positional analysis of guinea pig inner hair cell membrane … · 2017. 8. 25. · raybould et al.:...

JARO 02: 362–376 (2001)DOI: 10.1007/s101620010087

Positional Analysis of Guinea Pig Inner Hair Cell MembraneConductances: Implications for Regulation of theMembrane Filter

NICHOLAS P. RAYBOULD, DANIEL J. JAGGER, AND GARY D. HOUSLEY

Molecular Physiology Laboratory, Physiology Division, Faculty of Medical and Health Sciences,University of Auckland, Auckland, New Zealand

Received: 30 October 2000; Accepted: 12 April 2001; Online publication: 10 October 2001

ABSTRACT the P2X receptor conductance, averaged 31 nS (acti-vated by 400 mM ATP; about 275 mV) irrespective ofcell origin. Thus, regulation of intracellular Ca2+ and

In mammals, sound transduction by inner hair cells activation of P2X receptors by extracellular ATP pro-(IHC) generates a receptor potential whose amplitude vide capacity for local dynamic fine-tuning of the IHCand phase drive auditory nerve firing. The membrane membrane filter.filter properties that define the input-output function

Keywords: cochlea, P2X receptor, ATP, potassium chan-of IHC are derived from membrane conductance and

nels, tonotopic, tuningcapacitance. These elements of the membrane filterwere quantified using whole-cell voltage clamp of IHCfrom the four turns of the guinea pig cochlea. IHCmembrane properties were remarkably constant along

INTRODUCTIONthe cochlea, in contrast with all other auditory haircell systems, and suggests that extrinsic processes such

Sound transduction in the mammalian cochlea canas the active filter provided by the outer hair cells are

be separated into forward and reverse componentsmatched to a constant transfer function of the IHC.

(Mountain 1986; Dallos 1992; Ashmore and KolstonTwo outwardly rectifying K+ currents contribute to the

1994). Forward transduction is the conversion ofIHC membrane conductance. These combined cur-

sound-induced fluid vibration in the cochlea to arents activate at approximately 255 mV. IHC mean

phase-modulated receptor current passing throughinput resistance was 140 MV and capacitance was 10.0

the mechanoelectric transducer (MET) channels,pF, generating a membrane time constant of 1.4 ms

localized to the tips of the inner hair cell (IHC) andor a corner frequency of approximately 115 Hz, which

outer hair cell (OHC) stereocilia (Denk et al. 1995).is consistent with reported low-frequency roll-off of

This current produces a receptor potential across thethe IHC AC receptor potential in vivo. Approximately

hair cell basolateral membrane, determined by cell40% of the 313-1 nS total K+ conductance about 0 mV

capacitance and conductance (the membrane filter),was attributed to charybdotoxin-sensitive KCa channels

which initiates neurotransmitter release and drives(also sensitive to cell dialysis with the Ca2+ chelator

auditory nerve firing. This process is conserved fromBAPTA or removal of extracellular Ca2+). The only

lower-vertebrate sensory hair cell physiology (Art etknown ligand-activated conductance in mature IHC,

al. 1986; Art and Fettiplace 1987; Hudsepth and Lewis1988; Hudspeth 2000). Reverse transduction is specificto the mammalian cochlea and arises from the genera-

Correspondence to: Gary D. Housley, Ph.D. • Molecular Physiology tion of a phase-related movement of the cell wall ofLaboratory • Physiology Division • Faculty of Medical and Health the OHC (Ashmore 1987, 1994; Holley and AshmoreSciences • University of Auckland • Private Bag 92019 • Auckland •

1988; Kalinec et al. 1992; Santos–Sacchi 1992; DallosNew Zealand. Telephone: (64) (9) 3737 499; fax: (64) (9) 3737 599ext 6300; email: [email protected] and Evans 1995). This movement is derived from the

