25 results on '"Münkner, Stefan"'
Search Results
2. Calcium signaling in interdental cells during the critical developmental period of the mouse cochlea
- Author
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Schade-Mann, Thore, primary, Münkner, Stefan, additional, Eckrich, Tobias, additional, and Engel, Jutta, additional
- Published
- 2020
- Full Text
- View/download PDF
3. Elementary properties of CaV1.3 Ca2+ channels expressed in mouse cochlear inner hair cells
- Author
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Zampini, Valeria, Johnson, Stuart L., Franz, Christoph, Lawrence, Neil D., Münkner, Stefan, Engel, Jutta, Knipper, Marlies, Magistretti, Jacopo, Masetto, Sergio, and Marcotti, Walter
- Published
- 2010
- Full Text
- View/download PDF
4. Cochlea-Specific Deletion of Cav1.3 Calcium Channels Arrests Inner Hair Cell Differentiation and Unravels Pitfalls of Conditional Mouse Models
- Author
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Eckrich, Stephanie, primary, Hecker, Dietmar, additional, Sorg, Katharina, additional, Blum, Kerstin, additional, Fischer, Kerstin, additional, Münkner, Stefan, additional, Wenzel, Gentiana, additional, Schick, Bernhard, additional, and Engel, Jutta, additional
- Published
- 2019
- Full Text
- View/download PDF
5. Elementary properties of CaV1.3 Ca2+ channels expressed in mouse cochlear inner hair cells
- Author
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Zampini, Valeria, Johnson, Stuart L, Franz, Christoph, Lawrence, Neil D, Münkner, Stefan, Engel, Jutta, Knipper, Marlies, Magistretti, Jacopo, Masetto, Sergio, and Marcotti, Walter
- Subjects
Mice, Inbred C57BL ,Mice ,Hair Cells, Auditory, Inner ,Calcium Channels, L-Type ,Action Potentials ,Animals ,Calcium ,Calcium Signaling ,Ion Channel Gating ,Cells, Cultured ,Neuroscience ,Membrane Potentials - Abstract
Mammalian cochlear inner hair cells (IHCs) are specialized to process developmental signals during immature stages and sound stimuli in adult animals. These signals are conveyed onto auditory afferent nerve fibres. Neurotransmitter release at IHC ribbon synapses is controlled by L-type Ca(V)1.3 Ca(2+) channels, the biophysics of which are still unknown in native mammalian cells. We have investigated the localization and elementary properties of Ca(2+) channels in immature mouse IHCs under near-physiological recording conditions. Ca(V)1.3 Ca(2+) channels at the cell pre-synaptic site co-localize with about half of the total number of ribbons present in immature IHCs. These channels activated at about 70 mV, showed a relatively short first latency and weak inactivation, which would allow IHCs to generate and accurately encode spontaneous Ca(2+) action potential activity characteristic of these immature cells. The Ca(V)1.3 Ca(2+) channels showed a very low open probability (about 0.15 at 20 mV: near the peak of an action potential). Comparison of elementary and macroscopic Ca(2+) currents indicated that very few Ca(2+) channels are associated with each docked vesicle at IHC ribbon synapses. Finally, we found that the open probability of Ca(2+) channels, but not their opening time, was voltage dependent. This finding provides a possible correlation between presynaptic Ca(2+) channel properties and the characteristic frequency/amplitude of EPSCs in auditory afferent fibres.
- Published
- 2009
6. Cochlea-Specific Deletion of Cav1.3 Calcium Channels Arrests Inner Hair Cell Differentiation and Unravels Pitfalls of Conditional Mouse Models.
