Journal of Physiology Fuchs tonotopic the distribution underlie of resonant properties channels (Art & Fettiplace, 1987; BK the in sensitivity differences calcium and kinetic as well as both types of channel magnitude the in variations Tonotopic 1987). channels (Art (BK) potassium calcium-activated and channels calcium between interaction the by driven is resonance Electrical Sokolowski, 1990; Art Fuchs 1983; Hudspeth, & Lewis 1983; lower vertebrates (Crawford & Fettiplace, 1978; Ashmore, in cells hair auditory of mechanism tuning primary the is frequency, particular cell’s a at oscillate to potential membrane hair the of ability the resonance, Electrical 1990). (Roberts release neurotransmitter and excitability auditory sensory hair cells, regulating both the membrane Calcium channels are fundamental to signal processing in Neuroscience Center and Kresge Hearing Laboratories, Louisiana State University Health Sciences Center, New Orleans, LA 70112, M. E. Schnee and A. J. Ricci gated calcium currents in turtle auditory hair cells Biophysical and pharmacological characterization of voltage- J Physiol © The Physiological Society 2003 and is one of the questions addressed by this work. the hair cell calcium channel occur tonotopically is unknown, of properties steady-state or kinetics in variations similar (2003), t al. et 549.3 98 Hdpt & ei, 1988 Lewis, & Hudspeth 1988; eoac. bl-hpd otg dpnec ad oet estvte t itaellr calcium intracellular to chelators and sensitivities external barium modest ions suggest that and inactivation the was dependence calcium dependent. characterizing voltage to bell-shaped prior A depolarized resonance. or hyperpolarized was potential membrane the channel where Calcium cells. low-frequency for mV 2 ± _46 inactivation and did not cells significantly high-frequency alter hair for cell mV electrical resonant properties 2 elicited from protocols ± _40 was half-inactivation of voltage The inactivation. complete in result could depolarizations Long high- and low-frequency cells. The time course of inactivation 1mV. required Inactivation three was time observed constants ± in 1mV for both compared a to fit. _35 ± half-activating voltages of _43 with potentials, hyperpolarized at and faster slightly activated cells low-frequency cells, frequency no pharmacological differences were found between calcium currents obtained from high- and low- relative insensitivity to nimodipine suggest the channels were of the ms),hyperpolarized half-activationpotentialsanda 0.5 were present.Fastactivationrisetimes(< 8644), K Bay (nimodipine, non-L-type calcium channel antagonists support the conclusion that only L-type calcium channels dihydropyridines to benzothiazepines (diltiazem) and sensitivity acetonitrile derivatives (verapamil, D600) and the insensitivity to Pharmacological properties. pharmacological and low- inactivation activation, and on based Hz) characterized were 27 currents calcium ± and (317 technique, recording whole-cell the using clamped voltage high- were positions Hz) from 6 cells ± hair (115 frequency auditory turtle end, this To function. different different characteristic frequencies or if multiple channel types exist within a hair cell, each serving a present investigations focused on determining whether calcium channels vary between hair cells of Hair cellcalciumchannelsregulatemembraneexcitabilityandcontrolsynaptictransmission.The Health Sciences Center, New Orleans, LA 70112, USA. Corresponding author (Received 12 December 2002; accepted after revision 4 April 2003; first published online 9 May 2003) et al. , pp. 697–717 1986; Hudspeth, 1986; Art & Fettiplace, et al. 1995; Wu A. J. Ricci: Neuroscience Center and Kresge Hearing Laboratories, 2020 Gravier Street Suite D, LSU et al. 1995). Whether t al. et b Fcs & Fuchs ; 1988). t al. et Email: [email protected] omn rgo ad r mnaoy o channel main for two have mandatory channels calcium are L-type functioning. and region forming sometimes hnes r mliei, containing multimeric, are channels (Tsien inactivation slow show and at dihydropyridines to activate sensitive are potentials, typically depolarized channels calcium L-type review). for 2001 biophysically, Hille, (see classified molecularly and been pharmacologically have channels Calcium electrical resonance remains to be elucidated. neurotransmitter release are different from those linked to Wu 1988; (Sneary, frequency characteristic release sites, but not the density of channels, increases with 1995). The number of calcium channels and the number of al. are (Roberts sites release channels synaptic at presumably clustered, Calcium channels. calcium through cells hair entering calcium by driven is transmission Synaptic Ricci 90 Is & useh 19; ukr Fettiplace, & Tucker 1994; Hudspeth, & Issa 1990; t al. et g 00. hte clim hnes ikd to linked channels calcium Whether 2000). subunits. The a 1D (CaV 1.3 a ) variety. Although subunits make up the pore- et al. et DOI: 10.1113/jphysiol.2002.037481 a 1988). Calcium 1988). , b USA t al. et , www.jphysiol.org a 2 d 1996; and et Journal of Physiology uioy ail (Kollmar papilla auditory The channel electrical properties (Yang 1999). Inaddition,theseaccessoryproteinscanmodulate (Yang transmission synaptic of forms some regulate been linked to synaptic release proteins and an are thought to and (Koschak antagonists times dihydropyridine L-type the rise to insensitivity activation (submillisecond) fast curve, activation hyperpolarized a including properties The cells. epithelial the while heart, a the was channel L-type characterized and identified first expressed heterologously the from different somewhat Koschak 2000; (Zhang cochlea mammalian saccule and trout 2001), Yamoah, & (Rodriguez-Contreras subtypes based on based subtypes 698 et al. et papilla (Fuchs turtle papilla (Art been 1988 have Lewis, & (Hudspeth saccule channels L-type cells. identified in a variety of hair cell organs including the hair been frog have in channel calcium identified of types different Several those reported for expressed to comparethepropertiesofhaircellcalciumchannels was work present the of purpose Another inactivation. to nxetd oe fnig a te dniiain of identification the was calcium-dependent inactivation. finding novel the unexpected of are channels calcium the that hypothesis the support sensitivities pharmacological cells. and of hair properties properties Activation described. auditory also activation are channels L-type the turtle in in differences Tonotopic present are channels The data presented here will demonstrate that only L-type signal of also a focus of the aspects present work. different is and unknown, is for cells hair auditory turtle in processing responsible are types al. (Martini cells hair canal semicircular frog in identified been have currents R-type 2001). Yamoah, & Contreras (Su channels L-type only have to thought traditionally frog end-organ an in cells, hair recently saccule identified been have channels N-type 1999) and the frog semicircular canal (Prigioni 1990; Nakagawa 1991; Martini been described in vestibular hair cells (Rennie & Ashmore, Martini 1C type, which is found largely in skeletal muscle and muscle skeletal in largely found is which type, 1C 2000; Rispoli 2000; a 2001), guinea-pig cochlear hair cells (Bobbin cells hair cochlear guinea-pig 2001), D hne tp hs en dniid n h chick the in identified been has type channel 1D et al. t al. et 00.N,R and T-type channels have also 2000). N-, R- et al. et al. et al. et a et al. 01. eety the Recently 2001). et al. et 1D is found in neuronal cells and some and cells neuronal in found is 1D et al. a 2000; Rispoli 1990; Zidanic & Fuchs, 1995; Spassova 2001). Hair cell calcium channels are channels calcium cell Hair 2001). a subunits, the subunits, 1991; Oshima D hnes ae eea unusual several have channels 1D 1986; Art & Fettiplace, 1987), chick 2000). Whether different channel different Whether 2000). in vitro t al. et a 1D channels. , in particular with regard et al. a t al. et et al. et a D hnes ht are that channels 1D 1997 a et al. ; Roberts ; et al. 1C and the and 1C 2000). In particular, a 1999; Platzer 1999; D hnes have channels 1D a 1999). 95 Rodriguez- 1995; a , 1996; Zhang b D ait. An variety. 1D , rg saccule frog ), et al. et et al. M. E. Schnee and A. J. Ricci a 1D. The 1D. 1990), 1992; et al. et et al. et et al. et et al. et hme a efsda aeo .– lmin ml 0.5–1 of rate a at perfused was chamber recording The floss. dental of strands single three with place in a into placed and recording chamber with a trimmed coverslip at its of base. was The tissue was rinses papilla held multiple The solution. with external out washed was enzyme membrane the tectorial and removed The potency. enzyme on depending min, added tothesolutionandtissuewasincubatedfor5–20 xenl ebae a rmvd epsn te tectorial the ml exposing mg 0.02–0.04 removed, (Sigma) XXIV type Protease The membrane. was upward. facing membrane papilla auditory external the with pins minutien The tissue was pinned to the bottom of a Sylgard-coated dish with a hyperpolarized state, thus lengthening the viability of the tissue. potassium solution was used to maintain the cells in the papilla in ouin upeetd ih 0 n 100 with supplemented solution (Goodman & Art, 1996). conductance delayed-rectifier any block to included was (4-AP) CaCl eodns rm o-rqec cls 100 In cells, perfusion. low-frequency bath from recordings the for used was (Gilson) pump peristaltic 1 Fig. see example (for potassium current calcium-activated SK caesium-permeable the block H76adhdafnlomllt f25mso kg mosmol 275 of osmolality final a had and 7.6 pH to buffered was solution The Hepes. 10 and glucose 6 ascorbate, 25 % during the recording were also excluded. Cell capacitance Cell excluded. also were recording the during % 25 of loss a amplifier and circuit. Cells where series the resistance currents varied with by more performed than was the subtraction leak of Minimal inactivation. run-down produce were to pA tended 50 than greater currents excluded as they provided an uncontrolled leak source of calcium that with Cells resistance. series residual any was as off-line, corrected and measured was otg-lm peso 60 ≤ of speeds voltage-clamp M 5 pF to ( 0.2 1 compensation. ± Cell capacitance from was 11.6 ranged resistances Series the unmasking thus calcium conductance, current (Art potassium BK the block to was reduced to maintain a constant osmolality. Caesium was used al. (Ricci caesium by replaced was potassium experiments, clamp current- For 7.2. was pH the ascorbate; 2 and Hepes 10 BAPTA, uioy aile ee rprd s ecie previously described ( 1998). as sliders 1997, Red-eared Fettiplace, & prepared Ricci 1985; Fettiplace, & were (Crawford papillae Auditory Tissue preparation METHODS sd o cretcap esrmns Te nenl solution internal (m The contained measurements. current-clamp for used was EPC8 The recordings. all for used was Instruments) (Axon 1D Axopatch an or (Heka) EPC8 An 1997). Fettiplace, & (Ricci previously described been has as obtained were recordings cell recorded was carefully cleared 1 before the cell was patched. Whole- (Fig. access good ensure the of end back the pipette, makingaholefromwhich1–3cellscouldberemovedto the to from pressure applying papilla while the edge into abneural advanced was pipette blunt large A Recording procedures into external solution containing (m containing solution external into standards by and placed were organs ear Center inner The guidelines. NIH by established Sciences Health LSU Use Care at Animal Committee the by approved procedures using removed organs ear inner the and decapitated were inches) (3–5 cm 8–13 2000). Where higher BAPTA concentrations were used, CsCl used, were concentrations BAPTA higher Where 2000). 