to the ED with dizziness improves stroke detection and selection for subsequent preventive measures. MRI-based evaluation leads to lower long-term costs and higher cumulative QALYs. CLINICAL IMPACT. MRI, incorporating specialized protocols when available, is the preferred approach for evaluation of patients presenting to the ED with dizziness, to establish a stroke diagnosis and to select patients for secondary prevention measures.

CT With CTA Versus MRI in Patients Presenting to the Emergency Department With Dizziness: Analysis Using Propensity Score Matching.

BACKGROUND. CT with CTA is widely used to exclude stroke in patients with dizziness, although MRI has higher sensitivity. OBJECTIVE. The purpose of this article was to compare patients presenting to the emergency department (ED) with dizziness who undergo CT with CTA alone versus those who undergo MRI in terms of stroke-related management and outcomes. METHODS. This retrospective study included 1917 patients (mean age, 59.5 years; 776 men, 1141 women) presenting to the ED with dizziness from January 1, 2018, to December 31, 2021. A first propensity score matching analysis incorporated demographic characteristics, medical history, findings from the review of systems, physical examination findings, and symptoms to construct matched groups of patients discharged from the ED after undergoing head CT with head and neck CTA alone and patients who underwent brain MRI (with or without CT and CTA). Outcomes were compared. A second analysis compared matched patients discharged after CT with CTA alone and patients who underwent specialized abbreviated MRI using multiplanar high-resolution DWI for increased sensitivity for posterior circulation stroke. Sensitivity analyses were performed involving MRI examinations performed as the first or only neuroimaging examination and involving alternative matching and imputation techniques. RESULTS. In the first analysis (406 patients per group), patients who underwent MRI, compared with patients who underwent CT with CTA alone, showed greater frequency of critical neuroimaging results (10.1% vs 4.7%, p = .005), change in secondary stroke prevention medication (9.6% vs 3.2%, p = .001), and subsequent echocardiography evaluation (6.4% vs 1.0%, p < .001). In the second analysis (100 patients per group), patients who underwent specialized abbreviated MRI, compared with patients who underwent CT with CTA alone, showed greater frequency of critical neuroimaging results (10.0% vs 2.0%, p = .04), change in secondary stroke prevention medication (14.0% vs 1.0%, p = .001), and subsequent echocardiography evaluation (12.0% vs 2.0%, p = .01) and lower frequency of 90-day ED readmissions (12.0% vs 28.0%, p = .008). Sensitivity analyses showed qualitatively similar findings. CONCLUSION. A proportion of patients discharged after CT with CTA alone may have benefitted from alternative or additional evaluation by MRI (including MRI using a specialized abbreviated protocol). CLINICAL IMPACT. Use of MRI may motivate clinically impactful management changes in patients presenting with dizziness.

Ischemic Stroke, Inflammation, and Endotheliopathy in COVID-19 Patients.

[Figure: see text].

Stroke Code Presentations, Interventions, and Outcomes Before and During the COVID-19 Pandemic.

BACKGROUND: Anecdotal reports suggest fewer patients with stroke symptoms are presenting to hospitals during the coronavirus disease 2019 (COVID-19) pandemic. We quantify trends in stroke code calls and treatments at 3 Connecticut hospitals during the local emergence of COVID-19 and examine patient characteristics and stroke process measures at a Comprehensive Stroke Center (CSC) before and during the pandemic. METHODS: Stroke code activity was analyzed from January 1 to April 28, 2020, and corresponding dates in 2019. Piecewise linear regression and spline models identified when stroke codes in 2020 began to decline and when they fell below 2019 levels. Patient-level data were analyzed in February versus March and April 2020 at the CSC to identify differences in patient characteristics during the pandemic. RESULTS: A total of 822 stroke codes were activated at 3 hospitals from January 1 to April 28, 2020. The number of stroke codes/wk decreased by 12.8/wk from February 18 to March 16 (P=0.0360) with nadir of 39.6% of expected stroke codes called from March 10 to 16 (30% decrease in total stroke codes during the pandemic weeks in 2020 versus 2019). There was no commensurate increase in within-network telestroke utilization. Compared with before the pandemic (n=167), pandemic-epoch stroke code patients at the CSC (n=211) were more likely to have histories of hypertension, dyslipidemia, coronary artery disease, and substance abuse; no or public health insurance; lower median household income; and to live in the CSC city (P<0.05). There was no difference in age, sex, race/ethnicity, stroke severity, time to presentation, door-to-needle/door-to-reperfusion times, or discharge modified Rankin Scale. CONCLUSIONS: Hospital presentation for stroke-like symptoms decreased during the COVID-19 pandemic, without differences in stroke severity or early outcomes. Individuals living outside of the CSC city were less likely to present for stroke codes at the CSC during the pandemic. Public health initiatives to increase awareness of presenting for non-COVID-19 medical emergencies such as stroke during the pandemic are critical.