- Author
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Eckrich, Stephanie, Hecker, Dietmar, Sorg, Katharina, Blum, Kerstin, Fischer, Kerstin, Münkner, Stefan, Wenzel, Gentiana, Schick, Bernhard, and Engel, Jutta
- Subjects
HAIR cells ,CALCIUM channels ,CELL differentiation ,GREEN fluorescent protein ,COCHLEA physiology ,FLUORESCENT proteins - Abstract
Inner hair cell (IHC) Ca
v 1.3 Ca2+ channels are multifunctional channels mediating Ca2+ influx for exocytosis at ribbon synapses, the generation of Ca2+ action potentials in pre-hearing IHCs and gene expression. IHCs of deaf systemic Cav 1.3-deficient (Cav 1.3-/- ) mice stay immature because they fail to up-regulate voltage- and Ca2+ -activated K+ (BK) channels but persistently express small conductance Ca2+ -activated K+ (SK2) channels. In pre-hearing wildtype mice, cholinergic neurons from the superior olivary complex (SOC) exert efferent inhibition onto spontaneously active immature IHCs by activating their SK2 channels. Because Cav 1.3 plays an important role for survival, health and function of SOC neurons, SK2 channel persistence and lack of BK channels in systemic Cav 1.3-/- IHCs may result from malfunctioning neurons of the SOC. Here we analyze cochlea-specific Cav 1.3 knockout mice with green fluorescent protein (GFP) switch reporter function, Pax2::cre;Cacna1d-eGFPflex/flex and Pax2::cre;Cacna1d-eGFPflex/- . Profound hearing loss, lack of BK channels and persistence of SK2 channels in Pax2::cre;Cacna1d-eGFPflex/- mice recapitulated the phenotype of systemic Cav 1.3-/- mice, indicating that in wildtype mice, regulation of SK2 and BK channel expression is independent of Cav 1.3 expression in SOC neurons. In addition, we noticed dose-dependent GFP toxicity leading to death of basal coil IHCs of Pax2::cre;Cacna1d-eGFPflex/flex mice, likely because of high GFP concentration and small repair capacity. This and the slower time course of Pax2 -driven Cre recombinase in switching two rather than one Cacna1d-eGFPflex allele lead us to study Pax2::cre;Cacna1d-eGFPflex/- mice. Notably, control Cacna1d-eGFPflex/- IHCs showed a significant reduction in Cav 1.3 channel cluster sizes and currents, suggesting that the intronic construct interfered with gene translation or splicing. These pitfalls are likely to be a frequent problem of many genetically modified mice with complex or multiple gene-targeting constructs or fluorescent proteins. Great caution and appropriate controls are therefore required. [ABSTRACT FROM AUTHOR]- Published
- 2019
- Full Text
- View/download PDF
7. Deletion of the Ca2+-activated potassium (BK) alpha-subunit but not the BK-beta-1-subunit leads to progressive hearing loss
- Author
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Rüttiger, Lukas, Sausbier, Matthias, Zimmermann, Ulrike, Winter, Harald, Braig, Claudia, Engel, Jutta, Knirsch, Martina, Arntz, Claudia, Langer, Patricia, Hirt, Bernhard, Müller, Marcus, Köpschall, Iris, Pfister, Markus, Münkner, Stefan, Rohbock, Karin, Pfaff, Imke, Rüsch, Alfons, Ruth, Peter, Knipper, Marlies, and Latorre, Ramon
- Subjects
otorhinolaryngologic diseases ,sense organs ,ddc:610 - Abstract
The large conductance voltage- and Ca2+-activated potassium (BK) channel has been suggested to play an important role in the signal transduction process of cochlear inner hair cells. BK channels have been shown to be composed of the pore-forming alpha-subunit coexpressed with the auxiliary beta-1-subunit. Analyzing the hearing function and cochlear phenotype of BK channel alpha-(BKalpha–/–) and beta-1-subunit (BKbeta-1–/–) knockout mice, we demonstrate normal hearing function and cochlear structure of BKbeta-1–/– mice. During the first 4 postnatal weeks also, BKalpha–/– mice most surprisingly did not show any obvious hearing deficits. High-frequency hearing loss developed in BKalpha–/– mice only from ca. 8 weeks postnatally onward and was accompanied by a lack of distortion product otoacoustic emissions, suggesting outer hair cell (OHC) dysfunction. Hearing loss was linked to a loss of the KCNQ4 potassium channel in membranes of OHCs in the basal and midbasal cochlear turn, preceding hair cell degeneration and leading to a similar phenotype as elicited by pharmacologic blockade of KCNQ4 channels. Although the actual link between BK gene deletion, loss of KCNQ4 in OHCs, and OHC degeneration requires further investigation, data already suggest human BK-coding slo1 gene mutation as a susceptibility factor for progressive deafness, similar to KCNQ4 potassium channel mutations. © 2004, The National Academy of Sciences. Freely available online through the PNAS open access option.
- Published
- 2005
8. α2δ2 Controls the Function and Trans-Synaptic Coupling of Cav1.3 Channels in Mouse Inner Hair Cells and Is Essential for Normal Hearing.