362

RAYBOULD ET AL.: Positional Analysis of IHC Conductance 363

receptor potential-generated conformation changes of MgCl2 1.0, NaH2PO4 2.0, Na2HPO4 8.0, D-glucose 1,the unique OHC electromotile protein Prestin (Zheng pH 7.25 with NaOH, osmolarity 5 320 mOsm L21).et al. 2000) and is the active substrate of the “cochlear The otic capsule was split providing access to theamplifier” (Ashmore 1987, 1994; Kolston 1999). In cochlea, and a selected turn of organ of Corti wasconjunction with the passive mechanical filtering prop- microdissected from the modiolus. The isolated turnerties of the organ of Corti, the OHCs focus and was then incubated in 60 mL of standard solution con-enhance the sound energy to the IHCs in a frequency- taining 0.25 mg.mL21 trypsin (Sigma, St. Louis, MO,specific (tonotopic) manner (Kolston 1999). Trans- USA) for 10 min. Following trituration to aid dissocia-duction of this integrated stimulus by the IHC, the tion, the tissue was placed in an elongated 70 mL micro-primary sensory cell mediating auditory neurotrans- scope bath mounted on an inverted microscopemission (Ashmore 1994), generates the exquisite selec- (Nikon TMD, Tokyo, Japan) equipped with Nomarskitivity and sensitivity of the mammalian cochlea. differential interference contrast optics. Bath solu-

In lower vertebrates the absence of the cochlear tions were rapidly exchanged through superfusionamplifier tuning mechanism is mitigated by an electri- (500 mL/min21) by peristaltic pump (Gilson, Villiers-cal tuning of the hair cell membrane properties (Fetti- le-Bel, France). All experiments were performed atplace and Fuchs 1999 for a review). This mechanism room temperature (202228C).depends upon a tonotopic variation of Ca2+-activated IHCs were selected for study if they possessed theK+ channel (KCa) properties that optimize the trans- following morphological properties: uniform flaskduction of the stimulus for a given position along the shape, membrane birefringence, centrally locatedbasilar papilla (cochlea) (Wu et al. 1995; Navaratnam nucleus, intact stereocilia, and lack of Brownianet al. 1997; Rosenblatt et al. 1997; Jones et al. 1999a,b). motion in the cytoplasm (Fig. 1A). Images were cap-

Given the abstraction of the tuning process away tured continuously by video camera (Ikegami ICD 42E,from the IHCs in the mammalian cochlea, the present CCD, Tokyo, Japan) and stored on sVHS videotapestudy assessed the potential for the IHC membrane (Panasonic, Osaka, Japan). Cell dimensions (exclud-filter to provide tonotopic conditioning of the trans- ing stereocilia) were measured post hoc by image analy-duction current. The IHC membrane conductance, sis software (Image Pro Plus, version 1.0, Mediaand hence the membrane filter, is largely determined Cybernetics, Silver Spring, MD, USA) using an 8-bitby Ca2+-independent K+ (Kros and Crawford 1990) and videoframe grabber board (ITEX, Woburn, MA, USA).KCa conductances (Housley et al. 1993; Dulon et al. All measurements were made with 0.3 mm resolution1995; Appenrodt and Kros 1997; Kros et al. 1998). and corrected for the aspect ratio.In addition, IHC also exhibit an ATP-activated (P2X

Recording electrodes (2–5 MV; filamented borosili-receptor) conductance localized to the apical (endo-

