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1.
Hear Res ; 410: 108347, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34536825

RESUMO

ARHL has been thought to result from disordered hair cell function and their loss. ARHL has a significant genetic component. We sought to determine the expression in the cochlea of genes associated with single nucleotide polymorphisms linked to ARHL. We find widespread and varying expression of genes associated with these SNPs in subtypes of cells in the cochlea identified by single-cell RNA sequencing. Genes associated with SNPs with the highest significance were preferentially expressed highly in hair cells, while genes associated with SNPs with a lower significance were expressed more universally. In addition, we find significant overlap with genesets associated with Alzheimer's disease suggesting shared mechanisms, and genesets enriched for apical cell polarity and vesicle recycling suggesting mechanisms of cell death/ dysfunction with ageing.

2.
Sci Rep ; 11(1): 16149, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373481

RESUMO

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.

4.
Nat Commun ; 12(1): 2449, 2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33907194

RESUMO

In the developing auditory system, spontaneous activity generated in the cochleae propagates into the central nervous system to promote circuit formation. The effects of peripheral firing patterns on spontaneous activity in the central auditory system are not well understood. Here, we describe wide-spread bilateral coupling of spontaneous activity that coincides with the period of transient efferent modulation of inner hair cells from the brainstem medial olivocochlear system. Knocking out α9/α10 nicotinic acetylcholine receptors, a requisite part of the efferent pathway, profoundly reduces bilateral correlations. Pharmacological and chemogenetic experiments confirm that the efferent system is necessary for normal bilateral coupling. Moreover, auditory sensitivity at hearing onset is reduced in the absence of pre-hearing efferent modulation. Together, these results demonstrate how afferent and efferent pathways collectively shape spontaneous activity patterns and reveal the important role of efferents in coordinating bilateral spontaneous activity and the emergence of functional responses during the prehearing period.


Assuntos
Vias Auditivas/fisiologia , Cóclea/fisiologia , Vias Eferentes/fisiologia , Retroalimentação Fisiológica , Receptores Nicotínicos/genética , Estimulação Acústica , Animais , Vias Auditivas/citologia , Cóclea/citologia , Lateralidade Funcional/fisiologia , Expressão Gênica , Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/fisiologia , Colículos Inferiores/citologia , Colículos Inferiores/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Núcleo Olivar/citologia , Núcleo Olivar/fisiologia , Receptores Nicotínicos/deficiência
5.
Stroke ; 51(9): 2664-2673, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32755347

RESUMO

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.


Assuntos
Isquemia Encefálica/epidemiologia , Hemorragias Intracranianas/epidemiologia , Acidente Vascular Cerebral/epidemiologia , Tempo para o Tratamento/estatística & dados numéricos , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus , Isquemia Encefálica/diagnóstico , Isquemia Encefálica/fisiopatologia , Isquemia Encefálica/terapia , COVID-19 , Estudos de Coortes , Comorbidade , Connecticut/epidemiologia , Doença da Artéria Coronariana/epidemiologia , Infecções por Coronavirus/epidemiologia , Dislipidemias/epidemiologia , Serviços Médicos de Emergência , Grupos Étnicos , Feminino , Humanos , Hipertensão/epidemiologia , Renda , Seguro Saúde , Hemorragias Intracranianas/diagnóstico , Hemorragias Intracranianas/fisiopatologia , Hemorragias Intracranianas/terapia , Masculino , Pessoas sem Cobertura de Seguro de Saúde , Pessoa de Meia-Idade , Avaliação de Processos e Resultados em Cuidados de Saúde , Pandemias , Pneumonia Viral/epidemiologia , Estudos Retrospectivos , SARS-CoV-2 , Índice de Gravidade de Doença , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/fisiopatologia , Acidente Vascular Cerebral/terapia , Transtornos Relacionados ao Uso de Substâncias/epidemiologia , Telemedicina , Trombectomia , Terapia Trombolítica
6.
Physiol Rep ; 8(15): e14449, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32748549

RESUMO

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 calcium-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. Protein kinase A (PKA) activation increases the size of 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.

