HCN channels in the mammalian cochlea: Expression pattern, subcellular location, and age‐dependent changes

Luque, María Custodia Sánchez; Schrott‐Fischer, Anneliese; Dudás, József; Pechriggl, Elisabeth; Brenner, Erich; Rask‐Andersen, Helge; Liu, Wei; Glueckert, Rudolf

doi:10.1002/jnr.24754

Cited by 9 publications

(10 citation statements)

References 82 publications

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“…There is also evidence for the expression of HCN channels in cells outside of the nervous system and heart, such as the kidneys [ 317 ], pancreas [ 318 ], and bladder [ 319 ]. All HCN isoforms, except HCN3, are present in the retina [ 320 ]. HCN channels are localized mainly in the plasma membrane, although they have also been detected in the mitochondria of rat kidneys, human HEK 293 cells [ 321 ], and human cardiac tissue [ 321 ].…”

Section: Pharmacology Of Mitochondrial Hcn Channelsmentioning

confidence: 99%

Alternative Targets for Modulators of Mitochondrial Potassium Channels

et al. 2022

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Mitochondrial potassium channels control potassium influx into the mitochondrial matrix and thus regulate mitochondrial membrane potential, volume, respiration, and synthesis of reactive oxygen species (ROS). It has been found that pharmacological activation of mitochondrial potassium channels during ischemia/reperfusion (I/R) injury activates cytoprotective mechanisms resulting in increased cell survival. In cancer cells, the inhibition of these channels leads to increased cell death. Therefore, mitochondrial potassium channels are intriguing targets for the development of new pharmacological strategies. In most cases, however, the substances that modulate the mitochondrial potassium channels have a few alternative targets in the cell. This may result in unexpected or unwanted effects induced by these compounds. In our review, we briefly present the various classes of mitochondrial potassium (mitoK) channels and describe the chemical compounds that modulate their activity. We also describe examples of the multidirectional activity of the activators and inhibitors of mitochondrial potassium channels.

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Section: Pharmacology Of Mitochondrial Hcn Channelsmentioning

confidence: 99%

Alternative Targets for Modulators of Mitochondrial Potassium Channels

et al. 2022

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“…Human cell bodies could act as Ranvier nodes, and the electric coupling could synchronize APs, similar to the firing in Purkinje neurons ( Han et al, 2018 ). NKA α1 and β1 as well as HCN channels 1, 2, and 4 are richly expressed at adjoining cell membranes in the apical cochlea ( Liu et al, 2019 ; Luque et al, 2021 ). HCN channels may generate “pacemaker currents” in heart muscle cells and could act to boost coupling and synchronize firing, similar to that described in the brain.…”

Section: Discussionmentioning

confidence: 99%

Na/K-ATPase Gene Expression in the Human Cochlea: A Study Using mRNA in situ Hybridization and Super-Resolution Structured Illumination Microscopy

Liu

Rask‐Andersen

2022

Front. Mol. Neurosci.

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BackgroundThe pervasive Na/K-ATPase pump is highly expressed in the human cochlea and is involved in the generation of the endocochlear potential as well as auditory nerve signaling and relay. Its distribution, molecular organization and gene regulation are essential to establish to better understand inner ear function and disease. Here, we analyzed the expression and distribution of the ATP1A1, ATP1B1, and ATP1A3 gene transcripts encoding the Na/K-ATPase α1, α3, and β1 isoforms in different domains of the human cochlea using RNA in situ hybridization.Materials and MethodsArchival paraformaldehyde-fixed sections derived from surgically obtained human cochleae were used to label single mRNA gene transcripts using the highly sensitive multiplex RNAscope® technique. Localization of gene transcripts was performed by super-resolution structured illumination microscopy (SR-SIM) using fluorescent-tagged probes. GJB6 encoding of the protein connexin30 served as an additional control.ResultsSingle mRNA gene transcripts were seen as brightly stained puncta. Positive and negative controls verified the specificity of the labeling. ATP1A1 and ATP1B1 gene transcripts were demonstrated in the organ of Corti, including the hair and supporting cells. In the stria vascularis, these transcripts were solely expressed in the marginal cells. A large number of ATP1B1 gene transcripts were found in the spiral ganglion cell soma, outer sulcus, root cells, and type II fibrocytes. The ATP1B1 and ATP1A3 gene transcripts were rarely detected in axons.DiscussionSurgically obtained inner ear tissue can be used to identify single mRNA gene transcripts using high-resolution fluorescence microscopy after prompt formaldehyde fixation and chelate decalcification. A large number of Na/K-ATPase gene transcripts were localized in selected areas of the cochlear wall epithelium, fibrocyte networks, and spiral ganglion, confirming the enzyme’s essential role for human cochlear function.

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“…The antibodies used in these protocols were previously described, and positive and (Luque et al, 2021).…”

Section: Positive and Negative Controlsmentioning

confidence: 99%

“…Conservation of the 3D protein structure and especially the antibody epitopes in the cochlear tissue is important to obtain better results in immunostaining. We used young adult mice aged 21 days here because older mice presented lower staining intensities with these antibodies (Luque et al, 2021). 2.…”

Section: Tissue Preparation Cryoembedding and Sectioningmentioning

confidence: 99%

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Sequential Indirect Dual Immunohistochemistry with Primary Rabbit Antibodies on Cochlear Sections Using an Intermediate Heat‐Denaturation Step

Luque

Glueckert

2021

Current Protocols

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Advanced immunohistochemical (IHC) protocols aim to visualize different molecules in situ simultaneously. These techniques are of utmost importance as a first step in studying possible interactions of proteins at the subcellular level. Colocalized stains in tissue sections indicate proximity of two proteins of interest. Frequently, double staining protocols are restricted by the lack of primary antibodies generated in different animal species for indirect IHC visualization. Here, we present a detailed protocol for mouse inner ear tissue using two different primary rabbit antibodies directed against transmembrane ion channel proteins of cochlear neurons. The two antibodies are combined for fluorescence (confocal) as well as dual multiplex colorimetric visualization in two sequential single IHC stainings. A heat-denaturation step is performed in between. Primary antibody specificity is tested by preadsorption with the immunogenic peptide, and positive and negative tissue controls are performed to confirm the reliability of the antibody detection. We describe the whole procedure in detail beginning with tissue extraction of the mouse inner ear and continuing with chemical fixation, cryoembedding, and preparation for manual and fully automated immunostaining, including steps for heat-induced antigen retrieval. The potential to use antibodies from the same host species for single and double IHC staining opens up multiple possibilities for detecting different targets in the same tissue section using resources and materials that are widely available.

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HCN channels in the mammalian cochlea: Expression pattern, subcellular location, and age‐dependent changes

Abstract: This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Cited by 9 publications

References 82 publications

Alternative Targets for Modulators of Mitochondrial Potassium Channels

Alternative Targets for Modulators of Mitochondrial Potassium Channels

Na/K-ATPase Gene Expression in the Human Cochlea: A Study Using mRNA in situ Hybridization and Super-Resolution Structured Illumination Microscopy

Sequential Indirect Dual Immunohistochemistry with Primary Rabbit Antibodies on Cochlear Sections Using an Intermediate Heat‐Denaturation Step

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