cate glass, Harvard Apparatus GC120 TF-10, Eden-lymphatic) surface which modulates the driving forcebridge, Kent, UK) were filled with an internal solutionfor sound transduction (Housley et al. 1993, 1998a,(in mM: KCl 140; MgCl2 2.0, NaH2PO4 1.0, Na2HPO41999; Sugasawa et al. 1996; Housley 2000). The8.0, CaCl2 0.01, either 1,2-bis(o-aminophenoxy)e-observed constancy of the IHC membrane filter prop-thane-N,N,N8,N8-tetraacetic acid (BAPTA) 10 or ethyl-erties suggests that IHCs perform a low-pass transfer ofene glycol-bis(b -aminoethyl ether)-N,N,N8,N8-tetra-pretuned stimulus input (from the cochlear amplifier)acetic acid (EGTA) 0.5, D-glucose 3.0, pH 7.25 withthat may be locally regulated by variations in intracellu-KOH, osmolarity 5 310–320 mOsm L21). Whole-celllar Ca2+ or extracellular ATP.voltage clamp recordings were made using a patchclamp amplifier (Axopatch 200, Axon Instruments,Union City, CA, USA) controlled by computer softwareMETHODS(pClamp 5.0, Axon Instruments) via an interface(Tecmar TL 1, Labmaster Scientific Solutions, Solon,Guinea pig cochlear IHC were isolated and whole-Mentor, OH, USA). IHCs with stable current baselinescell patch clamp recordings were made as describedand zero-current potentials (Vz) more negative thanpreviously for OHCs (Raybould and Housley 1997).240 mV were selected for study. Application of a 10Experiments were performed with approval from themV hyperpolarizing step (50 ms) from a holdingUniversity of Auckland Animal Ethics Committee.potential of 260 mV permitted post hoc calculationGuinea pigs (160–1000 g, either sex, pigmented andof membrane resistance, capacitance (Cm), and seriesalbino) were euthanized by an overdose of anestheticresistance (Rs) from the charging transient (sampled(sodium pentobarbitone 150 mg.kg21, Abbott Labora-at 30 kHz). The mean series resistance derived aftertories, North Chicago, IL, USA) injected into the intra-capacitance compensation was 5.0 6 0.2 MV (n 5peritoneal cavity. The temporal bones were removed226). Recordings were corrected online for bathand placed in an artificial (standard) perilymph solu-

tion (composition in mM: NaCl 140, KCl 4, CaCl2 1.5, potentials and .95% series resistance-induced voltage

364 RAYBOULD ET AL.: Positional Analysis of IHC Conductance

FIG. 1. Ancillary activation of KCa conductance is mediated by Ca2+ we attribute to activation of additional inward current. B. Inclusionentry through the inner hair cell ATP-gated ion channels. A. In Ca2+- of 10 mM BAPTA in the internal solution suppressed the ATP-mediatedfree external solution or solution with endolymphatic Ca2+ levels (40 activation of the KCa conductance in external solution containingmM), bath-applied ATP (100 mM) produced a sustained inward current 1.5 mM Ca2+ (comparable to subsequent exposure to ATP under(Vh 5 260 mV) that was reversible with washout (internal solution nominally Ca2+-free condition; bath application of 100 mM ATP). C.includes 0.5 mM EGTA). Subsequent application of ATP with 1.5 Coapplication of the KCa channel blocker iberiotoxin (1 mM) withmM Ca2+ in standard external solution resulted in a biphasic current ATP (100 mM) from the same pipette barrel resulted in a delayedresponse, where the inward current was transient and later masked inhibition of the outward current in standard external solution. Insetby the developing outward current (right trace). Note that this outward image is a video micrograph of an inner hair cell.current declined prior to the end of the ATP application, an effect

error. Junction potential was not corrected for (esti- IHC slope conductances were derived from current-voltage relationships (I/V) obtained directly from volt-mated at 23 mV for the external/internal solutions

used). Data from voltage-step protocols was filtered age ramps (2100 to 150 mV; 1 s). These voltage rampswere repeated every 5 s in experiments to monitoronline using the patch clamp amplifier, 4-pole low-

pass Bessel filter (10 kHz) and digitized at 20 kHz. changes in membrane conductance. Initial superfu-sion of standard solution was followed by superfusionThe recording time constant was approximately 50 ms

based on the average of the R s 3 Cm data and the of nominally Ca2+-free solution (where 1.5 mM CaCl2was replaced with MgCl2; estimated at 3 mM Ca2+ basedlimiting factor of the temporal resolution of the volt-

age clamp. on published residual levels in buffer stocks). The Ca2+-free external solution along with BAPTA in the inter-In experiments studying KCa channel block, ATP