7.
Sci Rep ; 10(1): 14401, 2020 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-32848168

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

8.
Neuroscience ; 431: 128-133, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32061780

RESUMO

Prestin (SLC26a5) is an integral membrane motor protein in outer hair cells (OHC) that underlies cochlear amplification. As a voltage-dependent protein, it relies on intrinsic sensor charge to respond to transmembrane voltage (receptor potentials), thereby effecting conformational changes. The protein's electromechanical actively is experimentally monitored as a bell-shaped nonlinear capacitance (NLC), whose magnitude peaks at a characteristic voltage, Vh. This voltage denotes the midpoint of prestin's charge-voltage (Q-V) Boltzmann distribution and region of maximum gain of OHC electromotility. It is an important factor in hearing capabilities for mammals. A variety of biophysical forces can influence the distribution of charge, gauged by shifts in Vh, including prior holding voltage or membrane potential. Here we report that the effectiveness of prior voltage augments during the delivery of prestin to the membranes in an inducible HEK cell line. The augmentation coincides with an increase in prestin density, maturing at a characteristic membrane areal density of 870 functional prestin units per square micrometer, and is likely indicative of prestin-prestin cooperative interactions.


Assuntos
Células Ciliadas Auditivas Externas , Proteínas , Animais , Proteínas de Transporte de Ânions , Membrana Celular/metabolismo , Capacitância Elétrica , Células Ciliadas Auditivas Externas/metabolismo , Potenciais da Membrana , Técnicas de Patch-Clamp , Proteínas/metabolismo
9.
Sci Rep ; 9(1): 16460, 2019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31712635

RESUMO

Several studies have documented the early development of OHC electromechanical behavior. The mechanical response (electromotility, eM) and its electrical correlate (nonlinear capacitance, NLC), resulting from prestin's voltage-sensor charge movement, increase over the course of several postnatal days in altricial animals. They increase until about p18, near the time of peripheral auditory maturity. The correspondence of auditory capabilities and prestin function indicates that mature activity of prestin occurs at this time. One of the major requirements of eM is its responsiveness across auditory frequencies. Here we evaluate the frequency response of prestin charge movement in mice over the course of development up to 8 months. We find that in apical turn OHCs prestin's frequency response increases during postnatal development and stabilizes when mature hearing is established. The low frequency component of NLC, within in situ explants, agrees with previously reported results on isolated cells. If prestin activity is independent of cochlear place, as might be expected, then these observations suggest that prestin activity somehow influences cochlear amplification at high frequencies in spite of its low pass behavior.


Assuntos
Capacitância Elétrica , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas Externas/metabolismo , Audição/fisiologia , Mecanotransdução Celular , Proteínas Motores Moleculares/metabolismo , Órgão Espiral/metabolismo , Animais , Animais Recém-Nascidos , Vias Auditivas , Células Ciliadas Auditivas Externas/citologia , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Órgão Espiral/citologia
11.
J Neurosci Methods ; 313: 68-76, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30578868

RESUMO

BACKGROUND: Fluorescent proteins (FPs) have widespread uses in cell biology. However, the practical applications of FPs are significantly limited due to their rapid photobleaching and misfolding when fused to target proteins. NEW METHOD: Using a combination of novel and known mutations to eGFP, we developed a well folded and very photostable variant, SiriusGFP. RESULTS: The fluorescence spectrum indicated that the excitation and emission peaks of SiriusGFP were red-shifted by 16 and 8 nm, respectively. Co- operative effects of two key mutations, S147R and S205 V, contribute to its photostability. SiriusGFP tagged to the mitochondrial outer membrane protein Omp25 showed sustained fluorescence during continuous 3D-scanning confocal imaging (4D confocal) compared to eGFP-tagged Omp25. Furthermore, with super-resolution structured illumination microscopy (SIM) we demonstrate marked improvements in image quality and resolution (130 nm in XY axis, and 310 nm in Z axis), as well as, decreased artifacts due to photobleaching. COMPARISON WITH EXISTING METHOD(S): Compared to eGFP. SiriusGFP shows a 2-fold increase in photostability in vitro, and folds well when fused to the N- and C- termini of cytoplasmic and membrane proteins. While its quantum yield is ˜3 fold lower than eGFP, its decreased brightness was more than compensated by its increasedphotostability in different experimental paradigms allowing practical experimentation without dynamic adjustment of light intensity or fluorescence sampling times. CONCLUSIONS: We have developed a variant of eGFP, SiriusGFP, that shows over a two fold increase in photostability with utility in methods requiring sustained or high intensity excitation as in 4D confocal or SIM imaging.