2 22 MgCl 2.2 , M : 1 CC, MAP 5 raie hsht, 1 phosphate, creatine 5 MgATP, 3 CsCl, 110 ): 2 2 ah f yuae cetn, att and lactate creatine, pyruvate, of each 2 , rcey srpa elegans scripta Trachemys et al. 1993). A . h sae rud h cl being cell the around space The ). m B .Ajnto oeta f_ mV _4 of potential junction A s. ; Tucker & Fettiplace, 1996). A 1996). Fettiplace, & Tucker ; M M pmn Clice) to (Calbiochem) apamin ): 125 NaCl, 0.5 KCl, 2.8 KCl, 0.5 NaCl, 125 ): m V M 4-aminopyridine fe u t 7 % 70 to up after , aaae length carapace ), _1 J Physiol with external with n _1 75), giving = . The low- The . _1 549.3 was et Journal of Physiology nrae napiuefrtefrt1–5mn(i.1 (Fig. min 10–15 first the for amplitude currents in increased calcium configuration, whole-cell the reaching Upon °C. temperature of 19–22 a at performed were experiments All recording. the of end the at distance from the apical end where the lagena as is recorded located, cell, was the noted of location The observations). unpublished change in the voltage dependence of the current–voltage ( current–voltage the of dependence voltage the in change no h hi bnls eoe eodn (Assad recording before Crawford bundles hair whole-cell the onto the when were eliminated by spraying internal solution containing BAPTA recycling vesicle configuration was of obtained. Mechanoelectric transducer currents interruption the to due presumably recording, the throughout increase to tended J Physiol 549.3 et al. et tripling in size. or doubling often amplitude, in increased currents calcium configuration, whole-cell the of establishment ieatrrpuewsbs itdb igeepnnilfnto ihatm osato . . min 0.3 ± 4.3 of constant ( time a with function exponential single a by fitted best was rupture after time ah plcto o aai a 10n 100 at apamin of application Bath to 1996). accumulation Fettiplace, & calcium (Tucker conductance for potassium calcium-activated allowed SK caesium-permeant that the activate durations for mV _14 to depolarized and mV _84 at clamped rapid and reproducible solution exchange to cells. to exchange solution reproducible and rapid A Figure 1. Recording procedures and calcium current run-up 0% reduction in 10 peak current during this time. times after rupture of the patch. n , differentialinterferencecontrastimageoftherecordingelectrode andpapillaafterclearingthatensured 1991; Marquis & Hudspeth, 1997; and authors’ and 1997; Hudspeth, & Marquis 1991; = 10). After reaching a maximal current, recordings were stable for more than 1 h, showing a less than less a showing h, 1 than more for stable were recordings current, maximal a reaching After 10). = D , there was no shift in the plot of peak current against command potential ( Calcium currents in auditory hair cells C and M D t al. et are from the same cell. opeey lce te K urn i aot min. 4 about in current SK the blocked completely C – E ). No ). 1991; I – B V , to estimate drug delivery time, a hair cell was voltage was cell hair a time, delivery drug estimate to , ) dihydropyridine and barium application, so hand pipetting was pipetting hand so application, barium and dihydropyridine for pump the miniature using when precipitation with difficulty was There through coupled pump pipetted. hand were drugs else or Valves), (Lee switches solenoid peristaltic Gilson a using All drugs were bath applied. The application was either automated Drug application cells met these criteria. cells were % pA. required of More to the than reach 90 at least 250 low-frequency and current maximal pA 600 least at to run-up to uncontrolled variable intheseexperiments,high-frequencycellswererequired an was run-up Since break-through. after min about 15 stabilized, had current peak the until collected not were plots was observed during the run-up period. Due to run-up, data E , a plot of the normalized current against I – V ) for different C upon , 699 Journal of Physiology function of the form: the command potential. The curves were fitted with a Boltzmann remaining were plotted as peak current for a given by command voltage against contaminated this, of lieu be In currents. by transducer also mechanoelectric obscured could In tails were curves. the activation or addition, proper fast generate to too artefacts were capacitative currents tail Typically, with significance, statistically significant difference. statistical assess two-tailed Student’s otherwise, stated Unless where, ciain and activation rgn ss h LvnegMrurt loih fr fitting. for algorithm Levenberg-Marquardt as given are coefficients correlation appropriate, Where the uses Origin analysis. for (Microcal) Origin to exported and (CED) software Signal with collected were Data protocols. inactivation for traces illustrated are averages of four for activation protocols and single given with each set of data. Unless otherwise stated, current traces calcispetine, steady-state response. Calcium channel blockers a ensure to minutes several next the over made were recordings minbefore measuringaresponseand were appliedforatleast10 drugs general, In current. calcium the revealing thereby current, SK the of block complete for allowed min 4 average, On slowly. large molecular weight peptide (MW 2027) and so should diffuse a is Apamin recorded. was block SK until time and preparation 1 (Fig. applied was current potassium calcium-activated SK the activate to duration absent from the external solution. A depolarizing step of sufficient aspects of the hair cells, experiments were carried out with apamin basolateral the reach to drug a for took it time the determine To drug necessary time periods. any for application performed of the normal solution or turning the bath flow was off for bath application. Controlexperimentsshowednoeffectofeitherbath of 10-fold a substitution least At volume consistent. quite were results and observed isle i DS, ia cnetain big .1% No %. 0.01 < were being effects of concentrations DMSO drugs alone were final observed. necessary, DMSO, Where in dissolved Sigma. from cyclodextrin and nimodipine, v colchicine, D, 8644 Cytochalasin (mixed isomers), verapamil, K diltiazem, apamin, D600, Bay Sigma. or from purchased Fisher were Chemicals Laboratories. Alomone from l aaaepeetda en ± All data are presented as means Data analysis low-calcium a in first washed (50 was tissue the problems, these circumvent To tissue. the from released being was calcium if as In addition, exchange appeared to be very slow at the preparation, form. would precipitate a solution, external normal with contact in When difficulties. technical some included. gave also not exchange Barium were uncontrolled was precipitation of level the minbetweensolutionpreparationandapplication.Datawhere 5 than more no was there that so recorded were currents control and obtained been had recording quite whole-cell was the after nimodipine giving prepared problem, initially this circumvent preparation, To results. of variable min 30 within solution of out comes DMSO in dissolved Nimodipine out. carried was pipetting while off turned perfusion bath the and these for used 700 -conotoxin GVIA and DIDS were purchased from Calbiochem, m I M ) solution before barium was applied. No precipitation was max is the maximal current elicited, v d cntxn n MI, N-8 wr purchased were SNX-482 MVII, and -conotoxin I x = elcs h sepes f h pos Te same The plots. the of steepness the reflects I B max . pmn a te prue ot the onto perfused then was Apamin ). ( exp( + /(1 S . E V_V . M . The number of cells ( P Î )/ .1 niaig a indicating 0.01 < V Î d is the voltage of half- x ,(1) ), t test was used to used was test v M. E. Schnee and A. J. Ricci -agatoxin IVA, I – V 2 r values. curves n ) is where, Dose–response curves were fitted with a Hill equation: pharmacological different conditions. under or positions papilla different current. does allow for a simple not comparison between currents obtained at does maximum measurement of it yet inactivation, this or schemes gating about % assumptions require estimate, 90 formal to less 10 a Although from activate the to for taken the time current the as by measured were times compromised rise Activation is goals these types methodology channel used. of the Neither of characterization present. for frequency also different at and cells positions from responses to cell is hair plots compare these of purpose The positions. frequency between consistent be will errors these however shallower; be will curve some the of slope the and depolarizations, larger the for response peak or typically current were error associated with the method used transducer here will underestimate the potentials remaining unblocked residual SK or BK at these depolarized potentials. The by Reversal contaminated determined. be not could potentials clearly reversal the and isolated be not currents could tail because the here done by was current Neither potential. of maximal reversal use dividing the or hence currents for, tail accounted isopotential are force driving if in accurate changes only are curves Boltzmann function,Theoretically, where and fast to slow and at a given potential. current steady-state to peak of ratio the is index inactivation The of the form: mV. The equation was s depolarization to _14 current during a 1 in decay the were to equations exponential constants triple fitting time by obtained inactivation indicated, otherwise Unless represents the voltage of half-inactivation and oeta o _6±1m ( mV 1 resting a ± and _46 Hz of 27 potential ± 317 of frequency resonant resting a had and 2 resonance (Fig. obtained were potentials electrical of a measurements Using position. current-clamp 0.01) solution, intracellular ± potassium-based (0.34 low-frequency a and 0.01) ± (0.64 high- a from clamped patch were cells Hair Tonotopic variations in calcium current RESULTS nciain a osre i ms cls A reported As Complex cells. most mV. _10 in observed to was negative inactivation potentials at peaked and mV _40 to negative potentials at activated Currents 2 Fig. in given are solution intracellular caesium-based different ( significantly ( statistically mV were 0.5 ± measurements _51 of potential Hz and a resting 6 ± cells had a resonant frequency of 115 P B equation was used to fit normalized inactivation plots. where, 0.01). Examples of calcium currents obtained using a < I k max I is the half-blocking dose (IC dose half-blocking the is = 0 is the maximal block obtained. stecreta s, 1 at current the is I 0 + A A I 1 exp(_ 1 blocked , A 2 and / t I / max t 1 )+ A = 3 are the proportionality constants. B A max n t 2 1 exp(_ , 1, hl low-frequency while 11), = ( t x 2 n and H n A /( 50 ). High-frequency cells High-frequency ). ; i.2 Fig. 9; = t k ), / n t H t 2 n + 3 )+ H are the time constants time the are x is the Hill coefficient Hill the is n H A ) (2) )), 3 x exp(_ is the slope of the d J Physiol A t . Both ). / t 3 ,(3) ), 549.3 B . Î V Journal of Physiology J Physiol 549.3 . . mV 0.2 ± 4.2 and o lw ( low- for were currents Larger electrodes. caesium-based obtained from using high-frequency cells. above, shown protocol stimulus solution. corresponding intracellular potassium-based a using injections step current B pA 100 to response in position A Figure 2. Tonotopic variations in hair cell properties low-frequency cells ( o-rqec el.