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

The function of prestin (SLC26a5), an anion transport family member, has evolved to enhance auditory sensitivity and frequency selectivity by providing mechanical feedback via outer hair cells (OHC) into the organ of Corti. The frequency extent of this boost is governed by the voltage-dependent kinetics of the protein's charge movements, otherwise known as nonlinear capacitance (NLC) that we measure in membrane patches under voltage clamp. Here we extend our previous studies on guinea pig OHCs by studying the frequency response of NLC in the mouse OHC, a species with higher frequency auditory needs. We find that the characteristic frequency cut-off (F is ) for the mouse surpasses that of the guinea pig, being 27 kHz vs. 19 kHz, respectively; nevertheless, each shows significant activity in the ultrasonic range. We also evaluate the influence of anion binding on prestin frequency response. Several single point mutations within the chloride binding pocket of prestin (e.g., S396E, S398E) lack anion influence. In agreement, we show absence of anion binding through molecular dynamics (MD) simulations. NLC F is in the S396E knock-in mouse remains the same as controls, indicating that high frequency activity is likely governed by viscoelastic loads within the membrane characterized by stretched-exponential frequency roll-off. Accordingly, treatment with MßCD, which removes membrane cholesterol, possibly from prestin itself, and can alter membrane fluidity, augments NLC F is out to 39 kHz. Although interactions between membrane lipid and prestin have been suggested from structural studies to arise at their interfacial boundaries within the membrane, our MD simulations suggest that phospholipids can insert within transmembrane domains of prestin during voltage perturbation. Such novel lipid-protein interactions could account for our observed changes in the phase of prestin's voltage-sensor charge movements across frequency. We hypothesize that because prestin tertiary structures of all species studied to-date are indistinguishable, it is likely that any special auditory requirements of individual species for cochlear amplification have evolved to capitalize on prestin performance by modifying, not the protein itself, but the external loads on the protein, including those within the membrane and organ of Corti. Significance: Prestin is believed to provide cochlear amplification in mammals that possess a wide range of frequency sensitivities, yet its tertiary structure is indistinguishable among those species studied. We find that prestin kinetics is faster in mice than in guinea pigs, mice showing higher frequency auditory capabilities. Chloride binding is not influential, but membrane lipids/viscosity is. We suggest that the evolution of prestin's species performance involves modifications of impinging loads, not the protein itself.

Single-cell transcriptomic atlas reveals increased regeneration in diseased human inner ear balance organs.

Mammalian inner ear hair cell loss leads to permanent hearing and balance dysfunction. In contrast to the cochlea, vestibular hair cells of the murine utricle have some regenerative capacity. Whether human utricular hair cells regenerate in vivo remains unknown. Here we procured live, mature utricles from organ donors and vestibular schwannoma patients, and present a validated single-cell transcriptomic atlas at unprecedented resolution. We describe markers of 13 sensory and non-sensory cell types, with partial overlap and correlation between transcriptomes of human and mouse hair cells and supporting cells. We further uncover transcriptomes unique to hair cell precursors, which are unexpectedly 14-fold more abundant in vestibular schwannoma utricles, demonstrating the existence of ongoing regeneration in humans. Lastly, supporting cell-to-hair cell trajectory analysis revealed 5 distinct patterns of dynamic gene expression and associated pathways, including Wnt and IGF-1 signaling. Our dataset constitutes a foundational resource, accessible via a web-based interface, serving to advance knowledge of the normal and diseased human inner ear.

Clinical criteria to exclude acute vascular pathology on CT angiogram in patients with dizziness.