Assuntos
Proteínas de Fluorescência Verde , Microscopia de Fluorescência/métodos , Células HEK293 , Células HeLa , Humanos , Microscopia Confocal/métodos
13.
Sci Rep ; 7: 46619, 2017 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-28422190

RESUMO

Prestin in the lateral membrane of outer hair cells, is responsible for electromotility (EM) and a corresponding nonlinear capacitance (NLC). Prestin's voltage sensitivity is influenced by intracellular chloride. A regulator of intracellular chloride is a stretch-sensitive, non-selective conductance within the lateral membrane, GmetL. We determine that prestin itself possesses a stretch-sensitive, non-selective conductance that is largest in the presence of thiocyanate ions. This conductance is independent of the anion transporter mechanism. Prestin has been modeled, based on structural data from related anion transporters (SLC26Dg and UraA), to have a 7 + 7 inverted repeat structure with anion transport initiated by chloride binding at the intracellular cleft. Mutation of residues that bind intracellular chloride, and salicylate treatment which prevents chloride binding, have no effect on thiocyanate conductance. In contrast, other mutations reduce the conductance while preserving NLC. When superimposed on prestin's structure, the location of these mutations indicates that the ion permeation pathway lies between the core and gate ring of helices, distinct from the transporter pathway. The uncoupled current is reminiscent of an omega current in voltage-gated ion channels. We suggest that prestin itself is the main regulator of intracellular chloride concentration via a route distinct from its transporter pathway.


Assuntos
Ativação do Canal Iônico/genética , Transportadores de Sulfato , Animais , Células CHO , Cricetulus , Células HEK293 , Humanos , Transporte de Íons/genética , Mutação , Estrutura Secundária de Proteína , Transportadores de Sulfato/química , Transportadores de Sulfato/genética , Transportadores de Sulfato/metabolismo
14.
Antioxid Redox Signal ; 27(8): 489-509, 2017 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-28135838

RESUMO

AIMS: Acquired hearing loss is a worldwide epidemic that affects all ages. It is multifactorial in etiology with poorly characterized molecular mechanisms. Mitochondria are critical components in hearing. Here, we aimed to identify the mechanisms of mitochondria-dependent hearing loss using Fus1 KO mice, our novel model of mitochondrial dysfunction/oxidative stress. RESULTS: Using auditory brainstem responses (ABRs), we characterized the Fus1 KO mouse as a novel, clinically relevant model of age-related hearing loss (ARHL) of metabolic etiology. We demonstrated early decline of the endocochlear potential (EP) that may occur due to severe mitochondrial and vascular pathologies in the Fus1 KO cochlear stria vascularis. We showed that pathological alterations in antioxidant (AO) and nutrient and energy sensing pathways (mTOR and PTEN/AKT) occur in cochleae of young Fus1 KO mice before major hearing loss. Importantly, short-term AO treatment corrected pathological molecular changes, while longer AO treatment restored EP, improved ABR parameters, restored mitochondrial structure, and delayed the development of hearing loss in the aging mouse. INNOVATION: Currently, no molecular mechanisms linked to metabolic ARHL have been identified. We established pathological and molecular mechanisms that link the disease to mitochondrial dysfunction and oxidative stress. CONCLUSION: Since chronic mitochondrial dysfunction is common in many patients, it could lead to developing hearing loss that can be alleviated/rescued by AO treatment. Our study creates a framework for clinical trials and introduces the Fus1 KO model as a powerful platform for developing novel therapeutic strategies to prevent/delay hearing loss associated with mitochondrial dysfunction. Antioxid. Redox Signal. 27, 489-509.