(disodium salt, Sigma) was pressure applied via a dou- nal solution minimized background KCa channel acti-vation. ATP was applied by bath substitution at twoble-barreled glass pipette controlled by a computer-

gated solenoid (Mac Valve Pacific Ltd., Auckland, New concentrations: (1) 10 mM, close to the EC50 of theATP-gated currents in IHCs and (2) a supramaximalZealand) and co-applied with either of the blockers

iberiotoxin and charybdotoxin (Alomone Labs, Jeru- dose of 400 mM (Sugasawa et al. 1996). The I/V ofthe P2X receptor conductance was calculated by sub-salem, Israel).


traction of the control I/V in Ca2+-free solution from (Evans et al. 1996) necessitated the establishment ofthe I/V obtained during the superfusion of ATP in protocols for isolation of the individual conductances.Ca2+-free medium. Membrane conductance was deter- The experiments described below indicate that sec-mined from the I/V about 275 mV (G275mV) and about ondary activation of KCa conductance during ATP0 mV (G0mV) as the slope between 260 and 290 mV application could be blocked using nominally Ca2+-and 210 and 110 mV, respectively. free conditions, significant intracellular Ca2+ chela-

The kinetics of channel opening was determined tion, or selective KCa channel blockers. In addition,from the current responses elicited by a series of volt- these experiments established that under physiologi-age steps (2100 to 130 mV, 10 mV increments, 40 cal (endolymphatic) Ca2+ levels, ATP is unlikely toms, Vh 5 260 mV). The activation time constant of cause activation of KCa channels via Ca2+ entry throughthe outward current was best fitted by a single expo- the P2X receptors. However, ATP will depolarize thenential I(t) 5 A exp[-(t 2 K)/t] 1 c, where A is the IHCs and therefore recruit voltage-activated con-current amplitude, K is the time at the start of the ductances.fit, t is the time constant, c is the offset over the first In initial experiments with IHCs isolated from2 ms for fast channel kinetics or from 3 to 40 ms for throughout the organ of Corti, using standard solutionslower channel kinetics (Clampfit 6.0, Axon (containing 1.5 mM Ca2+), and with low intracellularInstruments). Ca2+ buffering (0.5 mM EGTA), bath application of

Differences were considered statistically significant extracellular ATP (100 mM) produced a biphasic cur-at the p , 0.05 level. We used the statistical package rent profile where the initial inward current was fol-SyStat (SYSTAT Inc., Evanston, IL, USA) to perform lowed by activation of an outward current (Fig. 1A,one-way analysis of variance (ANOVA) and paired and right trace). This substantial outward current has beenunpaired Student’s t-tests where appropriate. Data are attributed to secondary activation of KCa channels viapresented as mean 6 SEM. Ca2+ influx through the ATP-gated ion channels

(Housley et al. 1993; Dulon et al. 1995; Sugasawa etal. 1996). The mean delay between the onset of the

RESULTS inward current and subsequent inflection resultingfrom the activation of the KCa conductance was 866

The average IHC length was 28.6 6 0.2 mm and the6 99 ms (n 5 16). In nominally Ca2+-free solution,

average cell width was 15.8 6 0.2 mm, with no signifi- bath application of ATP typically produced a sustainedcant variation between cells from the four turns of the inward current response (Fig. 1A, left trace). Similarcochlea ( p . 0.05; one-way ANOVA; n 5 297). Under

results were obtained using an endolymphatic level ofwhole-cell voltage clamp, IHCs exhibited marked out-