*Points that are significantly different. low-frequency cells. * form: fits to the data. Half-activation voltages ( voltages Half-activation data. the to fits xmls f acu cret otie fo a ih (et ad o-rqec (ih) el ih the with cell (right) low-frequency and (left) high- a from obtained currents calcium of examples , , examples of electrical resonance obtained from hair cells at the high (left) and low (right) frequency (right) low and (left) high the at cells hair from obtained resonance electrical of examples , t , respectively ( Y = Y 0 9 _1 + , for high- and low-frequency cells, respectively ( A exp(_ n 1 ad ihfeuny ( high-frequency and 11) = r 2 9 .4 o ihfeunyclsad20±1 3±1 n 5 14 ± and 153 10 ± 1, 53 ± 0.94) for high-frequency cells and 260 = ). x / t E eeotie.Vle f20±1,2 n 0 7 ± 5 and 102 ± 10, 20 were ± obtained. Values of 260 ) , normalized plots from the data shown in Calcium currents in auditory hair cells C , %) a against plot command of potential current rise time (10–90 V Î ) were _35 ± 1 and _43 ± 1 mV and slopes were 4.7 ± 0.3 and 0.3 ± 4.7 were slopes and mV 1 ± _43 and 1 ± _35 were ) ª , n 2 cls Snl epnnil is f the of fits exponential Single cells. 12) = D , r I 2 – 0.99 for each). = V curves for 10 high-frequency ( D , symbols the same, with Boltzmann m s were obtained for m s ( r 2 0.99) for = ª ) and 10 Y 0 , A 701 Journal of Physiology 702 rsne(8 mV, presence (_84 generated from cells using holding potentials mV mV, of in _84 the or absence _64 (_84 holding potential of _84 mV ( mV _84 of potential holding 3 . Vfrtedfeec lta 8 V u odfeecsi lp 52±03mV 0.3 ± mV, but no mV differences for in the slope difference (5.2 plot at _84 0.3 ± _37 _64 mV in the presence and absence of 5 of absence and presence the in mV _64 A Figure 3. Nimodipine blocks the calcium current in a voltage-dependent manner the absence ( control response is seen, but nimodipine block was greater at a _64 mV holding potential. holding mV _64 a at greater was block nimodipine but seen, is response control clim urns lctd y eoaiig h cl t _4m fo hlig oetas f 8 V or mV _84 of potentials holding from mV _14 to cell the depolarizing by elicited currents calcium , ª ) and presence ( • 6 mV, ; _64 9 C 0 ) of 5 ) and _64 mV ( mV _64 and ) ) of 5 M. E. Schnee and A. J. Ricci m m M M nimodipine and the difference (nimodipine-sensitive, nimodipine. m M nimodipine for a high-frequency cell. No difference in the in difference No cell. high-frequency a for nimodipine D ). There was a slight shift in the in shift slight a was There ). C and D , normalized I – V curves for data obtained in V Î ª from _40 ± 0.3 to 0.3 ± _40 from 6 mV, ; _64 _1 ). No difference B , I 0 – V ), from a 1 curves ) and J Physiol 549.3 Journal of Physiology yeplrzd ee ta d hg-rqec cells high-frequency do 2 than (Fig. level more a hyperpolarized at activate cells low-frequency in currents that ( potential command against current peak of be Plots ‘Inactivation’. will entitled, section and the in characterized locations both from cells hair in observed & (Art faster are Fettiplace, 1987; Zidanic & Fuchs, 1995). Inactivation kinetics was the that suggested has work previous where mV, _45 than negative at more currents potentials from times rise measure to inability our to Hodgkin-Huxley-type kinetics, but this may simply be due by predicted response bell-shaped the show not did plots were measured cells (Art&Fettiplace,1987;ZidanicFuchs,1995).Our times rise comparable to those low-frequency reported previously for auditory hair The and high- for respectively. mV 14 cells, ± 153 and 7 ± 102 of values with times, rise the of dependence voltage the a single exponential equation demonstrated a difference in hyperpolarized levels, with half-activating voltages ( 2 Fig. (1); (eqn functions Boltzmann with fitted and replotted current, locations, frequency between difference the quantify better To types. channel different from contributions suggest might which humps slightly faster at 2 hyperpolarized potentials were (Fig. cells low-frequency from measured times rise property The a mV), _14 the of to channels L-type for commonly found depolarizations for ms 0.2 (< locations, papilla both at obtained currents for fast were urns hn o-rqec cls 07 .2nA 0.02 ± (0.75 cells low-frequency ( than maximal larger currents had cells high-frequency previously, J Physiol ciae rpdy wt rs tms ht decreased that times 2 (Fig. rise depolarization with exponentially with rapidly, activated Fettiplace, 1987; Art n 4 Vcmae o_5±1mV for high-frequency cells. Here too, 1 single Boltzmann functions provided good ± mV compared to _35 1 ± _43 5 a cmae t 03 .4n ( nA 0.04 ± 0.36 to compared as 75) = D 549.3 ). The of 5 min after drug that application. begins Traces 10 at the top are in the absence and the bottom in the presence representing the first and the thin lines Hz the stimulus last. protocol Time zero represents the start of the 1 ( mV _84 of potential holding a from response last and block was investigated by ms depolarizing at mV Hz. a a for Examples cell frequency 20 of to of the _14 1 first 6 V rsetvl) Vle for Values respectively). mV, _64 oetaso 8 mV( potentials of_84 ( 8 V ( mV _84 obtained for the two conditions). test potential. A single exponential ( exponential single A potential. test . 0.2 ± 2.2 . . mV 0.8 ± 4.2 in respective condition ( .0 .0 n . mV for 1 ± 0.001 and 8.8 ± 0.003 1.7 ± 0.2, respectively, for the holding potential of _64 mV ( mV _64 of potential holding the for respectively, 0.2, ± 1.7 current obtained in the presence of 5 . 0.1 ± 2.0 was observed for this cell. a further % decrease was of observed 9 while from mV a a holding further % potential reduction of of _64 32 r I 2 E – I 0.99). = – ). Low-frequency cells activated at more at activated cells Low-frequency ). V m V M curves weresmooth,withnoapparent lt a bevdfrtehligptnilo 6 mV, where plots the was observed for the holding potential of _64 et al. nimodipine (Nim.). I m m – ª M M V G ad 6 V ( mV _64 and ) _1 n . .,rsetvl,frahligptnilo 8 Vad9 ,18±0.2 ± 1.8 %, 4 ± 90 and mV _84 of potential holding a for respectively, 0.4, ± 2.1 and and 1.9 ± 0.2 were obtained for obtained were 0.2 ± 1.9 and , the voltage dependence of nimodipine block was investigated further by plotting the ratio of 1993; Ricci lt wr nraie t peak to normalized were plots . E ds–epne uvs ih hi crepnig is o h Hl euto fr holding for equation Hill the to fits corresponding their with curves dose–response , H ª , J n 6 mV( ) and_64 ). The continuous lines reflect the initial value. From a mV holding potential of _84 et al. I 9 , plotofthepeak currentagainsttimewherethe symbolscorrespondtotheir wt te orsodn ft o h Hl euto. ee vle o 1.0, of values Here, equation. Hill the to fit corresponding the with ) 2000). Currents Calcium currents in auditory hair cells C F n Y , plots normalized to the maximal blocking dose for holding potentials of . ie times Rise ). 0 a I 0 At & Art 10; = , B – m A Y 1D variety. 1D max V M C = and ) suggest ) te afbokn ds ad h Hl cefcet ee 9±4%, 4 ± 69 were coefficient Hill the and dose half-blocking the , nimodipine to control current at different test potentials against the 9 ). Fits to Y .Tenme fclstse sgvni aetei s(8 mV, ). Thenumberofcellstestedisgiveninparenthesisas(_84 V 0 d + x B Î , respectively ( A ) of max exp(_ , half-blocking dose and the Hill coefficient, respectively coefficient, Hill the and dose half-blocking , x / o novd lpso . . n . . mV 0.2 ± 4.2 and 0.3 ± 4.7 of Slopes involved. not fits tothedata,suggestingthatmultiplechanneltypesare hne tps Tbe1. ikl a a IC an had Nickel 1). (Table between selective types not were channel that doses at but current, the blocking at effective were cadmium and nickel Both current. inward the blocked cadmium and nickel cations divalent the of concentrations High calcium. by carried was current measured this the all that ensure answering to was question towards step first turtle A in cells? channels hair auditory calcium of types multiple there are A fundamental question addressed by the present work is: Pharmacological characterization of channels ocnrtos s ih s 30 requiring as weak, high was as current concentrations cell-calcium hair the the hair 3). cell Nimodipine-induced current block (Fig. of a Nimodipine, on tested was dihydropyridine, of form soluble relatively compounds. of class the by dihydropyridine blocked selectively are channels calcium L-type Dihydropyridines d H by calcium. No effect of DIDS was observed. also used to ensure that all the inward current was carried (data not shown). The chloride channel ions blocker DIDS was these by blocked selectively or unmasked not were currents additional that suggesting blockers, of presence the in amplitude in decrease the than other change little of the current ( naoie h cret Fg 3 (Fig. current the antagonize eiet bten hs psiiiis a better a possibilities, these between delineate compounds these the by (i.e. antagonized partially only was dihydropyridines) or second, that the channel type present types channel multiple that one drawn, be can conclusions and were not significantly different. respectively, cells, low-frequency and high- for measured .4±02m 0.2 ± 0.74 x )bs itdti ltwt auso . 