- Author
-
Fell, Barbara, Eckrich, Stephanie, Blum, Kerstin, Eckrich, Tobias, Hecker, Dietmar, Obermair, Gerald J., Münkner, Stefan, Flockerzi, Veit, Schick, Bernhard, and Engel, Jutta
- Subjects
CALCIUM channels ,HAIR cells ,OTOACOUSTIC emissions ,EXOCYTOSIS ,PRESYNAPTIC receptors ,GLUTAMATE receptors - Abstract
The auxiliary subunit α
2 δ2 modulates the abundance and function of voltage-gated calcium channels. Here we show that α2 δ2 mRNA is expressed in neonatal and mature hair cells. A functional α2 δ2-null mouse, the ducky mouse (du), showed elevated auditory brainstem response click and frequency-dependent hearing thresholds. Otoacoustic emissions were not impaired pointing to normal outer hair cell function. Peak Ca2+ and Ba2+ currents of mature du/du inner hair cells (IHCs) were reduced by 30-40%, respectively, and gating properties, such as the voltage of half-maximum activation and voltage sensitivity, were altered, indicating that Cav 1.3 channels normally coassemble with α2 δ2 at IHC presynapses. The reduction of depolarization-evoked exocytosis in du/du IHCs reflected their reduced Ca2+ currents. Ca2+ - and voltage-dependent K+ (BK) currents and the expression of the pore-forming BKα protein were normal. Cav 1.3 and Cav β2 protein expression was unchanged in du/du IHCs, forming clusters at presynaptic ribbons. However, the close apposition of presynaptic Cav 1.3 clusters with postsynaptic glutamate receptor GluA4 and PSD-95 clusters was significantly impaired in du/du mice. This implies that, in addition to controlling the expression and gating properties of Cav 1.3 channels, the largely extracellularly localized α2 δ2 subunit moreover plays a so far unknown role in mediating trans-synaptic alignment of presynaptic Ca2+ channels and postsynaptic AMPA receptors. [ABSTRACT FROM AUTHOR]- Published
- 2016
- Full Text
- View/download PDF
9. Elementary properties of CaV1.3 Ca2+channels expressed in mouse cochlear inner hair cells
- Author
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Zampini, Valeria, primary, Johnson, Stuart L., additional, Franz, Christoph, additional, Lawrence, Neil D., additional, Münkner, Stefan, additional, Engel, Jutta, additional, Knipper, Marlies, additional, Magistretti, Jacopo, additional, Masetto, Sergio, additional, and Marcotti, Walter, additional
- Published
- 2010
- Full Text
- View/download PDF
10. Synaptotagmin IV determines the linear Ca2+ dependence of vesicle fusion at auditory ribbon synapses
- Author
-
Johnson, Stuart L, primary, Franz, Christoph, additional, Kuhn, Stephanie, additional, Furness, David N, additional, Rüttiger, Lukas, additional, Münkner, Stefan, additional, Rivolta, Marcelo N, additional, Seward, Elizabeth P, additional, Herschman, Harvey R, additional, Engel, Jutta, additional, Knipper, Marlies, additional, and Marcotti, Walter, additional
- Published
- 2009
- Full Text
- View/download PDF
11. Tonotopic Variation in the Calcium Dependence of Neurotransmitter Release and Vesicle Pool Replenishment at Mammalian Auditory Ribbon Synapses
- Author
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Johnson, Stuart L., primary, Forge, Andrew, additional, Knipper, Marlies, additional, Münkner, Stefan, additional, and Marcotti, Walter, additional
- Published
- 2008
- Full Text
- View/download PDF
12. Deletion of the Ca 2 +-activated potassium (BK) α-subunit but not the BKβ1-subunit leads to progressive hearing loss
- Author
-
Rüttiger, Lukas, primary, Sausbier, Matthias, additional, Zimmermann, Ulrike, additional, Winter, Harald, additional, Braig, Claudia, additional, Engel, Jutta, additional, Knirsch, Martina, additional, Arntz, Claudia, additional, Langer, Patricia, additional, Hirt, Bernhard, additional, Müller, Marcus, additional, Köpschall, Iris, additional, Pfister, Markus, additional, Münkner, Stefan, additional, Rohbock, Karin, additional, Pfaff, Imke, additional, Rüsch, Alfons, additional, Ruth, Peter, additional, and Knipper, Marlies, additional
- Published
- 2004
- Full Text
- View/download PDF
13. Influence of fine structure and envelope variability on gap‐duration discrimination thresholds
- Author
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Münkner, Stefan, primary, Kohlrausch, Armin, additional, and Püschel, Dirk, additional
- Published
- 1996
- Full Text
- View/download PDF
14. Simulations of temporal resolution and integration data with a computer model using a modulation filter bank.
- Author
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Münkner, Stefan, primary and Kohlrausch, Armin, additional
- Published
- 1996
- Full Text
- View/download PDF
15. Elementary properties of CaV1.3 Ca2+ channels expressed in mouse cochlear inner hair cells.
- Author
-
Zampini, Valeria, Johnson, Stuart L., Franz, Christoph, Lawrence, Neil D., Münkner, Stefan, Engel, Jutta, Knipper, Marlies, Magistretti, Jacopo, Masetto, Sergio, and Marcotti, Walter
- Abstract
Mammalian cochlear inner hair cells (IHCs) are specialized to process developmental signals during immature stages and sound stimuli in adult animals. These signals are conveyed onto auditory afferent nerve fibres. Neurotransmitter release at IHC ribbon synapses is controlled by L-type Ca
V 1.3 Ca2+ channels, the biophysics of which are still unknown in native mammalian cells. We have investigated the localization and elementary properties of Ca2+ channels in immature mouse IHCs under near-physiological recording conditions. CaV 1.3 Ca2+ channels at the cell pre-synaptic site co-localize with about half of the total number of ribbons present in immature IHCs. These channels activated at about −70 mV, showed a relatively short first latency and weak inactivation, which would allow IHCs to generate and accurately encode spontaneous Ca2+ action potential activity characteristic of these immature cells. The CaV 1.3 Ca2+ channels showed a very low open probability (about 0.15 at −20 mV: near the peak of an action potential). Comparison of elementary and macroscopic Ca2+ currents indicated that very few Ca2+ channels are associated with each docked vesicle at IHC ribbon synapses. Finally, we found that the open probability of Ca2+ channels, but not their opening time, was voltage dependent. This finding provides a possible correlation between presynaptic Ca2+ channel properties and the characteristic frequency/amplitude of EPSCs in auditory afferent fibres. [ABSTRACT FROM AUTHOR]- Published