Ca210 [40 mM (Gill and Salt 1997); Fig. 1A, center trace],ward rectification of voltage-activated currents compa-

while subsequent exposure to ATP in 1.5 mM Ca210rable to previous reports of IHCs with low resting

resulted in a biphasic current response. With anintracellular Ca2+ levels (Dulon et al. 1995; Sugasawaincreased level of intracellular Ca2+ buffering (10 mMet al. 1996). IHC capacitance (Cm) had an average ofBAPTA), bath-applied ATP produced a sustained10.0 6 0.2 pF (n 5 226; measured between 260 andinward current with 1.5 mM Ca21

0 (n 5 10; Fig. 1B).270 mV) and was independent of cell origin ( p .In standard solution (with 0.5 mM EGTA in the0.05; n 5 125). The average initial zero-current poten-recording pipette solution), pipette co-application oftial (Vz) of the IHCs, determined upon breakthroughATP (100 mM) with iberiotoxin (1 mM), a potentto whole-cell recording, was independent of intracellu-blocker of slo-type KCa channels (Kaczorowski et al.lar Ca2+ buffering ( p . 0.05; Vz 5 256.8 6 1.0 mV, n1996; Toro et al. 1998), either failed to activate the5 69, 10 mM BAPTA; Vz 5 257.4 6 0.8 mV, n 5 84,outward KCa conductance (sustained inward current0.5 mM EGTA).response, n 5 5; data not shown) or produced a greatlyattenuated biphasic response (n 5 6; Fig. 1C).

Interaction of KCa and P2X receptor The role of the KCa conductance in the generationconductances of the biphasic current response to ATP was further

examined using charybdotoxin (ChTx; Kazorowski etThe aim of this study was to quantify the principalal. 1996). In standard solution, application of ATPvoltage- and ligand-activated conductances of IHCs in(100 mM) with ChTx (1 mM) from a single micropi-order to measure their contribution to the membranepette barrel produced a sustained or weakly desensitiz-filter with respect to cell origin along the organ ofing inward current of 21289 6 128 pA (n 5 31; Fig.Corti. The potential for interaction between KCa con-2A). After washout of the ChTx, 100 mM ATP in stand-ductance, activated by both voltage and Ca2+ (Toro etard solution (from the second barrel of the drugal. 1998), and ATP-activated (P2X receptor) conduc-

tance that exhibits a significant Ca2+ permeability pipette) produced a rapid biphasic current response


FIG. 2. The biphasic currentresponse to extracellular ATP ismediated by charybdotoxin (ChTx)-sensitive KCa channels. A. In stand-ard solution, co-application of 100mM ATP and 1 mM ChTx by micro-pipette produced a slowly desensi-tizing inward current response.Subsequent application of 100 mMATP alone produced a rapid bipha-sic current response (Vh 5 260mV). Note that the outward currentphase declined prior to the end ofthe ATP application, an effect weattribute to recruitment of unchar-acterized inward current. Dashedline indicates zero current. B. Chartrecord of IHC current duringrepeated voltage ramps (2100 to150 mV, 1s, 0.2 Hz, Vh 5 260 mV)during bath application of ATP (100mM), with and without ChTx (1mM). Note that the ATP-mediatedactivation of the KCa conductance,evident as an increase in peak out-ward current (traces 2–4), isblocked by ChTx (trace 7). C.Recruitment of KCa conductanceduring the ATP response in standardsolution is detailed by the current-voltage relationships (I /V ) plottedfrom traces 1–4 from B. ATP initiallyproduced a depolarizing shift in Vz