0.01, ± 0.3 of values with plot this fitted best )) ) or _64 mV ( mV _64 or ) r 2 r 2 0.98). = values of 0.989 and 0.99, respectively, were respectively, 0.99, and 0.989 of values xs (.. hnes estv o isniie to insensitive or sensitive channels (i.e. exist a D -ye hne) Bfr atmtn to attempting Before channel). L-type 1D M H hl amu t1m , whilecadmiumat1 – J V n ) are given with the thicker lines thicker the with given are ) J , use dependence of nimodipine Î 3). = a 3 mV and the slope 1 ± was _39 I – V plots were smooth and showed E m and M m M M F lce 4±3% 3 ± blocked 94 o substantially to and . w possible Two ). _1 50 were 703 of Journal of Physiology rpris(i.3 (Fig. properties activation their of comparisons direct allowing current, block; 3 nimodipine Fig. to sensitive current (i.e. difference aia cnrl urn. is o h Hl equation Hill the to Fits current. control 3 maximal Fig. in plotted are mV _64 and _84 Dose–response curves obtained from holding potentials of 1995). Fuchs, & (Zidanic type channel one of existence the for not (data different not shown); similar results from chick papilla were hair cells argued or nimodipine absence the of in presence times rise addition, In present. was type channel one only that suggesting potential, holding either at plots between difference no showed data these dihydropyridines by antagonized 3 (Fig. be could current ( % 2 ± 90 of block Changing ( % the mV holding gave 2 potential a to maximal _64 ± 68 was block maximal the where mV, use _84 and voltage both dependent. Initial experiments used be a holding potential of can block Dihydropyridine needed. was effect dihydropyridine the of understanding 704 B A wr nraie t ter epcie maximal respective their to normalized were ) , B and E ). C I n – and V = 5), suggesting that the majority of majority the that suggesting 5), = curves in the presence, absence and D ). Boltzmann fits (eqn (1)) to (1)) (eqn fits Boltzmann ). E nraie to normalized , M. E. Schnee and A. J. Ricci n 5). = en(2)) gave (eqn IC in the in major difference between holding potentials appears to be eainhp a fud pras ugsig that suggesting perhaps found, was relationship a is state this mV favoured holding at potential. that the _64 and binding drug for required is state that no 3 demonstrating channels, cell hair for plot this reproduces Figure less. was block the potentials depolarized or hyperpolarized either at but mV), 0 to (_4 sensitive most the against nimodipine was drug the where region a revealed potential command of presence of the in percentage & left the current (Xu plotting is, nimodipine That by 2001). Lipscombe, block to dependence voltage IC potentials, holding 3 (Fig. different at block maximal to normalized were curves dose–response block in similarity were not statistically holding different. To further demonstrate the for 0.2 ± values These respectively. mV, 1.7 _64 and and _84 of potentials 0.4 ± 2.1 of coefficient Hill a D hnes xrse i octs ae a have oocytes in expressed channels 1D 50 nor the Hill coefficient were different. Therefore, the Therefore, different. were coefficient Hill the nor B F n a 8644-treated ( K and Bay respectively ( 8644-treated mV cells, for K control and 8 Bay ± 93 fits with voltage dependence, 8644. Coninuous lines K represent exponential Bay current, demonstrating the shift in the 8644. K Bay B refer to protocol is shown at the top; more negative steps 8644. The stimulus K dihydropyridine agonist Bay absence (upper) and presence (lower) of the A agonist, increases hair cell calcium currents 8644, a dihydropyridine K Figure 4. Bay cells, respectively ( . . mV 0.2 ± 3.5 0.2 and mV and ± the slopes 2 were 4.9 ± _51 function, where the Continuous lines represent fits to a Boltzmann activation in the absence and presence of 1 by % plotting rise the time 10–90 of the current D in a hyperpolarized direction. In each panel, magnitude increased and voltage sensitivity shifted the control and U max ), resulting in overlapping plots where neither the neither where plots overlapping in resulting ), , , an example of a calcium current obtained in the -shape was observed, rather a simple exponential activation kinetics were slowed , as demonstrated I – , perhaps suggesting that a particular channel particular a that suggesting perhaps , V curves demonstrating that the current I – V 50 plots 8644. in the K presence of Bay C auso . . n . 0.2 ± 0.2 and 1.8 ± values of 2.2 r _1 , 2 I values were 0.99 and 0.96 for control o oto n a 8644-treated for K control and Bay – 9 V s are in the presence of 1 r plots normalized to maximum 2 V 0.99 for both fits, = Î auswr 3 0.3 and ± values were _37 n 4) cells, respectively). = d x f13±26 and ± , of 153 V Î n . 4). = m m J Physiol M M ª s are U m -shaped M 549.3 and G Journal of Physiology odn ptnil o _4 n _4m, respectively mV, _64 for and % 2 ( _84 ± of 10 and 5 potentials 1 of ± holding 4 presence of the decrease a in gave nimodipine depolarization of cycle last the to first the Comparing 2001). Yamoah, & Contreras (Rodriguez- experiments previous match to and current the of inactivation significant cause to not as so chosen 3 5 of presence (Fig. and nimodipine absence the in Hz 1 at mV _64 or _84 either of potential holding a from ms 20 for mV _14 protocol was used that stepped the membrane potential to Yamoah, 2001). To investigate use dependence, a stimulus for observed a suggests strong use dependence cells to nimodipine block hair similar to that saccule frog from evidence Recent the and channels native hair cell channel. expressed between exist differences J Physiol was found, whereby 5 high- as nimodipine frequency cells. to A similar and dependence on sensitivities holding potential investigated same the also showed were cells Low-frequency cells. high-frequency from are presented data nimodipine The than saccule hair cells. nimodipine by block of dependence use less much show acu urnsi hw nFg 4 cell Fig. hair in shown on is currents action calcium the 8644 K of Bay time of example open An channel. the prolonging by channels L-type 8644isadihydropyridinethat actsasanagonistfor K Bay not are high-frequency cells. that values from recorded measurements mV, from different _64 statistically ( at % 3 blocked ± was 88 while current mV, _84 at current the n = 5). These data indicate that turtle auditory hair cells hair auditory turtle that indicate data These 5). = 549.3 a C hnes RdiuzCnrrs & (Rodriguez-Contreras channels 1C H – m J ). The frequency of stimulus was stimulus of frequency The ). M ioiiebokd6 % of 3 ± nimodipine blocked 64 A , where the current the where , Calcium currents in auditory hair cells n ) f the of 5) = m m M M and the slowed were kinetics activation increased, was magnitude uvs o cnrl n ByK84 wt Boltzmann with 8644 K Bay in shift a and demonstrated functions control normalized for the curves Fitting observed. was amplitude ( % 11 ± 46 A direction. oe f h frt acu canl naoit available antagonists channel calcium first the of some Verapamil and D600 are acetonitrile derivatives that were L-type antagonists were other used. reason, this For present. type only the are that conclusively demonstrate not do data is block nimodipine As incomplete andhighdoses were neededfortheblock, papilla. auditory turtle the in the of probably channels, L-type The dihydropyridine data support the hypothesis that only Non-dihydropyridine L-type blockers the channels present in hair cells were of the L-type. suggest reported effects the so channel, calcium of classes lwdb a 64(i.4 (Fig. 8644 K Bay by slowed the fit complex to resulting sufficient be not would function Boltzmann single a unaffected, currents non-L-type leaving currents, 8644 is Since predicted to K shift the cells. activation of Bay the L-type hair auditory turtle in present was channel of class one only that hypothesis the support also curves 4 (Fig. significant a rsneo a 64(i.4 (Fig. 8644 K Bay of presence the in slower were mV _30 to hyperpolarized potentials at kinetics activation that demonstrates potential against 5 V n a hne n lp fo 35±02 to 0.2 ± 3.5 from slope mV in 0.2 change ± a 4.9 and mV 2 ± _51 D and 1D I – V a relationship shifted in a more hyperpolarized C -ye acu canl, u nt other not but channels, calcium L-type 1C _1 ( n C ) bt vle big statistically being values both 4), = I . ige otmn ft t the to fits Boltzmann Single ). – V n uv. ciain ieis were kinetics Activation curve. ) nrae n ek current peak in increase 4) = A D and ). Bay K 8644 affects both affects 8644 K Bay ). a V 1D variety, are present are variety, 1D D Î ). Plotting rise time rise Plotting ). from _37 ± 0.3 to 0.3 ± _37 from a 1D channels 1D I – I – 705 V V Journal of Physiology auso 9 0ad35±25 ± 20 and 375 IC ± values with of 192 currents calcium the antagonized completely D600 and verapamil Both 5. Fig. in given are equations Striessnig Dose–response curves with their corresponding 1987; fits to Hill (Sperelakis, channel calcium of types other over channels L-type for selective (Lee &Tsien,1983).Theyarestillthoughttoberelatively 706 ht o dss ossety oetae te current fourth (1 calciseptine A compound, the previously. reported potentiated not effect an consistently amplitude, doses low that in unusual somewhat was result diltiazem The obtained. & Perez, 1987). was ineffective at blocking the hair cell current (Hamilton sensitive site and so it is not surprising that this compound Perez, & dihydropyridine- the for competes also Taicatoxin 1987). (Hamilton current at cell ineffective hair the was antagonizing blocker L-type another Taicatoxin, difference between the of reflection a be also might calcispetine a based on drug potency, may be different between act at, or near the dihydropyridine binding site, a site that, the antagonizing Weille at (de current ineffective was channels, calcium IC An diltiazem. for equation Hill the to fit corresponding 1998). Striessnig 1987; (Sperelakis, current cell hair the blocks channels calcium L-type 5 for selective relatively (Fig. respectively 0.2, be ± to known also derivative benzothiazepine a Diltiazem, 1.6 and 0.4 ± 1.7 1D subunits (Yasuda subunits 1D 50 f37±22 ± of 367 iue5 Figure a 1C and 1C and diltiazem, respectively, with 0.3 for verapamil, D600 ± 0.2 and 2.5 ± 0.4, D600 1.6 and diltiazem, respectively. ± Hill coefficients were 1.7 Dose–response curves, with corresponding fits to Hill equations, are shown for verapamil ( Figure 5. Traditional L-type channel blockers can completely antagonize the calcium current response. The scale bar is 500 pA and 20 ms. The value of value The ms. 20 and pA 500 is bar scale The response. trace isthelargestcurrentin mV in the absence and _84 presence of the three highest drug concentrations (averages of four). The control ( diltiazem lcigdssotie rmftigHl qain ee12±2,35±2 n 6 22 ± 25 and 367 ± 20, 375 blocking doses obtained ± from fitting Hill equations were 192 m C M a et al. et hw te oersos cre and curve dose–response the shows n ilcefceto . 0.3 were ± and a Hill coefficient of 2.5 1D subunits at the same binding site. binding same the at subunits 1D C et al. et . nes r cret eiie fo dplrztos o 1 V rm hlig oeta of potential holding a from mV _14 to depolarizations from elicited currents are Insets ). 1991). Calciseptine is thought to thought is Calciseptine 1991). m M 1993). The lack of effect of effect of lack The 1993). ), a peptide toxin of L-type of toxin peptide a ), m M and Hill coefficients of A and r 2 t al. et values of 0.997, 0.998 and 0.99, respectively. B , butinthediltiazemtraceitappears thatthelowdosepotentiates A M. E. Schnee and A. J. Ricci a and 1998). 1C and et al. et B ). 