- 2010
- Full Text
- View/download PDF
16. Synaptotagmin IV determines the linear Ca2+ dependence of vesicle fusion at auditory ribbon synapses.
- Author
-
Johnson, Stuart L., Franz, Christoph, Kuhn, Stephanie, Furness, David N., Rüttiger, Lukas, Münkner, Stefan, Rivolta, Marcelo N., Seward, Elizabeth P., Herschman, Harvey R., Engel, Jutta, Knipper, Marlies, and Marcotti, Walter
- Subjects
HAIR cells ,COCHLEA ,MAMMALS ,SYNAPSES ,NEURAL transmission ,SYNAPTIC vesicles ,RODENTS ,OLFACTORY receptors - Abstract
Mammalian cochlear inner hair cells (IHCs) are specialized for the dynamic coding of continuous and finely graded sound signals. This ability is largely conferred by the linear Ca
2+ dependence of neurotransmitter release at their synapses, which is also a feature of visual and olfactory systems. The prevailing hypothesis is that linearity in IHCs occurs through a developmental change in the Ca2+ sensitivity of synaptic vesicle fusion from the nonlinear (high order) Ca2+ dependence of immature spiking cells. However, the nature of the Ca2+ sensor(s) of vesicle fusion at hair cell synapses is unknown. We found that synaptotagmin IV was essential for establishing the linear exocytotic Ca2+ dependence in adult rodent IHCs and immature outer hair cells. Moreover, the expression of the hitherto undetected synaptotagmins I and II correlated with a high-order Ca2+ dependence in IHCs. We propose that the differential expression of synaptotagmins determines the characteristic Ca2+ sensitivity of vesicle fusion at hair cell synapses. [ABSTRACT FROM AUTHOR]- Published
- 2010
- Full Text
- View/download PDF
17. Persistence of Cav1.3 Ca2+ Channels in Mature Outer Hair Cells Supports Outer Hair Cell Afferent Signaling.
- Author
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Knirsch, Martina, Brandt, Niels, Braig, Claudia, Kuhn, Stephanie, Hirt, Bernhard, Münkner, Stefan, Knipper, Marlies, and Engel, Jutta
- Subjects
HAIR cells ,NEURONS ,EXOCYTOSIS ,MICE ,RATS ,MESSENGER RNA ,PROTEINS ,SYNAPSES - Abstract
Outer hair cells (OHCs) are innervated by type II afferent fibers of as yet unknown function. It is still a matter of debate whether OHCs perform exocytosis. If so, they would require presynaptic Ca
2+ channels at their basal poles where the type II fibers make contacts. Here we show that L-type Ca2+ channel currents (charge carrier, 10mM Ba2+ ) present in neonatal OHCs [postnatal day 1 (P1) to P7] decreased from ∼170 to ∼50 pA at approximately the onset of hearing. Ba2+ currents could hardly be measured in mature mouse OHCs because of their high fragility, whereas in the rat, the average Ba2+ current amplitude of apical OHCs was 58±9 pA (n=20, P19 -P30) compared with that of the inner hair cells (IHCs) of 181±50 pA (n = 24, P17-P30). Properties of Ba2+ currents of mature OHCs resembled those of neonatal OHCs. One exception was the voltage dependence of activation that shifted between birth and P12 by + 9mV toward positive voltages in OHCs, whereas it remained constant in the IHCs. Cav 1.3-specific mRNA was detected in mature OHCs using cell-specific reverse transcription (RT)-PCR and in situ hybridization. Cav 1.3 protein was stained exclusively at the base of mature OHCs, in colocalization with the ribbon synapse protein CtBP2 (C-terminal binding protein 2)/RIBEYE. When current sizes were normalized to the estimated number of afferent fibers or presynaptic ribbons, comparable values for IHCs and OHCs were obtained, a finding that together with the colocalization of Cav 1.3 and CtBP2/RIBEYE protein strongly suggests a role for Cav 1.3 channels in exocytosis of mature OHCs. [ABSTRACT FROM AUTHOR]- Published
- 2007
- Full Text
- View/download PDF
18. Thyroid Hormone Deficiency Affects Postnatal Spiking Activity and Expression of Ca2+ and K+ Channels in Rodent Inner Hair Cells.
