22/05/2019 Neurobiology of Hearing Salamanca, 22nd May 2019 3. Cochlear amplification: outer hair cells Jonathan Ashmore Neuroscience, Physiology and Pharmacology University College London [email protected] 1 Mechano-electrical transduction in hair cells • depends on a flow of K+ into the cells • adapts on a time scale of milliseconds • depends on mechanical coupling no 2nd messengers direct • depends on a fast channel opening (<1µs) • depends on an unknown channel? 2 1 22/05/2019 The mammalian cochlea is a mechanical spectrum analyser A Stape Basilar membrane s Fluid Fluid in motion at Round rest window B IHC 3 rows OHCs BM stiffness high low 3 y/x relative x amplitude y spring & mass frequency 4 2 22/05/2019 y/x relative x amplitude y + viscous damping frequency 5 We can use this idea to make an in-silico model of the cochlea See: http://147.162.36.50/cochlea/cochleapages/theory/index.htm 6 3 22/05/2019 Problem: cochlear bandwidths are narrow.. Solution: a ‘cochlear amplifier’ to counteract viscosity effects (Gold 1948, Davis 1983) responsible for sound amplification (100 x) responsible for frequency selectivity ‘linked’ to otoacoustic emissions Use the outer hair cells to compensate for fluid viscosity 7 the amplifier 8 4 22/05/2019 Kv7.4 / KCNQ4 – one of the main K channels in OHCs Mutations in the gene define a deafness locus DFNA2 9 Inside +ve Inside -ve Outer hair cells have an additional property: they change length 10 5 22/05/2019 Two ways to make a cylindrical cell change length solute influx PI PO Area constant water efflux Volume constant Vo Vm (The cochlear motor) 11 Outer hair cells in situ distort the cochlear partition Extracellular current 12 6 22/05/2019 The ‘motor’ occupies most of the OHC lateral membrane 100x magnification: 8nm diameter particles B Kachar - NIDCD 13 The motor is local and has an electrical ‘fingerprint’ 14 7 22/05/2019 The outer hair cell is an ultrafast motor / actuator an area motor high copy number (>107 /cell) fast cycle time if driven ( > 50 kHz) operation associated with a gating charge 15 The molecular motor, prestin, is a membrane transporter Homology model: Gorbunov et al., 2014 Genomic identity: SLC26A5 - a low efficiency Cl-HC03 antiporter? (SLC26 is a superfamily of anion-bicarbonate exchangers) Originally reported by Zheng et al, 2000 16 8 22/05/2019 Prestin transfected HEK cells Zheng et al,. 2000 17 Prestin KO mouse does not have amplification WT prestin KO • = post-mortem Liberman et al., Nature 2002 Mellado Lagarde et al., Curr Biol 2008 18 9 22/05/2019 2015: crystal structure: of a bacterial SLC26 (from a thermophilic bacterium, Deinococcus geothermalis) Geertsma, et al., Nat. Struct. Mol. Biol. 2015 From Reithmeier & Moreas with permission 19 Mammalian prestin (SLC26A5) is a tetramer = dimer of dimers outside nanometres membrane cytoplasm Mio et al, J Cell Biol. 2008 20 10 22/05/2019 The SLC26 superfamily and relatives in Drosophila & C. elegans They are bicarbonate exchangers (Red blood cells have bicarbonate exchangers in the SLC2 family) Mount and Romero Eur J Physiol (2004) 21 A simple model for prestin action extended =0exp(-bV) fast 1 μs compact - - = intracellular anion (Cl or HCO3 ) ‘Molecular crowding’ leads to macroscopic effects. 22 11 22/05/2019 Out In slow expanded state =0exp (-bV) fast compact state conventional 4 state carrier model 23 A nagging problem Ikeda et al, 1991: OHCs regulate their intracellular pH Prestin is a member of the SLC26A anion-bicarbonate family of transporters 24 12 22/05/2019 - - Cl controls HCO3 loading in prestin expression systems prestin pHluorin membrane peptide - pHluorin pH pH pH ? - - - CO2 HCO3 HCO3 Cl 25 HCO3 is transported by prestin under Cl control Cl = 6mM Cl =140 mM Mistrik et al, 2012 26 13 22/05/2019 Out In slow expanded state =0exp (-bV) fast compact state = aspirin conventional 4 state carrier model -1 low turnover rate ~900 s (=0.1% of other Cl-HCO3 transporters) 27 Conclusions 1) The mammalian OHC is an ultrafast actuator 2) The ‘motor’ molecule is also low efficiency Cl -HCO3 exchanger 3) The ‘motor’ arises from part of a transport cycle and creates a ‘cochlear amplifier’ Unresolved: how is it inserted and (?) is there turnover ? what is the mechano-enzyme structure of prestin? how is it coupled into the cell cytoskeleton? 28 14.