50 the presence of other calcium channel types. directly assess to tools pharmacological additional of use other of reports the to prompted cells, hair in present types channel calcium addition in antagonism, channel complete for required concentrations high the However, calcium L-type the of to probably most attributed channels, be can current calcium the all not if most, that suggest data pharmacological The Non-L-type antagonists n -ye hnes A 1 At channels. L-type on sites binding verapamil or dihydropyridine with interact GVIA is a potent N-type channel antagonist that does not v interact with L-type channels (Safa IVA is a potent blocker of P/Q-type channels that does not naoit (Bourinet antagonist v including 1, Table in listed are used blockers Additional current. the antagonizing at ineffective also was agatoxin All the L-type antagonists used have binding sites on the on sites binding have used antagonists L-type the All I hyperpolarized times; rise activation ms) 0.5 (< rapid on: the particular, L-type In blockers. only channel L-type by blocked be could current antagonists; of all and current the blocking at effective were antagonists presence and hypothesis absence this supporting fit functions Boltzmann single Data include: cells. hair turtle auditory in present are channels calcium L-type only that suggest data pharmacological and biophysical the Both antagonizing the current. – cntxn VA Tbe1 o summary). for 1 (Table GVIA -conotoxin -conotoxin MV11A and SNX-482, a purported R-type purported a SNX-482, and MV11A -conotoxin V n curves and relative insensitivity to dihydropyridines. to insensitivity relative and curves for each dose is given in parenthesis. Half- parenthesis. in given is dose each for a 1D type of L-channel is implicated based implicated is L-channel of type 1D t al. et m 01. oe a efcie at effective was None 2001). m A M M ), D600 ( for verapamil, hr ws o fet of effect no was there a D ait (al 1). (Table variety 1D et al. B ) and 2001). At 0.3 I – v V -Conotoxin J Physiol v plots in the in plots -Agatoxin 549.3 m M , Journal of Physiology ohwti h iet n xenlya 5m 25 at externally and pipette the within both included was TEA current. other some by contamination in decay measured the of artefact an not and inactivation was amplitude current that ensure to important was characterized in auditory hair cells previous to this work, it 1and2).Sinceinactivationhasnotbeen examples inFigs (see cells low-frequency and high- both from recorded currents in observed was inactivation Time-dependent Inactivation addressing one that section assumption channel type is present. the next with analysed is The inactivation subunits. accessory a J Physiol calcium entry and is dependent on the calcium channel. calcium the on dependent is and entry calcium by slowed and diltiazem, suggesting that inactivation is a function of was inactivation chemicals that antagonized addition, the channel, such as verapamil In channels. inactivation, slowed 8644 suggesting thatinactivationwasapropertyofthecalcium K Bay inactivation. altering voltage- a DIDS, 1). dependent chloride-channel blocker was also (Fig. ineffective in inactivation of component time-dependent the altering without SK the to ascribed block the SK potassium conductance, reduced the current eoeiatvto.Aai 10n remove inactivation. Apamin (100 not did channels, BK activating in calcium for substitute not should and currents potassium block barium, should which external described, be will As inactivation. on was observed. 4-AP was also used externally with no effect any residual potassium currents. No effect on inactivation subunit (Striessnig (white, dashed) exponential fit. single (black), double (light grey) and triple mV with depolarization corresponding to _14 ms ( 36 ± 174 exponential relationship with a time constant of recovery from inactivation and has an time 0 and at varying times (P mV ms at depolarization delivered to a _14 20 A determinant of inactivation Figure 6. The interpulse interval is a critical interpulse duration (P normalized to the control at time 0 against inactivation. both peak current and time-dependent continuous line is shown to point out the loss of averaging was used in these protocols). The time-dependent component of inactivation (no current responses used to illustrate the loss of a and the lower trace is an expansion of the first two protocol, the middle traces are currents elicited inactivation. The upper panel shows the stimulus interpulse interval needed to preserve initial pulse, was used to determine the required D s. depolarizing Both mV a for cell 25 to _14 channels can inactivate is illustrated by , a protocol that held mV the and cell at _84 are single-episode responses. 549.3 B n , a plot of the peak current 21). = et al. 2 C ) illustrates the time to , the s response to a 1 1998) and reveal nothing about 2 ) following the D , that all M ), which was used to Calcium currents in auditory hair cells C and M to block to nciain Wt sot uss iatvto de not does appear inactivation to be very robust; however, 6 as pulses, shown in Fig. short With inactivation. time-dependent any show plot not does thus and inactivated remains The current the IPI to short current. a ms with that 450 demonstrates constant-amplitude of a IPI an maintain required of depolarizations ms constant 20 ( time ms 36 a ± 174 with 6 relationship (Fig. exponential IPI against current normalized of plot A was stimulus. prior the by induced inactivation the amplitude current The reduced, suggesting that channels had not recovered from pulse. depolarizing the lower panel). That is, there was no decay in current during urns ih o iedpnet nciain Fg 6 (Fig. inactivation with time-dependent no mV with _14 currents to ms 20 for varying 6 times between pulses (Fig. depolarized and mV _84 of potential holding a used that developed was protocol large currents. To better quantify the importance of IPIs, a dependent inactivation, in spite of having stable, relatively time- show not would cells These IPIs. ms 50 brief, used interpulse interval(IPI).Initially,protocolswererunthat the was inactivation maintaining in factor relevant most The amplitude. current large reasonably a maintaining despite inactivation, for lose could depolarized time of were periods extended that cells in currents or pA) 30 (> current leak with cells labile, be often could Inactivation of artefact an not was and contamination by some other channels current. calcium the of Together, these data suggest that inactivation is a function n 1, niaig ht rtcl using protocols that indicating 21), = A ). A short IPI yielded B rvas an reveals ) C and A 707 , Journal of Physiology 708 ierrltosi ihasoeo . 0.1 linear and relationship a with ± a slope of 1.0 from the Boltzmann fits to the data shown in shown data the to fits Boltzmann the from F E mV. mV and +76 mV, _14 (top) and a low-frequency (bottom) cell. Traces shown are for mVareshown forahigh- prepulses to _84 mVfromaholdingpotentialof_84 mspulseto_10 ms,followedbya20 for 20 between prepulse values. ecnae urn iatvtd gis peus drto hs n xoeta rltosi wt a time a with ( ms relationship exponential 8 an ± has 35 of constant duration prepulse against inactivated current percentage a showed typically pulses ms 20 rebound the effect to where the response current The maximum was unaffected. greater was after depolarizing process prepulses the than control. of the dependence that voltage but durations, pulse longer for greater was decrease current of magnitude the that demonstrate B mV ( bottom to +86 top). Inactivation is measured current as the the decrease above in shown current amplitude protocol during stimulus the test The pulse. mV. records illustrates the _84 shape of of the potential protocol holding used. The the traces mV to shown and are return mV, for a _14 prepulses then to _84 and ms mVfor20 mV,followedbyatestpulse to_14 mV,incrementedby10 potentials between_114and96 A between hair cells of different papillary locations Figure 7.Calciumchannelinactivationhasabell-shaped voltagedependenceandissimilar lp au a mV 2 ± slope value was 4 low-frequency cells, respectively). cells, low-frequency , the normalized plots of test current against prepulse , potential were bell-shaped for both frequency positions. a lt f ek urn drn te tp o 1 V gis te orsodn peus ptnil to potential prepulse corresponding the against mV _14 to step the during current peak of plot a , , examples of a hair cell’s ms current prepulse response to to a stimulus protocol using either a 200 or a 20 V Î fiatvto esrdfo h otmn ist h aaws_0±2ad_6±2mV, and the 2 of ± inactivation measured 2 from and the _46 Boltzmann fits ± to the data was _40 n _1 D is given by points in the plot). A similar plot of the of plot similar A plot). the in points by given is , currents in response to a protocol that varied the mV prepulse from _114 to 104 for both high- and low-frequency cells ( G , a plot of the of plot a , M. E. Schnee and A. J. Ricci B y (data between _114 and _4 mV fit) showed no difference no showed fit) mV _4 and _114 between (data itreto 9±3mV ( 3 ± -intercept of _9 V Î for inactivation against the against inactivation for r 2 for fits were 0.99 and 0.97 for high- and r 2 0.88). = V Î for inactivation, measured inactivation, for V Î for activation gives a gives activation for C , a plot of J Physiol 549.3 Journal of Physiology low-frequency cells. Values of 6 ± 1 and 6 ± 1 ms for ms 1 ± 6 and 1 ± 6 of time Values cells. low-frequency in difference No constants of inactivation was observed between high- and data. the describe adequately triple and double exponential fits. Only the fit to three single, time constants could with current inactivating an h kntc o iatvto wr qie complex, course of current 6 decay. Figure quite were required three time constants to reflect accurately the time inactivation s of reductions in current in response to depolarizations of 1 kinetics The calcium the channels present all could be inactivated. that demonstrate also data These used. D J Physiol frequency cells, respectively ( respectively cells, frequency rtcl ht aid h mmrn ptnil between potential prepulse membrane a the varied using that studied protocol was Inactivation cell. given cells to recover sufficiently to do multiple experiments on a allow to developed were Protocols recover. to minutes of Often cells that s were would depolarized require for tens 1 mV. low- _14 and high- to was for depolarizations % for 4 respectively, locations ± positions, frequency 42 and frequency 2 ± different 37 at comparable observed of inactivation of cells amount between total The 7 %). ± 3 (55 ± cells 41 high-frequency than inactivation fast fast component, where low-frequency cells exhibited more the by contributed inactivation of proportion the in was The only difference observed between frequency positions 19±12m for ms 122 ± 1179 3±5m ad 7±1 for 14 ± 77 and ms 5 ± 73 , inactivation could be quite profound if long pulses were Q Fettiplace, 1987) according to the equation time course of the decay in the oscillations (Art & The quality ( Hz for left and and 312 right responses, respectively. (right). The frequencies of the oscillations were 290 pA) to was elicit injected electrical (50 resonance voltage (left) compared to the same cell where current pA) to depolarize of the 75 cell to its best resonant mV pA, and depolarizing then total injected _84 (50 pA) was first injected (_25 to hyperpolarize the cell to (averages of 20) from a hair cell where current mV. condition of _84 mV. _44 frequency and onset, was 5.5 and 5.2, respectively. the exponential decay of the oscillations at current (bottom). from a mV mV holding (top) potential or of _44 _84 depolarizing voltage steps mV between _64 and 0 A experiments in both voltage- and current-clamp calcium channel inactivation was investigated Figure 8. The physiological significance of holding potential ( in the magnitude of the calcium current when the , hair cell currents measured in response to =[( 549.3 p f 0 0 t B 0 s represent the return to the control ) , 2 Q I + – ) of the resonance measured from the t V 0 ˝ is the time constant measured from plot of data in t ] V 3 Î , where h ee band o hg- n low- and high- for obtained were ) was varied from _84 to C , current-clamp responses f 0 n is the resonant C A = 12 and 6, respectively). 