- Author
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Brandt, Niels, Kuhn, Stephanie, Münkner, Stefan, Braig, Claudia, Winter, Harald, Blin, Nikolaus, Vonthein, Reinhard, Knipper, Marlies, and Engel, Jutta
- Subjects
THYROID hormones ,HAIR cells ,CORTI'S organ ,CALCIUM ions ,LABORATORY rodents ,ACTION potentials ,ELECTROPHYSIOLOGY ,IMMUNOCYTOCHEMISTRY - Abstract
Thyroid hormone (TH) is essential for the development of hearing. Lack of TH in a critical developmental period from embryonic day 17 to postnatal day 12 (P12) in rats and mice leads to morphological and functional deficits in the organ of Corti and the auditory pathway. We investigated the effects of TH on inner hair cells (IHCs) using patch-clamp recordings, capacitance measurements, and immunocytochemistry in hypothyroid rats and athyroid Pax8
-/- mice. Spontaneous and evoked Ca2+ action potentials (APs) were present in control IHCs from P3-P11 rats and vanished in parallel with the expression of a rapidly activating Ca2+ and voltage-activated K+ (BK) conductance. IHCs of hypothyroid rats and athyroid Pax8-/- mice displayed APs until the end of the third postnatal week because of threefold elevated Ca2+ currents and missing expression of BK currents. After the fourth postnatal week, some IHCs showed BK currents whereas adjacent IHCs did not, demonstrated by electrophysiology and immunocytochemistry. To test whether the prolonged spiking activity during TH deficiency may be transmitted at IHC synapses, capacitance measurements were performed in parallel to analysis of otoferlin expression, a protein thought to play an essential role in exocytosis of IHCs. Strikingly, otoferlin was absent from IHCs of hypothyroid rats but not of Pax8-/- mice, although both cell types showed exocytosis with an efficiency typical for immature IHCs. These results demonstrate for the first time a TH-dependent control of IHC spiking activity before the onset of hearing attributable to effects of TH on Ca2+ and BK channels. Moreover, they question an indispensable role of otoferlin for exocytosis in IHCs. [ABSTRACT FROM AUTHOR]- Published
- 2007
- Full Text
- View/download PDF
19. Deletion of the Ca²+;-activated potassium (BK) α-subunit but not the BKβ1-subunit leads to progressive hearing loss.
- Author
-
Rüttiger, Lukas, Sausbier, Matthias, Zimmermann, Ulrike, Winter, Harald, Braig, Claudia, Engel, Jutta, Knirsch, Martina, Arntz, Claudia, Langer, Patricia, Hirt, Bernhard, Muller, Marcus, Köpschall, Iris, Pfister, Markus, Münkner, Stefan, Rohbock, Karin, Pfaff, Lmke, R&uum;sch, Alfons, Ruth, Peter, and Knipper, Marlies
- Subjects
GENETIC transduction ,MICROBIAL genetics ,HEARING disorders ,HAIR cells ,HEARING ,CELL death - Abstract
The large conductance voltage- and Ca
2+ -activated potassium (BK) channel has been suggested to play an important role in the signal transduction process of cochlear inner hair cells BK channels have been shown to be composed of the pore-forming α-subunit coexpressed with the auxiliary β1-subunit Analyzing the hearing function and cochlear phenotype of BK channel α- (BKα-/- ) and β1-subunit (BKβ1-/- ) knockout mice, we demonstrate normal hearing function and cochlear structure of BKβ1-/- mice. During the first 4 postnatal weeks also, BKα-/- mice most surprisingly did not show any obvious hearing deficits. High-frequency hearing loss developed in BKα-/- mice only from ≈8 weeks postnatally onward and was accompanied by a lack of distortion product otoacoustic emissions, suggesting outer hair cell (OHC) dysfunction. Hearing loss was linked to a loss of the KCNQ4 potassium channel in membranes of OHCs in the basal and midbasal cochlear turn, preceding hair cell degeneration and leading to a similar phenotype as elicited by pharmacologic blockade of KCNQ4 channels. Although the actual link between BK gene deletion, loss of KCNQ4 in OHCs, and OHC degeneration requires further investigation, data already suggest human BK-coding slo1 gene mutation as a susceptibility factor for progressive deafness, similar to KCNQ4 potassium channel mutations. [ABSTRACT FROM AUTHOR]- Published
- 2004
- Full Text
- View/download PDF
20. Cochlea-Specific Deletion of Ca v 1.3 Calcium Channels Arrests Inner Hair Cell Differentiation and Unravels Pitfalls of Conditional Mouse Models.