BACKGROUND: Patients presenting to the emergency department (ED) with dizziness may be imaged via CTA head and neck to detect acute vascular pathology including large vessel occlusion. We identify commonly documented clinical variables which could delineate dizzy patients with near zero risk of acute vascular abnormality on CTA. METHODS: We performed a cross-sectional analysis of adult ED encounters with chief complaint of dizziness and CTA head and neck imaging at three EDs between 1/1/2014-12/31/2017. A decision rule was derived to exclude acute vascular pathology tested on a separate validation cohort; sensitivity analysis was performed using dizzy "stroke code" presentations. RESULTS: Testing, validation, and sensitivity analysis cohorts were composed of 1072, 357, and 81 cases with 41, 6, and 12 instances of acute vascular pathology respectively. The decision rule had the following features: no past medical history of stroke, arterial dissection, or transient ischemic attack (including unexplained aphasia, incoordination, or ataxia); no history of coronary artery disease, diabetes, migraines, current/long-term smoker, and current/long-term anti-coagulation or anti-platelet medication use. In the derivation phase, the rule had a sensitivity of 100% (95% CI: 0.91-1.00), specificity of 59% (95% CI: 0.56-0.62), and negative predictive value of 100% (95% CI: 0.99-1.00). In the validation phase, the rule had a sensitivity of 100% (95% CI: 0.61-1.00), specificity of 53% (95% CI: 0.48-0.58), and negative predictive value of 100% (95% CI: 0.98-1.00). The rule performed similarly on dizzy stroke codes and was more sensitive/predictive than all NIHSS cut-offs. CTAs for dizziness might be avoidable in 52% (95% CI: 0.47-0.57) of cases. CONCLUSIONS: A collection of clinical factors may be able to "exclude" acute vascular pathology in up to half of patients imaged by CTA for dizziness. These findings require further development and prospective validation, though could improve the evaluation of dizzy patients in the ED.

Hair cell BK channels interact with RACK1, and PKC increases its expression on the cell surface by indirect phosphorylation.

Large conductance (BK) calcium activated potassium channels (Slo) are ubiquitous and implicated in a number of human diseases including hypertension and epilepsy. BK channels consist of a pore forming α-subunit (Slo) and a number of accessory subunits. In hair cells of nonmammalian vertebrates these channels play a critical role in electrical resonance, a mechanism of frequency selectivity. Hair cell BK channel clusters on the surface and currents increase along the tonotopic axis and contribute significantly to the responsiveness of these hair cells to sounds of high frequency. In contrast, messenger RNA levels encoding the Slo gene show an opposite decrease in high frequency hair cells. To understand the molecular events underlying this paradox, we used a yeast two-hybrid screen to isolate binding partners of Slo. We identified Rack1 as a Slo binding partner and demonstrate that PKC activation increases Slo surface expression. We also establish that increased Slo recycling of endocytosed Slo is at least partially responsible for the increased surface expression of Slo. Moreover, analysis of several PKC phosphorylation site mutants confirms that the effects of PKC on Slo surface expression are likely indirect. Finally, we show that Slo clusters on the surface of hair cells are also increased by increased PKC activity and may contribute to the increasing amounts of channel clusters on the surface of high-frequency hair cells.

CDK5 interacts with Slo and affects its surface expression and kinetics through direct phosphorylation.

Large-conductance calcium-activated potassium (BK) channels are ubiquitous and play an important role in a number of diseases. In hair cells of the ear, they play a critical role in electrical tuning, a mechanism of frequency discrimination. These channels show variable kinetics and expression along the tonotopic axis. Although the molecular underpinnings to its function in hair cells are poorly understood, it is established that BK channels consist of a pore-forming α-subunit (Slo) and a number of accessory subunits. Here we identify CDK5, a member of the cyclin-dependent kinase family, as an interacting partner of Slo. We show CDK5 to be present in hair cells and expressed in high concentrations in the cuticular plate and in the circumferential zone. In human embryonic kidney cells, we show that CDK5 inhibits surface expression of Slo by direct phosphorylation of Slo. Similarly, we note that CDK5 affects Slo voltage activation and deactivation kinetics, by a direct phosphorylation of T847. Taken together with its increasing expression along the tonotopic axis, these data suggest that CDK5 likely plays a critical role in electrical tuning and surface expression of Slo in hair cells.

