Journal: Cell reports
Article Title: Optimized Tuning of Auditory Inner Hair Cells to Encode Complex Sound through Synergistic Activity of Six Independent K + Current Entities.
doi: 10.1016/j.celrep.2020.107869
Figure Lengend Snippet: Figure 4. 4-AP Potentiates K+ Currents at Negative Membrane Potentials in IHCs (A and B) Application of 4-AP (5 mM) induced a hyperpolarization of the membrane potential (A) in control IHC and (B) in IHCs treated with TEA, XE991, and E-4031. (C) Representative recordings of IHC currents elicited with the indicated voltage steps before and after 1 min of extracellular 4-AP (after TEA, XE991, and E-4031). 4-AP inhibited currents at +30 mV (indicated by asterisk), but potentiated currents at 60 mV (indicated by open circle). For the time course of 4-AP effects, see Figure S5A. (D–F) 4-AP induced a hyperpolarizing shift in the voltage dependence of currents (after TEA, XE991, and E-4031). (D) Boltzmann fits to averaged recordings before (black) and after (red) application of 4-AP in IHCs. (E and F) Mean Vh (E) and slope factors (F) of activation derived from analysis shown in (D). For absolute current amplitudes of recordings shown in (D), see Figure S5B. (G) RT-PCR detected mRNA for Kv12.1, Kv12.2, and Kv12.3 channels in cochlear lysates (M, nucleotide marker with size; positive controls: Kv7.4 and GAPDH). **p % 0.01; ***p % 0.001. Concentrations of applied substances: 4-AP (5 mM), TEA (5 mM), XE991 (10 mM), and E-4031 (20 mM). Sample sizes (n) are given in (A)–(F). Data are presented as means ± SEMs.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit polyclonal anti-Kv11.1 Alomone Labs Cat #: APC-016 Rabbit polyclonal anti-Kv11.2 Alomone Labs Cat #: APC-114 Rabbit polyclonal anti-Kv11.3 Enzo Life Sciences Cat #: ALX-215-053-R100 Rabbit polyclonal anti-Kv12.1 Alomone Labs Cat #: APC-113 Mouse monoclonal anti-MyosinVIIa Santa Cruz Biotechnology Cat #: sc-74516 Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 Thermo Fischer Cat #: A11008 Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 633 Thermo Fischer Cat #: A21050 Chemicals, Peptides, and Recombinant Proteins Tetraethylammonium chloride Tocris Cat #: 3068 Iberiotoxin Tocris Cat #: 1086 XE991 dihydrochloride Tocris Cat #: 2000 E-4031 dihydrochloride Tocris Cat #: 1808 4-AP Tocris Cat #: 0940 NS1643 Tocris Cat #: 3062 Experimental Models: Cell Lines Chinese hamster ovary (CHO) dhFR- cells ATCC CRL-9096 Experimental Models: Organisms/Strains C57BL/6NCrl mice Charles River Strain Code 027 C57Bl6/NJ mice The Jackson Laboratory Stock No: 005304 Kcnh2tm1a(EUCOMM)Wtsi KO first allele ES cells European Conditional Mouse Mutagenesis Program (EUCOMM) MGI ID MGI:1341722 Prestin-Cre (BAC transgenic) mice Tian et al., 2004 N/A Oligonucleotides Primers for RT-PCR, see Table S1 (last page of supplementary pdf) This Paper N/A Recombinant DNA Kv1.8-pRFP-N1 This paper Uniprot: B2RQA1 Kv11.1a-pcDNA3.1 Dr. CK Bauer Uniprot: O08962 Kv11.2-pcDNA3.1 Dr. CK Bauer Uniprot: O54853 Kv11.3-pcDNA3.1 Dr. CK Bauer Uniprot: O54852 Kv12.1-pcDNA3.1-IRESeGFP Dr. T Jegla Uniprot: Q96L42 Lyn11-GFP-pcDNA3.1 Dr. D. Oliver Uniprot: P07948 Software and Algorithms PatchMaster (HEKA) HEKA Elektronik https://www.heka.com/downloads/ IgorPro Wavemetrics https://www.wavemetrics.com/downloads/ current Other Inner hair cell biophysical model codes This Paper https://www.mechanicsofhearing.org/apg/ DAPI Sigma-Aldrich Cat #: D9542 e1 Cell Reports 32, 107869, July 7, 2020
Techniques: Membrane, Control, Activation Assay, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Marker
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