6, and 12 = t illustrates an example of showing reduction 2 n 93±12 and 132 ± 903 and Calcium currents in auditory hair cells t vs. 1 , on a given cell with this short-duration pulse. sufficient to quantify and multiple protocols could be run were amplitudes inactivation because protocol standard prepulse longer to ms was chosen as the protocols. response A prepulse duration of 20 in prevalent less was this from inactivation,seenasanincreaseinthepeakcurrent; ms protocols often resulted in an 20 overshoot in recovery xoetal a te rple uain a increased was increased 7 duration (Fig. inactivating prepulse the current as of exponentially fraction the while i.7 in Fig. given is durations prepulse different using obtained inactivation data of comparison A constants. time faster the by driven favour were would pulses prepulses shorter These duration tested. shorter recover, adequately to cells for allow not did prepulses long since However, time measured the on constants, were required to be of based several seconds duration. simply which, duration s IPI was used. Initial experiments varied the prepulse A 3 mV. mV from a holding potential of _84 test pulse to a _14 by followed increments, mV 10 in mV +114 and _114 potential 7 gave bell-shaped curves (Fig. current at the mV test against potential the of prepulse _14 7 Fig. shift in shift 7 Fig. in plot the of portion first the to functions Boltzmann fitting by ms prepulse. Half-inactivating voltages were obtained 200 & Eckert,1978).Moreinactivationwasobservedwiththe (Brehm inactivation calcium-dependent implies typically A A C – for a 20 and a 200 ms prepulse. Plotting the peak the Plotting prepulse. ms 200 a and 20 a for .A a ese nteeapesoni i.8 ). As can be seen in the example shown in Fig. afiatvtn otg a bevd(i.7 (Fig. observed was voltage half-inactivating C . An example of the data obtained is given in given is obtained data the of example An . B normalized to the maximal current. No current. maximal the to normalized B ). This bell shape C B 709 ), , Journal of Physiology 710 a 4 n 5 V epciey h lpsfrrcrsotie n5m 5 in obtained records for slopes The respectively. mV, 1 ± _53 and 1 ± _42 was 5m n m and 5 A Figure 9. Inactivation persists with barium as the charge carrier aim ee . . ad . . mV 0.2 ± 2.6 and 0.5 ± 3.4 were barium 0.98. panel. demonstrating the increase in maximal current and a leftward shift in the plot. Symbols are the same for each nciainfr5m inactivation for 5 normalized to peak current (data from G F from and fitted with single Boltzmann functions. The from fits to data shown in shown data to fits from urn–rple otg po dmntaig h efcs f iaet o seis n inactivation. on species ion divalent of effects the demonstrating plot voltage current–prepulse , , examples of currents elicited in s response depolarization mV to from in a _84 the 1 to presence _14 of 2.8 inactivation persists in the presence of , barium. For clarity, the Boltzmann fits to the inactivation currents M A acu n m 5 and calcium E to better illustrate the effects on inactivation. , M I – acu r5m calcium or 5 V plots generated from the data shown in M acu n m calcium and 5 M M aimwr n mV 1 ± 7 and 1 ± 4 were barium barium (single traces). A . D , I – V M. E. Schnee and A. J. Ricci lt o ciainuig5m 5 using activation for plots F M ) are shown without corresponding data points. The voltages of half- aimwr 4 n 6 mV, respectively . The slopes for 1 ± 1 and _62 ± barium were _47 _1 rsetvl. l ft had fits All respectively. , V Î B o eod bandi m for records obtained in 5 , expanded view of the initial portion of currents elicited C D , fast and intermediate time constants of inactivation normalized to peak current for each ionic condition _1 , respectively. All fits had fits All respectively. , M calcium ( calcium r 2 M aus rae ta 0.98. than greater values ª acu n m calcium and 5 n m 5 and ) M r 2 acu n m 5 and calcium values greater than greater values M barium ( barium M barium 9 M ) J Physiol 549.3 Journal of Physiology ee 4 ad 5 V o hg- n low- mV and 1 ± high- (4 between observed for differences were mV No slopes 2 ± respectively. _50 cells, and frequency 2 ± _44 were ihfeunycls(i.7 high-frequency cells (Fig. cells low-frequency than potentials negative more at the inactivated consistently that demonstrates functions f h cl ad acu cret wr eiie b the 8 by elicited (Fig. were protocol currents standard calcium and cell membrane the of on inactivation were voltageclampednearthemeasuredrestingpotential of role cells First, performed. were experiments two excitability, the assess To Physiological significance 7 fast Fig. in the of current mVagainstprepulsepotentialaregiven test amplitude at_14 preponderance of plots the Mean inactivation. to of component likely most due cells, low-frequency in greater was inactivation protocol, 1 (Fig. cells Inactivation was present in both high- and low-frequency Tonotopic differences J Physiol erae n urn apiue ih o hf in shift no with amplitude current in decrease holding a from mV. The elicited potential of _84 traces current in component kinetic obvious the to compared as mV _44 of potential ms pulse it from was a not holding observed during the 20 time courseofinactivationwasslowedtothepointwhere The activation. for channels profound of availability a the on effect have can inactivation that demonstrating mV, smaller _84 of potential holding a from obtained those than significantly were mV _44 of potential holding 7 Fig. from data the Replotting more. rebounded cells low-frequency control, where rebound, a currents elicited after positive prepulses were greater than show cells both although addition, consistently inactivated more than high-frequency cells. In rqec ws ossety ihr y 9±8% ( % 8 ± 29 8 by higher (Fig. consistently was cell frequency the of potential resting compared to simply eliciting a resonant response from the then and mV eliciting aresonantresponse nearitsrestingpotentialwas _84 near to cell hair the hyperpolarizing mode, etpae 17; Art 1978; Fettiplace, & (Crawford cells hair these in found mechanism tuning major the resonance, electrical investigated that experiment second conclusion The physiologically. the regulating amplitude for calcium-current supports mechanism major and a be 7 can inactivation Fig. in shown locations. frequency both at inactivation regulating was mechanism y a and 0.1 ± 1.0 of slope a with continuum linear a forms afatvto vlae ( voltage half-activation in difference the with well correlated voltage inactivation in accordwiththe voltage dependence. The reduction in current amplitude is itret f 9±3m, ugsig ht similar a that suggesting mV, 3 ± _9 of -intercept 549.3 sn a oasu-ae itaellr solution, intracellular potassium-based a using E , where it can be seen that low-frequency cells low-frequency that seen be can it where , A n Fg 7 Fig. and V Î E for inactivationobtainedfromdata and fitting them with Boltzmann with them fitting and t al. et I V E – F A Î . sn te 0m stimulus ms 20 the Using ). V ). Half-inactivating potentials Fg 7 Fig. ; . urns lctd rm a from elicited Currents ). lt nFg 8 plots in Fig. 96. n current-clamp In 1986). _1 G . h dfeec in difference The ). . h distribution The ). C . h resonant The ). Calcium currents in auditory hair cells B illustrate the V n =8) Î or was assessed from the decay in the onset oscillations ( oscillations onset the in decay the from assessed was resonance the of quality The capacitance. membrane the of charging additional the to due probably most was and resting cell’s be predicted if additional calcium channels were recruited the would what to opposite the from mV, _84 from than elicited position was resonance when equation the from obtained and decay) of constant time the being slowed in barium as compared to calcium. A summary of summary A calcium. to compared as barium in slowed significantly were constants time intermediate and fast are currents 9 shown in normalized Fig. the of ms 60 first The barium. was barium or calcium m and 5 at incorporated solution external the from removed was Magnesium 9. Fig. in given is calcium for barium substituting by obtained data the of summary A barium. of presence the in increase should amplitudes permeates L-type channels better than calcium, so current it that in type channel for test a also is barium addition, calcium for well biochemically and thus inactivation substitute should be reduced. In not does typically barium. Barium with replaced was calcium further, hypothesis in blockers increase intracellular calcium(Brehm&Eckert,1978).Totestthis an to by driven sensitivity was inactivation its that suggests and inactivation of course time the as well as plots inactivation the of shape bell The Underlying mechanisms resonance. more calcium channels than are required to elicit electrical effect on resonant properties, implying that hair cells have effect of doubling the calcium-current amplitude had little the compared, were data the However potential. resting longer from 0.99 ± 0.06 to 1.75 ± 0.07 nA ( nA 0.07 ± 1.75 to 0.06 ± 0.99 from a qioal usiue o acu Fg 9 (Fig. calcium for substituted equimolarly was not shown). The values of values The shown). not The and where, n _3±1m ad h soe wr 34±05 and 0.5 ± 3.4 were slopes the mV 0.2 and ± 2.9 mV 1 ± _53 and frequency: if frequency: in change the from part, in least at comes, variability The decreased. quality the cells two in and increased the quality cells two in effect, significant no showed cells eight constant ofdecay.Resultsweremorevariable,fourout o el ahdi . m for cells bathed in 2.8 9 voltage eliciting the maximal current are shown in Fig. eand bt a soe i te rsne f barium of 9 presence the (Fig. in slowed was Inactivation but (2001). remained, Yamoah & Rodriguez-Contreras by suggested as channel, and ion the between interaction epciey Smlr eut hv be rpre on the in shift ion-dependent reported been The 1987). Fettiplace, & (Art have cells hair turtle dissociated results Similar respectively. I E – ) and the activation kinetics slowed in barium (data barium in slowed kinetics activation the and ) V t A 0 f 0 plot shifted in 9 a hyperpolarized direction (Fig. rmahligptnilo 8 mV than from at a their holding potential of _84 – Q s h rsnn feuny and frequency resonant the is C . epne t 1s eoaiain o the of depolarizations s 1 to Responses ). =(( _1 t B 0 to illustrate the effect on inactivation. The o 5m 5 for is compared, six out of eight cells had a had cells eight of out six compared, is p f 0 t M 0 . The maximum current increased current maximum The . ) 2 M + M acu,5m calcium, 5 V ˝ acu ad m 5 and calcium Î I (r & etpae 1987) Fettiplace, & (Art ) – o ciainwr 4 1 ± _42 were activation for V lt ih ipiae an implicate might plot n = 5) when barium when 5) = M acu r5m calcium or 5 t 0 s h time the is M A and barium, D t 711 D ). A M 0 Journal of Physiology concentration m of 1 or 30 mV using depolarizations an to intracellular _10 BAPTA with higher concentrations of m BAPTA. Responses in 30 initial amplitude of the measured peak current was also increased auso 3 n 2 mV m for 1 and 2 30 ± 1 and _25 ± values of _39 BAPTA ( 2 and in 7 Fig. inactivation ( min. Activation ( 0.5 falling phase a ± time constant of 12.8 min and the the rising 0.7 phase having a ± time constant of 2.7 pA. The sum of 500 two exponentials was used to fit this data, with reached a peak current and then ran down over time by almost m 30 A inactivation of the calcium currents Figure 10. Calcium buffers alter, but do not prevent 712 epciey n lpso . . n . . mV 0.3 ± 0.1 and 4.7 respectively, ± and slopes of 4.1 1m (1 % 1 ± 1 magnitude to of 16 inactivation ± was reduced from 23 protocols were as ms mV, described prepulses. respectively. earlier The Inactivation using 20 3 ± 2 and _26 ± voltages of _43 similarly shifted in a depolarized direction, with half-inactivation respectively. Boltzmann functions to the inactivation curves AT nrae rm1m BAPTA increased from min 1 as the concentration 0.7 of ± 2 to 2.7 ± decreased from 7 time constant of the exponential fit to the data shown in 0m 30 voltages for activation and the persistence of inactivation even at , the time course of run-up of the calcium current is decreased in M M M vs. BAPTA ( BAPTA ( 9 0m 30 )). Boltzmann fits to the normalized C D M ) , respectively, show a trend towards depolarized n n I BAPTA). – 2fr1m 12 for 1 = 6) m as compared to = 1 V plots generated from the protocols described M D M Replotting the . and ( ª M o3 m ) to 30 BAPTA ( E , s current responses to 1 M ª M ( ) and V 9 BAPTA ( Î ). In addition, the of activation I – M n= V BAPTA, curves gave _1 o 0m 6 for 30 n M. E. Schnee and A. J. Ricci , 12). The = A M B ) and M V Î 5m and 2.8 in constants time between found was difference h iecntnsi hw sabrgahi i.9 Fig. in graph bar a as shown is constants time the BAPTA, m 30 and 10 both With shown). BAPTA the 10 complex effect of demonstrates increased BAPTA (Fig. recording whole-cell obtaining after a commonmechanism.Peakcurrentplottedagainsttime run-up and inactivation to calcium buffers might indicate the of sensitivity in similarity The respectively. BAPTA, 2 9, 6 7 ad 18±18m wr obtained were ms 118 m ± 2.8 1128 and for 178 ± 961 192, ± 920 of constants time Slow treatment. any between constant rm7±2t n hn3±1mni ,1 n 0m min in 1, 10 and 30 1 ± 1 and then 3 ± 2 to 5 ± from 7 4 ad 6 V ih lps f . . and 0.5 ± 4.0 of slopes with mV 1 ± 7±1mV _61 and 1 ± _47 f75±05mV ( 0.5 ± of 7.5 line is a 0.05 linear and regression an having intercept ± a slope of 1 line represents an intercept of 0 and a slope of 1. The continuous against the 9 5m 0m 30 lt ad h iatvto pos hfe twrs more 10 towards (Fig. shifted values depolarized plots inactivation the and plots activation the Both unknown. is run-down this of cause bu 1 i ad hn a rdcd y 2% 1 m (10 % 12 by reduced was then and min 12 about BAPTA solutions, the calcium current tended to peak after under varying conditions of external divalent cations and cations divalent external of conditions varying under 10 Fig. in shown as inactivation for that against activation for voltage the of plot the in found curve mirrors that of the these curveswereunaffected.Theshiftintheinactivation been implicated(Bodding&Penner,1999).Theslopes of has magnesium and GTP involving mechanism a where on effects Similar activation have been reported observed. for adrenal chromaffin cells, was values slope between ( respectively described earlier is 9 given in Fig. protocol prepulse standard the from obtained properties inactivation steady-state the of summary A respectively. o netgt frhr h clim eedne of dependence chelator BAPTA was increased calcium from m 1 to 10 or 30 the inactivation, the further concentration of the intracellular calcium investigate To substitute for calcium. to appears barium but calcium, by driven is inactivation that suggest results these inspection, first At respectively. n 5m 5 and erae rm2 n1m 1 in % 1 ± 23 from decreased index inactivation The 10. Fig. in summarized are results 3 t _6±3m fr , 0 n 3 m 30 and 10 1, for mV 3 ± _26 to 5 to ± 2 _33 ± _43 from varied voltages mV, half-inactivation 2 while ± 4 to _25 ± 1 to _28 ± activation varied from _39 ihfeuny,1 m , high frequency), 10 M M M external barium ( calcium. No difference was found in the slowest time BAPTA. The time constant of the run-up decreased M n _1 M V ) n 6 (0m (30 % 63 and 9) = Î aim Te otg o hl-nciain was half-inactivation of voltage The barium. fiatvto o m of inactivation for 1 acu, m 5 calcium, o 5m 5 for n r 2 = 12, 9 and 6, respectively). No difference No respectively). 6, and 9 12, = 0.83). = 8 M M ) has a linear relationship. The dashed BAPTA ( I acu ad m 5 and calcium M – B V acu ad m 5 and calcium and plot. Confirmation of this is 2 M M F ,3 m ), 30 BAPTA ( M C and BAPTA to 16 ± 1 % in % 1 ± 16 to BAPTA ). The values of values The ). BAPTA, E . The data obtained data The . G M o m for 5 ª BAPTA ( A M , low frequency, : 1 J Physiol intracellular n M M M vs. ) The 6). = M • BAPTA, barium, barium, calcium 0m 30 C ) and M V . No . The . 549.3 Î of M M M Journal of Physiology to be driven by calcium entry. value near the cell’s resting potential. Inactivation appears the channelsarepredictedto beinactivatedbyapotential channels. Third, haircellcalciumchannels inactivate.Abouthalfof high-frequency and than times potentials rise faster hyperpolarized having at activating channels low-frequency with channels, calcium activation the the of properties in exists difference tonotopic a Second, cells. hair papilla auditory turtle are in present channels functionally calcium L-type only First, investigations. present the from come have findings important Several DISCUSSION compared also 10 were mV (Fig. _10 to depolarizations s 1 plot, to linear same the Responses mechanism. underlying common a along suggesting fall all buffering internal J Physiol 0m 30 msin1and 3 ± 0.5 to22 ± increased byBAPTAfrom5.5 a nrae o ohte1 n 0m 30 and 10 the both for increased was constant was reduced from 903 ± 132 to 245 ± 22 ms in ms 22 m ± 30 245 to 132 ± 903 from reduced was constant hltr ,‚dfurBPA DB hs a has calcium (DFB) 5,5‚-difluoroBAPTA The chelator inactivation. of mechanism the decipher Two additional experiments were performed to attempt to channel and the slow component the being to further removed. close being components one fast the calcium, being possibility by driven is inactivation that hypothesis oprdt h m 1 the to compared 3 n 635 nakn o te nciain y niiig the inhibiting by reduce normally the total inactivation would recorded. inactivation that channel the of the the rephosphorylation reflect The of may channel. inactivation unmasking the of of robustness dephosphorylation increased the to due requires phosphorylationandthatinactivationis,inpart, current pass to channel the of ability the that suggesting 11 (Fig. robustly inactivated current calcium the Under conditions these activity. for MgATP require ATP on depending processes most intra- as inactive, the ATP renders pipette from cellular magnesium of that Removal possibility inactivation. the for required for was dephosphorylation or tested phosphorylation experiment second A implicating a calcium-dependent inactivation process. min, 11 first the in completely almost down run currents salsmn o te hl-el ofgrto are corresponding configuration their with whole-cell illustrated the of establishment i.11 Fig. buffer. Anexampleofthistypeexperimentisshownin calcium levels would be expected to approach the elevate baseline intracellular calcium levels. At steady state, incorporating DFB into the patch pipette it was possible to M M M wees ht o BPA s 6 n 160 is BAPTA for that whereas , BAPTA. The proportion of fast:slow inactivation fast:slow of proportion The BAPTA. 549.3 AT, epciey Hwvr te lws time slowest the However, respectively. BAPTA, A D C . h fs tm cntn ws significantly was constant time fast The ). hr rcrig a 10 40 n 60s after s 660 and 430 100, at recordings where and D ), but did not recover from inactivation, from recover not did but ), M BAPTA. These results support the support results These BAPTA. M I Calcium currents in auditory hair cells – V BAPTA when BAPTA K lt. The plots. d f about of K M d of the By . acu cret Fg ) ByK84 ptnits the potentiates 8644 K Bay antagonists 3). L-type Non-dihydropyridine 4). (Fig. current (Fig. whole-cell the current dihydro- of calcium % 90 doses, than more high block can and pyridines potentials depolarized At release. for driving both electrical resonance and neurotransmitter responsible is channel this that implying channels, L-type through only carried is current calcium cell hair auditory Several pieces of data support the conclusion that the turtle Types of channel present acu ufr( m calcium buffer (1 intracellular calcium is elevated by using a low-affinity Figure 11. Calcium currents run down pA to when near 0 below. protocol given at the top and current responses over time shown A no shift in activation curve was observed ( the whole-cell configuration with corresponding D dependent processes will fail without the presence of magnesium. included, also demonstrated strong run-down behaviour. ATP- filled with an internal solution where magnesium was not the recording. No shift in activation was observed. , examples of the time course of run-down, with the stimulus , examples of currents elicited at different times after establishing B , I – V plots show the decrease in current amplitude during M difluoroBAPTA, n 3). = n =3) I – C V , electrodes, plots. Again, 713 Journal of Physiology conductances may be limited (Hamilton & Perez, 1987). el urn. actxn lo id na te dihydro- the near binds pyridine siteandsoitseffectivenessatblocking also Taicatoxin current. cell an of L-type antagonistthatwasineffectiveinblockingthehair example another with is Taicatoxin but efficacy. channels different of types both more antagonize pyridines be may in that it calciseptine has no effect on the light, discriminating indifferentiatingbetweenL-typechannels this In channel. eue de o hs ae ifrne Nimodipine difference. same this insensitivity is to a hallmark of the due also reduced was affinity calciseptine that possible is it binding, dipine potency was limited, perhaps due to a difference in f20±0.1 ± 2.0 of IC an with current the antagonized Nimodipine 2). Fig. the to similar for reported kinetics again levels, current peak at ms 0.5 than 2001). Activationkineticswerefast,withrisetimesofless Koschak 2; (Fig. channels a the to respect with direction hyperpolarized a in shifted the of channels L-type expressed to similarities have channels calcium cell Hair channels Native channel compared with expressed (Yasuda site binding pyridine binds at the ineffective in blocking any part of the current. Calciseptine exception, since it is an L-type channel antagonist that was an is Calciseptine present. are channels L-type only that conclusion the support data these Together 1995). Fuchs, & (Zidanic type channel second a of presence the suggest changes in current rise times were observed that might also all fitted functions Boltzmann Single unmasked. or blocked selectively was type channel produced shifts in agents the pharmacological the of None 9). (Fig. L-type of calcium than permeable more signature is barium that is channels telltale a calcium; for substituted equimolarly was barium when doubled nearly magnitude current the finally, And the 1). of (Table part current macroscopic any antagonizing in ineffective were types Selective 5). blockers (Fig. for other current channel types the including N, R, of T and % P/Q 90 than more block 714 variety of L-type channels. the of are channels calcium the that conclusion the supports data the of preponderance the differences, small these Despite different. were kinetics the and channels, was not nearly as robust as that cell exhibited by expressed it inactivation, hair of degree significant a the exhibited channels although addition, In 2001). expressed Lipscombe, some for reported xii a exhibit not did block nimodipine The 3). Fig. 2001; Lipscombe, . 