- Author
-
Eckrich S, Hecker D, Sorg K, Blum K, Fischer K, Münkner S, Wenzel G, Schick B, and Engel J
- Abstract
Inner hair cell (IHC) Ca
v 1.3 Ca2+ channels are multifunctional channels mediating Ca2+ influx for exocytosis at ribbon synapses, the generation of Ca2+ action potentials in pre-hearing IHCs and gene expression. IHCs of deaf systemic Cav 1.3-deficient (Cav 1.3-/- ) mice stay immature because they fail to up-regulate voltage- and Ca2+ -activated K+ (BK) channels but persistently express small conductance Ca2+ -activated K+ (SK2) channels. In pre-hearing wildtype mice, cholinergic neurons from the superior olivary complex (SOC) exert efferent inhibition onto spontaneously active immature IHCs by activating their SK2 channels. Because Cav 1.3 plays an important role for survival, health and function of SOC neurons, SK2 channel persistence and lack of BK channels in systemic Cav 1.3-/- IHCs may result from malfunctioning neurons of the SOC. Here we analyze cochlea-specific Cav 1.3 knockout mice with green fluorescent protein (GFP) switch reporter function, Pax2::cre;Cacna1d-eGFPflex/flex and Pax2::cre;Cacna1d-eGFPflex/- . Profound hearing loss, lack of BK channels and persistence of SK2 channels in Pax2::cre;Cacna1d-eGFPflex/- mice recapitulated the phenotype of systemic Cav 1.3-/- mice, indicating that in wildtype mice, regulation of SK2 and BK channel expression is independent of Cav 1.3 expression in SOC neurons. In addition, we noticed dose-dependent GFP toxicity leading to death of basal coil IHCs of Pax2::cre;Cacna1d-eGFPflex/flex mice, likely because of high GFP concentration and small repair capacity. This and the slower time course of Pax2 -driven Cre recombinase in switching two rather than one Cacna1d-eGFPflex allele lead us to study Pax2::cre;Cacna1d-eGFPflex/- mice. Notably, control Cacna1d-eGFPflex/- IHCs showed a significant reduction in Cav 1.3 channel cluster sizes and currents, suggesting that the intronic construct interfered with gene translation or splicing. These pitfalls are likely to be a frequent problem of many genetically modified mice with complex or multiple gene-targeting constructs or fluorescent proteins. Great caution and appropriate controls are therefore required.- Published
- 2019
- Full Text
- View/download PDF
21. α2δ2 Controls the Function and Trans-Synaptic Coupling of Cav1.3 Channels in Mouse Inner Hair Cells and Is Essential for Normal Hearing.
- Author
-
Fell B, Eckrich S, Blum K, Eckrich T, Hecker D, Obermair GJ, Münkner S, Flockerzi V, Schick B, and Engel J
- Subjects
- Animals, Calcium metabolism, Calcium Channels genetics, Calcium Signaling physiology, Female, Ion Channel Gating physiology, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Calcium Channels metabolism, Calcium Channels, L-Type metabolism, Hair Cells, Auditory, Inner physiology, Hearing physiology, Synapses physiology, Synaptic Transmission physiology
- Abstract
The auxiliary subunit α
2 δ2 modulates the abundance and function of voltage-gated calcium channels. Here we show that α2 δ2 mRNA is expressed in neonatal and mature hair cells. A functional α2 δ2-null mouse, the ducky mouse (du), showed elevated auditory brainstem response click and frequency-dependent hearing thresholds. Otoacoustic emissions were not impaired pointing to normal outer hair cell function. Peak Ca2+ and Ba2+ currents of mature du/du inner hair cells (IHCs) were reduced by 30-40%, respectively, and gating properties, such as the voltage of half-maximum activation and voltage sensitivity, were altered, indicating that Cav 1.3 channels normally coassemble with α2 δ2 at IHC presynapses. The reduction of depolarization-evoked exocytosis in du/du IHCs reflected their reduced Ca2+ currents. Ca2+ - and voltage-dependent K+ (BK) currents and the expression of the pore-forming BKα protein were normal. Cav 1.3 and Cav β2 protein expression was unchanged in du/du IHCs, forming clusters at presynaptic ribbons. However, the close apposition of presynaptic Cav 1.3 clusters with postsynaptic glutamate receptor GluA4 and PSD-95 clusters was significantly impaired in du/du mice. This implies that, in addition to controlling the expression and gating properties of Cav 1.3 channels, the largely extracellularly localized α2 δ2 subunit moreover plays a so far unknown role in mediating trans-synaptic alignment of presynaptic Ca2+ channels and postsynaptic AMPA receptors., Significance Statement: Inner hair cells possess calcium channels that are essential for transmitting sound information into synaptic transmitter release. Voltage-gated calcium channels can coassemble with auxiliary subunit α2 δ isoforms 1-4. We found that hair cells of the mouse express the auxiliary subunit α2 δ2, which is needed for normal hearing thresholds. Using a mouse model with a mutant, nonfunctional α2 δ2 protein, we showed that the α2 δ2 protein is necessary for normal calcium currents and exocytosis in inner hair cells. Unexpectedly, the α2 δ2 protein is moreover required for the optimal spatial alignment of presynaptic calcium channels and postsynaptic glutamate receptor proteins across the synaptic cleft. This suggests that α2 δ2 plays a novel role in organizing the synapse., (Copyright © 2016 the authors 0270-6474/16/3611024-13$15.00/0.)- Published