Assuntos
Orelha Interna/fisiopatologia , Perda Auditiva/diagnóstico por imagem , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Acetilcisteína/administração & dosagem , Acetilcisteína/farmacologia , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Modelos Animais de Doenças , Orelha Interna/diagnóstico por imagem , Orelha Interna/efeitos dos fármacos , Orelha Interna/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Técnicas de Inativação de Genes , Perda Auditiva/tratamento farmacológico , Perda Auditiva/genética , Perda Auditiva/metabolismo , Humanos , Camundongos , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Estresse Oxidativo/efeitos dos fármacos , PTEN Fosfo-Hidrolase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Gânglio Espiral da Cóclea/diagnóstico por imagem , Gânglio Espiral da Cóclea/efeitos dos fármacos , Gânglio Espiral da Cóclea/metabolismo , Gânglio Espiral da Cóclea/fisiopatologia , Serina-Treonina Quinases TOR/metabolismo
15.
Cell Rep ; 16(9): 2281-8, 2016 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-27545877

RESUMO

Human mutations in the cytoplasmic C-terminal domain of Slack sodium-activated potassium (KNa) channels result in childhood epilepsy with severe intellectual disability. Slack currents can be increased by pharmacological activators or by phosphorylation of a Slack C-terminal residue by protein kinase C. Using an optical biosensor assay, we find that Slack channel stimulation in neurons or transfected cells produces loss of mass near the plasma membrane. Slack mutants associated with intellectual disability fail to trigger any change in mass. The loss of mass results from the dissociation of the protein phosphatase 1 (PP1) targeting protein, Phactr-1, from the channel. Phactr1 dissociation is specific to wild-type Slack channels and is not observed when related potassium channels are stimulated. Our findings suggest that Slack channels are coupled to cytoplasmic signaling pathways and that dysregulation of this coupling may trigger the aberrant intellectual development associated with specific childhood epilepsies.


Assuntos
Membrana Celular/metabolismo , Proteína do X Frágil de Retardo Mental/genética , Proteínas dos Microfilamentos/genética , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Canais de Potássio/genética , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Técnicas Biossensoriais , Bitionol/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Membrana Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Proteína do X Frágil de Retardo Mental/antagonistas & inibidores , Proteína do X Frágil de Retardo Mental/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Transporte de Íons/efeitos dos fármacos , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/metabolismo , Mutação , Proteínas do Tecido Nervoso/agonistas , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Fosforilação , Canais de Potássio/agonistas , Canais de Potássio/metabolismo , Canais de Potássio Ativados por Sódio , Cultura Primária de Células , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Tiazolidinas/farmacologia , Xenopus laevis
16.
Methods Mol Biol ; 1427: 95-107, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27259923

RESUMO

We describe a protocol to screen for protein-protein interactions using the Gal-4-based yeast two-hybrid system. In this protocol, we describe serial transformation of bait into an already constructed cDNA library in yeast AH109 cells. We find this method gives the most number of true interactions. Where a premade library in yeast cells is not available, the method outlined can be quickly adapted. AH109 cells can be first transformed with bait containing a vector followed by selection of yeast containing the bait. A second transformation of yeast cells is then accomplished with the cDNA library. The method is quick and can lead to the discovery of significant interactions.


Assuntos
Cóclea/metabolismo , Mapeamento de Interação de Proteínas/métodos , Leveduras/genética , Animais , Biblioteca Gênica , Humanos , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido
17.
Cell ; 165(2): 434-448, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26997484

RESUMO

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.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Canais de Potássio Shaw/metabolismo , Ataxias Espinocerebelares/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Sequência de Aminoácidos , Membrana Celular/metabolismo , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo , Canais de Potássio Shaw/química , Canais de Potássio Shaw/genética , Transdução de Sinais , Proteínas rac de Ligação ao GTP/metabolismo
18.
Biol Open ; 4(2): 197-205, 2015 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-25596279