0.3 ± 2.7 1C type channel and are comparable to expressed to comparable are and channel type 1C m M U a I m sae vlae eedne a hs been has as dependence, voltage -shaped – subunit at a region overlapping the dihydro- V M o expressed for plots that might be expected if a particular for the hair cell channel as compared to compared as channel cell hair the for a 1D channels (Koschak channels 1D a 1D variety. The variety. 1D et al. et a 1D channel, while dihydro- I t al. et a – a 2001; Xu & Lipscombe, & Xu 2001; 1D subunit of the L-type V D hnes X & (Xu channels 1D a D hnes X & (Xu channels 1D plots. In addition, no addition, In plots. 93. ic nimo- Since 1993). I – V a et al. et plots were plots M. E. Schnee and A. J. Ricci 1D a 1D-type 2001; a a a 1D 1D 1D 50 hw iatvto ta ws trbtd o n R-type an to (Martini attributed current, was that inactivation shown have types cell hair Other conductance. additional some by contamination of artefact an not is inactivation that suggest data these Together important. is accumulation calcium that that suggesting current, calcium the antagonized blockers by reduced significantly was inactivation and none possibility was effective this in altering inactivation. address In addition, to performed were experiments pharmacological of variety A inactivating. not was itself some otheroutwardcurrentandthatthecalcium by Another contaminated were data present the more that inactivation. is possibility currents calcium-induced the to make susceptible here measured amplitudes current larger the that possible also is It current. calcium usually is that inactivation irreversible calcium despite the presence of of a reasonable amplitude as loss a stress, in metabolic results loading of cells states dissociated different at to are compared cells the that as possible is It 1998). Roberts, & (Armstrong papilla intact an that the difference observed here comes from recording in current so properties inactivation alters digestion maximal enzymatic that and observed possible also is It underestimated. likely been most amplitudes have not inactivation may time-dependent used, was pulses between was very sensitive inactivation to between the IPI of used, so that if presence too differences short a the time example, technical For experiments. are there is that It mystery. a possible is not do types cell hair auditory and other inactivation show cells turtle Why inactivation. of component significant a of presence the is work previous The major difference between the data presented here and type is conserved across different species. 2002) and the data presented here suggest that this channel al. pig and mouse inner hair cells (Zhang cells (Nakagawa diinl hne tps ae en dniid n turtle in auditory hair cells. identified been have types channel additional cells (Kollmar cells importance of the the channelbeingof Fuchs 1987; Fettiplace, & (Art cells hair auditory from obtained data with agreement in also is potentials negative at and in turtle (Art cells. ThatthechannelsareofL-typehasbeensuggested with agreement previous in work regarding calcium largely channels in auditory are hair here presented data The Comparisons with other hair cell types ugs ta lwfeuny hnes r dfeet from different are channels low-frequency that suggest steady-state the and times rise current the Both Tonotopic differences al. et 2000; Engel 1997 et al. et b ; Spassova ; 1990; Engel 1990; et al. et al. et et al. et al. a 1986), chick (Fuchs t al. et 1D subtype of channel in cochlear hair 2002). That currents activated rapidly 1997 1991; Chen et al. et et al. et a b 1D type.Evidenceexistsforthe ; Platzer ; 00 Perin 2000; 2001), guinea-pig outer hair outer guinea-pig 2001), 2002) and is consistent with consistent is and 2002) et al. et al. et et al. et al. 1995) andguinea- t al. et 2000; Engel 2000; 1999; Platzer 1990; Kollmar J Physiol 01. No 2001). I – V plots et al. et 549.3 et Journal of Physiology os o hv a ao efc o eetia resonance. electrical on effect major a have the not does shaping in inactivation that suggest results Current-clamp cell? the hair play of resonance) electrical inactivation (i.e. response physiological does role What the calcium channels, reprime making more available to to open. by necessary be induced might stimulation efferent hyperpolarization the is where regulation system, this efferent of transmitter mediator and possible excitability A release. cell for hair mechanism both powerful regulating a suggests cell’s potential hair the resting at inactivated be the might of current half calcium to up that conclusion The inactivated. was long current the of % 50 about that potential resting cell’s hair the demonstrated at and current the of most examples inactivate can depolarizations inactivation, of Although the protocols used for analysis evoked low levels positions would also support this hypothesis. frequency different at cells hair between comparable was 2000). The present results demonstrating that inactivation the (Ricci tonotopically and increases hot-spots area of number surface the that but positions, between frequency constant remains channels calcium the of that density suggested has work previous However, more. be expected that cells with larger currents would inactivate that inactivation appears to be driven by calcium, it might Given magnitude. current the twice approximately have cells high-frequency high- that fact the and despite cells frequency low- between comparable was Inactivation Physiological significance of inactivation (Ricci channels BK the for work has demonstrated a similar difference in values of Previous measurements. between consistent was it mV, 7 steady-state the difference Although between frequency positions was modest, about channels. high-frequency J Physiol uiir subunits, the auxiliary in Differences exist. possibilities additional Several a the the of function a be not may properties cell hair in difference that implying 2001), Lipscombe, & (Xu properties resting in a difference the potential. Recent evidence expressing for splice variants of the responsible ultimately the for responsible hyperpolarized part, in least at is, current calcium cells. It is probable that the hyperpolarized high-frequency to compared potential resting polarized hyper- a have cells low-frequency that demonstrate also ie a dtriig h udryn mechanism underlying the determining responsible for these differences. at aimed (Yang activation properties in differences cause to purported been have syntaxin like proteins accessory addition, In responsible. be also might differences Post-translational properties. 1D and not all have been expressed (Safa expressed been have all not and 1D activation in difference no reported channel 1D a subunit. However, many splice variants exist for the 549.3 I – V t al. et uv o te K hne, hc is which channel, BK the of curve b or 99. uue xeiet are experiments Future 1999). et al. et a 2 d mgt le activation alter might , 2000). The present data present The 2000). Calcium currents in auditory hair cells I – V et al. et plot of the 2001). et al. et V Î possibility. this investigate to underway presently are Experiments does and/or that simplest these channels regulate synaptic strength. The that the BK channel activates channels? whenever a calcium channel calcium ensure to present are channels these that are possibilities additional close the its its of to activate due to proximity (Roberts channel sufficient potassium is corresponding channel calcium one on number channel calcium membrane excitability may the suggest that at any given time, of effect of lack The of tonotopic position (Wu position tonotopic of of calcium channels to potassium channels is 2:1 regardless excitability. Previous work has demonstrated that the ratio that further and addition of calcium resonance, channels has little effect on electrical membrane drive to needed of implying that the hair cell quality has more calcium channels than and frequency the resonance. However, no be effect on both resonance was observed, would alter to and be predicted potentials could current physiological the at of half inactivated as much as that suggest properties inactivation steady-state The conclusion. this of component hair cell’s receptor potential. Current-clamp data support temporal the inactivation does not play a significant role in shaping the Therefore, affected. Neither the frequency nor the quality of the resonance was h fc o 3 m 30 of face the in experiments where barium replaced calcium and also in reduced reduced but persistent was Inactivation was intra- calcium. cellular involvement current the implicating calcium pharmacologically, the even when or reduced eliminated was component, fast the Inactivation, particularly occurred. inactivation before current the current such that the by steady-state properties required activation of driven was Inactivation process. dependent calcium- a of typical is that dependence voltage shaped calcium-dependent a bell- the had Inactivation equivocal. somewhat is process implicating Evidence calcium. intracellular by regulated is inactivation the of part least, at that, suggest do phosphorylation but possibilities, three these between distinguish or cannot here presented data dependent The dependent. voltage dependent, calcium be can channels calcium L-type of Inactivation inactivation? for responsible mechanisms the are What Mechanisms of inactivation can substitute for calcium, albeit with a will limit the effectiveness of calcium buffers. Also, barium The Calcium summatingbetween channelsofcloseproximity clustering. channel manipulations. to part, in these due, be may to insensitivity sensitive less somehow calcium was involved but that the calcium dependence was concentrations will be determined by the sum of ion entry higher levels. First, thechannelsareclustered andsolocal these reach can barium of concentration the that possible that of calcium (Ferreira M et al. nrclua BPA sgetn that suggesting BAPTA, intracellular 1990). What then is the function et al. et al. et 1997). Three factors make it 1995; Ricci 1995; K d that is 100 times et al. et 2000). 715 Journal of Physiology Assad JA, Shepherd GM & Corey DP (1991). Tip-link integrity and Ashmore JF (1983). Frequency tuning in a frog vestibular organ. Art JJ, Wu YC & Fettiplace R (1995). The calcium-activated Art JJ, Fettiplace R & Wu YC (1993). The effects of low calcium on Art JJ & Fettiplace R (1987). 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