- 2016
- Full Text
- View/download PDF
22. Synaptotagmin IV determines the linear Ca2+ dependence of vesicle fusion at auditory ribbon synapses.
- Author
-
Johnson SL, Franz C, Kuhn S, Furness DN, Rüttiger L, Münkner S, Rivolta MN, Seward EP, Herschman HR, Engel J, Knipper M, and Marcotti W
- Subjects
- Age Factors, Animals, Animals, Newborn, Biophysics, Cattle, Cells, Cultured, Chromaffin Cells, Cochlea cytology, Cochlea metabolism, Electric Stimulation methods, Exocytosis genetics, Exocytosis physiology, Gene Expression Regulation, Developmental physiology, Gerbillinae, Green Fluorescent Proteins genetics, Linear Models, Membrane Potentials genetics, Mice, Mice, Knockout, Microscopy, Electron, Transmission methods, Patch-Clamp Techniques, Rats, Rats, Wistar, Synapses ultrastructure, Synaptic Vesicles ultrastructure, Synaptotagmin I genetics, Synaptotagmin I metabolism, Synaptotagmin II genetics, Synaptotagmin II metabolism, Synaptotagmins deficiency, Time Factors, Transfection methods, Calcium metabolism, Hair Cells, Auditory cytology, Synapses physiology, Synaptic Vesicles physiology, Synaptotagmins physiology
- Abstract
Mammalian cochlear inner hair cells (IHCs) are specialized for the dynamic coding of continuous and finely graded sound signals. This ability is largely conferred by the linear Ca(2+) dependence of neurotransmitter release at their synapses, which is also a feature of visual and olfactory systems. The prevailing hypothesis is that linearity in IHCs occurs through a developmental change in the Ca(2+) sensitivity of synaptic vesicle fusion from the nonlinear (high order) Ca(2+) dependence of immature spiking cells. However, the nature of the Ca(2+) sensor(s) of vesicle fusion at hair cell synapses is unknown. We found that synaptotagmin IV was essential for establishing the linear exocytotic Ca(2+) dependence in adult rodent IHCs and immature outer hair cells. Moreover, the expression of the hitherto undetected synaptotagmins I and II correlated with a high-order Ca(2+) dependence in IHCs. We propose that the differential expression of synaptotagmins determines the characteristic Ca(2+) sensitivity of vesicle fusion at hair cell synapses.
- Published
- 2010
- Full Text
- View/download PDF
23. Persistence of Ca(v)1.3 Ca2+ channels in mature outer hair cells supports outer hair cell afferent signaling.
- Author
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Knirsch M, Brandt N, Braig C, Kuhn S, Hirt B, Münkner S, Knipper M, and Engel J
- Subjects
- 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester pharmacology, Age Factors, Analysis of Variance, Animals, Animals, Newborn, Barium pharmacology, Calcium Channel Agonists pharmacology, Calcium Channels, L-Type genetics, Chlorides metabolism, Dose-Response Relationship, Radiation, Electric Stimulation methods, Hair Cells, Auditory, Inner drug effects, Hair Cells, Auditory, Inner physiology, Hair Cells, Auditory, Inner radiation effects, Hair Cells, Auditory, Outer growth & development, In Situ Hybridization methods, Membrane Potentials drug effects, Membrane Potentials physiology, Mice, Patch-Clamp Techniques methods, Rats, Calcium Channels, L-Type metabolism, Gene Expression Regulation, Developmental physiology, Hair Cells, Auditory, Outer metabolism, Signal Transduction physiology
- Abstract
Outer hair cells (OHCs) are innervated by type II afferent fibers of as yet unknown function. It is still a matter of debate whether OHCs perform exocytosis. If so, they would require presynaptic Ca2+ channels at their basal poles where the type II fibers make contacts. Here we show that L-type Ca2+ channel currents (charge carrier, 10 mM Ba2+) present in neonatal OHCs [postnatal day 1 (P1) to P7] decreased from approximately 170 to approximately 50 pA at approximately the onset of hearing. Ba2+ currents could hardly be measured in mature mouse OHCs because of their high fragility, whereas in the rat, the average Ba2+ current amplitude of apical OHCs was 58 +/- 9 pA (n = 20, P19-P30) compared with that of the inner hair cells (IHCs) of 181 +/- 50 pA (n = 24, P17-P30). Properties of Ba2+ currents of mature OHCs resembled those of neonatal OHCs. One exception was the voltage dependence of activation that shifted between birth and P12 by +9 mV toward positive voltages in OHCs, whereas it remained constant in the IHCs. Ca(v)1.3-specific mRNA was detected in mature OHCs using cell-specific reverse transcription (RT)-PCR and in situ hybridization. Ca(v)1.3 protein was stained exclusively at the base of mature OHCs, in colocalization with the ribbon synapse protein CtBP2 (C-terminal binding protein 2)/RIBEYE. When current sizes were normalized to the estimated number of afferent fibers or presynaptic ribbons, comparable values for IHCs and OHCs were obtained, a finding that together with the colocalization of Ca(v)1.3 and CtBP2/RIBEYE protein strongly suggests a role for Ca(v)1.3 channels in exocytosis of mature OHCs.