Extracellular chloride regulation of Kv2.1, contributor to the major outward Kv current in mammalian outer hair cells.

Outer hair cells (OHC) function as both receptors and effectors in providing a boost to auditory reception. Amplification is driven by the motor protein prestin, which is under anionic control. Interestingly, we now find that the major, 4-AP-sensitive, outward K(+) current of the OHC (I(K)) is also sensitive to Cl(-), although, in contrast to prestin, extracellularly. I(K) is inhibited by reducing extracellular Cl(-) levels, with a linear dependence of 0.4%/mM. Other voltage-dependent K(+) (Kv) channel conductances in supporting cells, such as Hensen and Deiters' cells, are not affected by reduced extracellular Cl(-). To elucidate the molecular basis of this Cl(-)-sensitive I(K), we looked at potential molecular candidates based on Cl(-) sensitivity and/or similarities in kinetics. For I(K), we identified three different Ca(2+)-independent components of I(K) based on the time constant of inactivation: a fast, transient outward current, a rapidly activating, slowly inactivating current (Ik(1)), and a slowly inactivating current (Ik(2)). Extracellular Cl(-) differentially affects these components. Because the inactivation time constants of Ik(1) and Ik(2) are similar to those of Kv1.5 and Kv2.1, we transiently transfected these constructs into CHO cells and found that low extracellular Cl(-) inhibited both channels with linear current reductions of 0.38%/mM and 0.49%/mM, respectively. We also tested heterologously expressed Slick and Slack conductances, two intracellularly Cl(-)-sensitive K(+) channels, but found no extracellular Cl(-) sensitivity. The Cl(-) sensitivity of Kv2.1 and its robust expression within OHCs verified by single-cell RT-PCR indicate that these channels underlie the OHC's extracellular Cl(-) sensitivity.

Physiology and biophysics of outer hair cells: The cells of Dallos.

The outer hair cell (OHC) is celebrated on the 21st birthday of prestin's identification and the year of this molecular motor's sub-nanometer structural solution. Dogmatic conceptions of OHC performance have been challenged by decades of biophysical interrogations that must be influential on hearing, but which have received little attention by cochlear modelers. Here we point to these interrogations and present a compilation of articles in a Special Issue of Hearing Research that reconsiders the OHC's role in cochlear amplification, as well as the cell's basic physiology. We are getting closer to understanding these special cells of Dallos.

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

The mammalian outer hair cell (OHC) protein prestin (Slc26a5) differs from other Slc26 family members due to its unique piezoelectric-like property that drives OHC electromotility, the putative mechanism for cochlear amplification. Here, we use cryo-electron microscopy to determine prestin's structure at 3.6 Å resolution. Prestin is structurally similar to the anion transporter Slc26a9. It is captured in an inward-open state which may reflect prestin's contracted state. Two well-separated transmembrane (TM) domains and two cytoplasmic sulfate transporter and anti-sigma factor antagonist (STAS) domains form a swapped dimer. The transmembrane domains consist of 14 transmembrane segments organized in two 7+7 inverted repeats, an architecture first observed in the bacterial symporter UraA. Mutation of prestin's chloride binding site removes salicylate competition with anions while retaining the prestin characteristic displacement currents (Nonlinear Capacitance), undermining the extrinsic voltage sensor hypothesis for prestin function.

Pilot MRI-based strategies to improve the detection of stroke in patients with dizziness/vertigo.

Posterior strokes are frequently misdiagnosed as they present with non-specific complaints such as dizziness/vertigo. Emergency department (ED) practice often relies on CT/CTA to "exclude" infarct in such patients, providing false reassurance due to lower sensitivity of CT (42%) for stroke in the posterior circulation. We describe a pilot at our institution using a specialized MRI protocol with 95% sensitivity for posterior stroke, which may be used in place of CT/CTA or conventional MRI for stroke evaluation. Further development of this approach may help reduce the high rate of missed posterior stroke in patients presenting with dizziness.

Outer hair cell function is normal in ßV spectrin knockout mice.

Reports have proposed a putative role for ßV 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 ßV 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 ßV spectrin is important for hearing, affecting neuronal structure and function. Significantly, these data support that ßV spectrin as is not functionally important to OHCs as has been previously suggested.