(standard: trace 1 5 251 mV, ATP;trace 2 5 239 mV) with subsequenthyperpolarizing shifts arising fromrecruitment of the KCa conductance(trace 3 5 248 mV, trace 4 5 260mV; detail shown in the inset).Traces in the inset were obtainedby subtracting control trace (1) fromtraces 2–4. D. Detail of the I /Vrecords obtained during ChTxblock (100 nM in the bath) of theKCa conductance shown in B (trace5, control Vz 5 255 mV; ChTxblock, trace 6, Vz 5 246 mV) andsubsequent ATP-gated inward cur-rent during ChTx block (pipetteapplication: 100 mM ATP, 1 mMChTx). In the presence of ChTx, ATPcaused a sustained depolarizingshift in Vz to 241 mV (trace 7).Upper inset: The ChTx-sensitive KCa

current (IKCa derived by subtractingtrace 6 (ChTx block) from trace 5(standard). Lower inset: ATP-gatedcurrent (IATP) derived by subtractingtrace 6 (ChTx block) from trace 7(ATP 1 ChTx). Note that the reversalpotential of the ATP-gated current(with ChTx) is close to 0 mV.


in 11/25 cells (Fig. 2A) (peak inward current 5 21108 ATP-gated channel activation to quantify IHC mem-brane conductance properties with respect to cell ori-6 96 pA, n 5 25).gin in each of the four turns of the guinea pig cochlea.The secondary activation of the KCa conductance

during the biphasic ATP response was evident from aprogressive leftward shift toward the calculated EK Voltage-activated potassium conductances with(290 mV) in the current–voltage (I/V) relationship respect to cell originof the IHCs (measured using repeated voltage ramps,

In the guinea pig cochlea, the basal (first) turn2150 to 1100 mV, 1 s, 0.2 Hz) in standard solutionencodes frequencies approaching 45 kHz, whereas the(Figs. 2B and C). The derived I/V of the ATP-activatedapical (turn 4) region encodes low-frequency soundconductance (bath application) illustrates the progres-(,250 Hz, Pujol et al. 1992). Voltage ramps (2150 tosive recruitment of the KCa conductance following an1100 mV, 1 s) applied within 5 s of breakthroughinitial inwardly rectifying P2X receptor conductanceto whole-cell configuration (prior to 10 mM BAPTA(Fig. 2C, inset). This rectification resulted in an ATP-dialysis) revealed an outwardly rectifying conductanceinduced increase of the average IHC slope conduc-with a mean threshold of activation of 256 6 2.1tance about 275 mV from 17.3 6 3.1 to 83.5 6 10.0mV (n 5 69; Fig. 3). This represented the combinednS (n 5 24). The slope of the I/V’s measured aboutactivation of the KCa conductance, described above,0 mV (standard 5 202.1 nS, ATP 5 210.5 nS) did notand Ca2+-insensitive outwardly rectifying K+ conduc-vary significantly ( p . 0.05, paired t-test; see Fig. 2C).tance (Kros and Crawford 1990).In six of these experiments, we subsequently super-

There was no evidence for a systematic variationfused ChTx (100 nM) to assess the KCa conductance.in IHC voltage-activated potassium conductances withTwo experiments were subsequently excluded becauserespect to cell origin. Membrane conductances aboutthe baseline conductance did not fully recover from275 or 0 mV measured immediately after establishingthe preceding ATP challenge. In the remaining fourwhole-cell recording showed no difference withcells, ChTx produced a 45% reduction in mean sloperespect to turn of origin ( p . 0.05, one-way ANOVA;conductance about 0 mV from 169.1 6 11.7 to 93.3 6Figs. 4A and B). Conductance measured about 27511.9 nS, with no significant effect on the conductancemV (G275mV) ranged from 1.6 to 24.4 nS with a meanabout 275 mV (standard 5 18.3 6 4.6 nS to standardof 7.3 6 1.2 nS (n 5 69; Figs. 4A and C), equivalent