- Published
- 2007
- Full Text
- View/download PDF
24. Thyroid hormone deficiency affects postnatal spiking activity and expression of Ca2+ and K+ channels in rodent inner hair cells.
- Author
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Brandt N, Kuhn S, Münkner S, Braig C, Winter H, Blin N, Vonthein R, Knipper M, and Engel J
- Subjects
- Animals, Animals, Newborn, Calcium Channels genetics, Female, Gene Expression Regulation physiology, Hair Cells, Auditory, Inner growth & development, Hypothyroidism genetics, Mice, Mice, Transgenic, Potassium Channels genetics, Pregnancy, Rats, Rats, Wistar, Action Potentials physiology, Calcium Channels biosynthesis, Hair Cells, Auditory, Inner metabolism, Hypothyroidism metabolism, Potassium Channels biosynthesis
- Abstract
Thyroid hormone (TH) is essential for the development of hearing. Lack of TH in a critical developmental period from embryonic day 17 to postnatal day 12 (P12) in rats and mice leads to morphological and functional deficits in the organ of Corti and the auditory pathway. We investigated the effects of TH on inner hair cells (IHCs) using patch-clamp recordings, capacitance measurements, and immunocytochemistry in hypothyroid rats and athyroid Pax8-/- mice. Spontaneous and evoked Ca2+ action potentials (APs) were present in control IHCs from P3-P11 rats and vanished in parallel with the expression of a rapidly activating Ca2+- and voltage-activated K+ (BK) conductance. IHCs of hypothyroid rats and athyroid Pax8-/- mice displayed APs until the end of the third postnatal week because of threefold elevated Ca2+ currents and missing expression of BK currents. After the fourth postnatal week, some IHCs showed BK currents whereas adjacent IHCs did not, demonstrated by electrophysiology and immunocytochemistry. To test whether the prolonged spiking activity during TH deficiency may be transmitted at IHC synapses, capacitance measurements were performed in parallel to analysis of otoferlin expression, a protein thought to play an essential role in exocytosis of IHCs. Strikingly, otoferlin was absent from IHCs of hypothyroid rats but not of Pax8-/- mice, although both cell types showed exocytosis with an efficiency typical for immature IHCs. These results demonstrate for the first time a TH-dependent control of IHC spiking activity before the onset of hearing attributable to effects of TH on Ca2+ and BK channels. Moreover, they question an indispensable role of otoferlin for exocytosis in IHCs.
- Published
- 2007
- Full Text
- View/download PDF
25. Deletion of the Ca2+-activated potassium (BK) alpha-subunit but not the BKbeta1-subunit leads to progressive hearing loss.
- Author
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Rüttiger L, Sausbier M, Zimmermann U, Winter H, Braig C, Engel J, Knirsch M, Arntz C, Langer P, Hirt B, Müller M, Köpschall I, Pfister M, Münkner S, Rohbock K, Pfaff I, Rüsch A, Ruth P, and Knipper M
- Subjects
- Animals, Calcium metabolism, Cochlea metabolism, Gene Deletion, Hair Cells, Auditory, Outer abnormalities, Hearing Loss metabolism, Immunohistochemistry, Large-Conductance Calcium-Activated Potassium Channel alpha Subunits, Mice, Phenotype, Potassium Channels metabolism, Hearing Loss genetics, Potassium Channels genetics
- Abstract
The large conductance voltage- and Ca2+-activated potassium (BK) channel has been suggested to play an important role in the signal transduction process of cochlear inner hair cells. BK channels have been shown to be composed of the pore-forming alpha-subunit coexpressed with the auxiliary beta1-subunit. Analyzing the hearing function and cochlear phenotype of BK channel alpha-(BKalpha-/-) and beta1-subunit (BKbeta1-/-) knockout mice, we demonstrate normal hearing function and cochlear structure of BKbeta1-/- mice. During the first 4 postnatal weeks also, BKalpha-/- mice most surprisingly did not show any obvious hearing deficits. High-frequency hearing loss developed in BKalpha-/- mice only from approximately 8 weeks postnatally onward and was accompanied by a lack of distortion product otoacoustic emissions, suggesting outer hair cell (OHC) dysfunction. Hearing loss was linked to a loss of the KCNQ4 potassium channel in membranes of OHCs in the basal and midbasal cochlear turn, preceding hair cell degeneration and leading to a similar phenotype as elicited by pharmacologic blockade of KCNQ4 channels. Although the actual link between BK gene deletion, loss of KCNQ4 in OHCs, and OHC degeneration requires further investigation, data already suggest human BK-coding slo1 gene mutation as a susceptibility factor for progressive deafness, similar to KCNQ4 potassium channel mutations., (Copyright 2004 The National Academy of Sciencs of the USA)
- Published
- 2004
- Full Text
- View/download PDF
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