ß4-subunit increases Slo responsiveness to physiological Ca2+ concentrations and together with ß1 reduces surface expression of Slo in hair cells.

Changing kinetics of large-conductance potassium (BK) channels in hair cells of nonmammalian vertebrates, including the chick, plays a critical role in electrical tuning, a mechanism used by these cells to discriminate between different frequencies of sound. BK currents are less abundant in low-frequency hair cells and show large openings in response to a rise in intracellular Ca(2+) at a hair cell's operating voltage range (spanning -40 to -60 mV). Although the molecular underpinnings of its function in hair cells are poorly understood, it is established that BK channels consist of a pore-forming α-subunit (Slo) and a number of accessory subunits. Currents from the α (Slo)-subunit alone do not show dramatic increases in response to changes in Ca(2+) concentrations at -50 mV. We have cloned the chick ß(4)- and ß(1)-subunits and show that these subunits are preferentially expressed in low-frequency hair cells, where they decrease Slo surface expression. The ß(4)-subunit in particular is responsible for the BK channel's increased responsiveness to Ca(2+) at a hair cell's operating voltage. In contrast, however, the increases in relaxation times induced by both ß-subunits suggest additional mechanisms responsible for BK channel function in hair cells.

Prestin surface expression and activity are augmented by interaction with MAP1S, a microtubule-associated protein.

Prestin is a member of the SLC26 family of anion transporters that is responsible for outer hair cell (OHC) electromotility. Measures of voltage-evoked charge density (Q(sp)) of prestin indicated that the protein is highly expressed in OHCs, with single cells expressing up to 10 million molecules within the lateral membrane. In contrast, charge density measures in transfected cells indicated that they express, at best, only a fifth as many proteins on their surface. We sought to determine whether associations with other OHC-specific proteins could account for this difference. Using a yeast two-hybrid technique, we found microtubule-associated protein 1S (MAP1S) bound to prestin. The interaction was limited to the STAS domain of prestin and the region connecting the heavy and light chain of MAP1S. Using reciprocal immunoprecipitation and Forster resonance energy transfer, we confirmed these interactions. Furthermore, co-expression of prestin with MAP1S resulted in a 2.7-fold increase in Q(sp) in single cells that was paralleled by a 2.8-fold increase in protein surface expression, indicating that the interactions are physiological. Quantitative PCR data showed gradients in the expression of prestin and MAP1S across the tonotopic axis that may partially contribute to a previously observed 6-fold increase in Q(sp) in high frequency hair cells. These data highlight the importance of protein partner effects on prestin.

State dependent effects on the frequency response of prestin's real and imaginary components of nonlinear capacitance.

The outer hair cell (OHC) membrane harbors a voltage-dependent protein, prestin (SLC26a5), in high density, whose charge movement is evidenced as a nonlinear capacitance (NLC). NLC is bell-shaped, with its peak occurring at a voltage, Vh, where sensor charge is equally distributed across the plasma membrane. Thus, Vh provides information on the conformational state of prestin. Vh is sensitive to membrane tension, shifting to positive voltage as tension increases and is the basis for considering prestin piezoelectric (PZE). NLC can be deconstructed into real and imaginary components that report on charge movements in phase or 90 degrees out of phase with AC voltage. Here we show in membrane macro-patches of the OHC that there is a partial trade-off in the magnitude of real and imaginary components as interrogation frequency increases, as predicted by a recent PZE model (Rabbitt in Proc Natl Acad Sci USA 17:21880-21888, 2020). However, we find similar behavior in a simple 2-state voltage-dependent kinetic model of prestin that lacks piezoelectric coupling. At a particular frequency, Fis, the complex component magnitudes intersect. Using this metric, Fis, which depends on the frequency response of each complex component, we find that initial Vh influences Fis; thus, by categorizing patches into groups of different Vh, (above and below - 30 mV) we find that Fis is lower for the negative Vh group. We also find that the effect of membrane tension on complex NLC is dependent, but differentially so, on initial Vh. Whereas the negative group exhibits shifts to higher frequencies for increasing tension, the opposite occurs for the positive group. Despite complex component trade-offs, the low-pass roll-off in absolute magnitude of NLC, which varies little with our perturbations and is indicative of diminishing total charge movement, poses a challenge for a role of voltage-driven prestin in cochlear amplification at very high frequencies.