1 ChTx 5 14.8 6 4.5 nS; Figs. 2B and D). Theseto an input resistance of approximately 140 MV . Mem-changes in membrane conductance with the additionbrane conductance about 0 mV (G0mV) ranged fromof ChTx to the external solution were associated with52.5 to 634 nS with a mean of 313 6 16.3 nS (n 5 69;

a depolarizing shift in Vz from a mean of 258.3 6 1.2Figs. 4B and D), equivalent to an input resistance of

to 250.8 6 2.0 mV. The ChTx-sensitive (KCa) conduc- only 3 MV . The modes of the histograms for IHCtance was isolated by subtracting the I/V trace conductance about 0 mV were between 200 and 400obtained during ChTx perfusion from the control I/ nS for all four turns (Fig. 4B). Intraturn variabilityV (Figs. 2B and D). The activation threshold of this here may mask interturn differences, but the histo-conductance, determined from the derived I/V (Fig. gram profiles make this unlikely.2D, upper inset) as the intersection of the tangents of Repeated voltage ramps (2100 to 150 mV, 1 s, 0.2the upward slope of the current trace with the slope Hz) showed a stabilizing reduction in KCa conductanceof the trace negative to 260 mV, had a mean of 252.3 over approximately 7 min after breakthrough to whole-6 1.9 mV (n 5 4). In comparison, addition of 100 nM cell configuration (Fig. 3A), consistent with previouslyiberiotoxin to standard solution produced a smaller reported buffering of intracellular Ca2+ by BAPTA in(25%) reduction in membrane conductance about 0 IHC following the onset of voltage clamp (SugasawamV from a mean of 120.7 6 30.0 to 89.7 6 13.2 nS et al. 1996). The reduction in membrane conductance(n 5 4). was not observed when the lower-affinity Ca2+ chelator

Bath application of 100 mM ATP produced a sus- EGTA (0.5 mM) was included in the recording pipette.tained inward-current response in the presence of 1 This is consistent with the differential effect of thesemM ChTx (n 5 23), with an increase in slope conduc- two Ca2+ buffers on the ATP-induced secondary activa-tance about 275 mV of 35.8 6 2.3 nS (n 5 4). The tion of KCa channels (compare Figs. 1A and B). WithI/V of the net ATP-gated current showed pronounced BAPTA dialysis, the mean steady-state membrane con-inward rectification, reversing close to 0 mV (Fig. 2D, ductance about 0 mV was 188.0 6 13.0 nS (n 5 62;lower inset). Fig. 4D), indicating, as found with the ChTx experi-

These experiments established the considerable ments, that the KCa conductance accounts for approxi-dynamic range of IHC conductance regulated by intra- mately 40% of the outwardly rectifying conductance.cellular Ca2+ and extracellular ATP. Subsequent experi- The conductance measured about 275 mV slightly

increased under these conditions (steady-state meanments utilized these protocols for isolating KCa and


FIG. 3. Voltage-activated andATP-activated conductances of anisolated IHC. A. Chart record show-ing the current trace during thesequential superfusion of standardsolution, Ca2+-free solution, 10 mMATP in Ca2+-free solution, Ca2+-freewash, 400 mM ATP in Ca2+-freesolution, and Ca2+-free washout(voltage ramp protocol: 2100 to150 mV, 1 s, 0.2 Hz, Vh 5 260mV). A progressive reduction in theoutward component of the current–voltage (I /V ) relationship is evidentduring the initial recording phasein standard solution because ofdialysis of the IHCs by 10 mMBAPTA (comparison of t2 with t1,where t1 was obtained approxi-mately 200 s after establishingwhole-cell recording). Subsequentsuperfusion of Ca2+-free solutionproduced a further small reductionin the outward component of the I /V (trace 3). Application of 10 mMATP in Ca2+-free solution producedan inward current of 2608 pA (Vh

5 260 mV), seen as a downwardshift in the current trace (trace 4)that was reversible with washout.Subsequent application of a supra-maximal concentration of ATP (400mM) produced an inward current of2998 pA (trace 5). B. I /V relation-ships of the voltage-activated andATP-activated conductances (traceslabeled in A). Inset shows theinward rectification of the net P2Xreceptor conductance, determinedby subtraction of the I /V in Ca2+-free solution from the I /V with ATPin Ca2+-free solution.