RESUMO

Prestin is targeted to the lateral wall of outer hair cells (OHCs) where its electromotility is critical for cochlear amplification. Using MDCK cells as a model system for polarized epithelial sorting, we demonstrate that prestin uses tyrosine residues, in a YXXΦ motif, to target the basolateral surface. Both Y520 and Y667 are important for basolateral targeting of prestin. Mutation of these residues to glutamine or alanine resulted in retention within the Golgi and delayed egress from the Golgi in Y667Q. Basolateral targeting is restored upon mutation to phenylalanine suggesting the importance of a phenol ring in the tyrosine side chain. We also demonstrate that prestin targeting to the basolateral surface is dependent on AP1B (µ1B), and that prestin uses transferrin containing early endosomes in its passage from the Golgi to the basolateral plasma membrane. The presence of AP1B (µ1B) in OHCs, and parallels between prestin targeting to the basolateral surface of OHCs and polarized epithelial cells suggest that outer hair cells resemble polarized epithelia rather than neurons in this important phenotypic measure.

19.
PLoS One ; 9(6): e99095, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24901231

RESUMO

BACKGROUND: Chloride is the major anion in cells, with many diseases arising from disordered Cl- regulation. For the non-invasive investigation of Cl- flux, YFP-H148Q and its derivatives chameleon and Cl-Sensor previously were introduced as genetically encoded chloride indicators. Neither the Cl- sensitivity nor the pH-susceptibility of these modifications to YFP is optimal for precise measurements of Cl- under physiological conditions. Furthermore, the relatively poor photostability of YFP derivatives hinders their application for dynamic and quantitative Cl- measurements. Dynamic and accurate measurement of physiological concentrations of chloride would significantly affect our ability to study effects of chloride on cellular events. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we developed a series of YFP derivatives to remove pH interference, increase photostability and enhance chloride sensitivity. The final product, EYFP-F46L/Q69K/H148Q/I152L/V163S/S175G/S205V/A206K (monomeric Cl-YFP), has a chloride Kd of 14 mM and pKa of 5.9. The bleach time constant of 175 seconds is over 15-fold greater than wild-type EYFP. We have used the sensor fused to the transmembrane protein prestin (gerbil prestin, SLC26a5), and shown for the first time physiological (mM) chloride flux in HEK cells expressing this protein. This modified fluorescent protein will facilitate investigations of dynamics of chloride ions and their mediation of cell function. CONCLUSIONS: Modifications to YFP (EYFP-F46L/Q69K/H148Q/I152L/V163S/S175G/S205V/A206K (monomeric Cl-YFP) results in a photostable fluorescent protein that allows measurement of physiological changes in chloride concentration while remaining minimally affected by changes in pH.


Assuntos
Proteínas de Bactérias/metabolismo , Cloretos/metabolismo , Gerbillinae/metabolismo , Proteínas Luminescentes/metabolismo , Animais , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Ânions/química , Proteínas de Bactérias/genética , Concentração de Íons de Hidrogênio , Luz , Proteínas Luminescentes/genética , Mutação , Fotodegradação , Estabilidade Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética
20.
PLoS One ; 8(6): e66078, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23762468

RESUMO

Prestin (SLC26a5) is the outer hair cell integral membrane motor protein that drives cochlear amplification, and has been described as an obligate tetramer. We studied in real time the delivery of YFP-prestin to the plasma membrane of cells from a tetracycline-inducible cell line. Following the release of temperature block to reinstate trans Golgi network delivery of the integral membrane protein, we measured nonlinear capacitance (NLC) and membrane fluorescence during voltage clamp. Prestin was delivered exponentially to the plasma membrane with a time constant of less than 10 minutes, with both electrical and fluorescence methods showing high temporal correlation. However, based on disparity between estimates of prestin density derived from either fluorescence or NLC, we conclude that sub-tetrameric forms of prestin contribute to our electrical and fluorescence measures. Thus, in agreement with previous observations we find that functional prestin is not an obligate tetramer.


Assuntos
Proteínas de Transporte de Ânions/metabolismo , Membrana Celular/metabolismo , Rede trans-Golgi/metabolismo , Proteínas de Transporte de Ânions/química , Proteínas de Transporte de Ânions/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Capacitância Elétrica , Eletrofisiologia , Fluorescência , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Multimerização Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transportadores de Sulfato
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