5 14.0 6 0.9 nS, p , 0.05, unpaired t-test, n 5 62; solution ( p , 0.05, paired t-test, n 5 56; Fig. 4D). Therelatively small background conductance at 275 mVFig. 4C). The lack of turn-dependent variation in

steady-state membrane conductance about both 275 showed variable changes with the switch to the Ca2+-free solution (average 5 16.25 6 1.2 nS, n 5 56; Fig.and 0 mV is shown in Figure 7.

Subsequent superfusion of these IHCs with nomi- 4C). The residual Ca2+-insensitive voltage-activatedconductance determined at both 275 and 0 mV wasnally Ca2+-free solution produced a mean depolarizing

shift in Vz of 6.4 mV (mean Vz 5 250.4 6 1.0 mV, independent of cell origin ( p . 0.05, one-way ANOVA;Fig. 7).n 5 56, p , 0.05). These changes in Vz in Ca2+-free

solution were independent of IHC origin ( p . 0.05, The preceding experiments showed that there wasno variation in magnitude of the voltage-activated con-one-way ANOVA). There was a significant ( p , 0.05)

depolarizing shift in the threshold voltage of the out- ductances with respect to cell origin. However, we alsoconsidered the possibility that a variation in the kineticwardly rectifying conductance, to an average onset in

Ca2+-free solution of 246.8 6 2.8 mV (n 5 56). properties of the voltage-activated channels could pro-vide a basis for IHC tuning [as noted for KCa channelsTogether, these data are consistent with the further

suppression of a residual KCa conductance. in lower-vertebrate hair cells (Fettiplace and Fuchs,1999)]. To address this issue, we undertook a separateRemoval of Ca21

0 decreased the membrane conduc-tance about 0 mV to 160.2 6 11.7 nS, a significant series of experiments to characterize the activation

kinetics of IHC membrane currents in response todecline compared with levels at steady state in standard


FIG. 4. Summary of the voltage-activatedconductances of IHCs isolated from all fourturns of the cochlea. A, B. Comparison of thedistribution of slope conductance measuredabout 275 mV (G275mV) and 0 mV (G0mV)within 5 s of attaining whole-cell recordingshows no systematic variation across thecochlear turns. C. Slope conductance about275 mV (G275mV) increased during dialysis ofthe cell with BAPTA and exposure to Ca2+-freesolution [std (t0) 5 initial conductance; std(t1) 5 conductance 60–180 s after std (t0); std(t2) 5 steady state immediately prior to Ca2+-free solution]* indicates significant difference( p , 0.05, unpaired t-test for adjacent bars.D. Slope conductance about 0 mV (G0mV)decreased during BAPTA dialysis and superfu-sion of Ca2+-free solution. Each bin in C andD represents the mean 6 SEM for a total of56–69 IHCs.

depolarizing voltage steps. These experiments used and D, inset). The latter IHCs had activation thresholdsof approximately 230 mV, with a voltage-dependentthe lower cytosolic Ca2+ buffering (0.5 mM EGTA) to

preserve the KCa conductance. IHC from turns 1–3 activation best fitted (over 40 ms) by slower singleexponential functions with time constants that ranged(n 5 38) and 14/23 cells from turn 4 exhibited fast

activation kinetics (Figs. 5A, B, and D) best fitted by from 51.8 6 5.4 ms at 220 mV to 10.6 6 0.7 ms at130 mV (Fig. 5D, inset). During the initial 2 ms, thea single exponential function over the first 2 ms (activa-

tion threshold approximately 255 mV). Five turn 4 activation kinetics of IHCs from turns 1 and 2 werefaster than those of IHCs from turns 3 and 4 ( p ,IHCs exhibited a variable mixture of fast- and slow-

activating outward currents (obvious as a pronounced 0.05, unpaired t-test; Fig. 5D; analysis excludes the fourslowly activating turn 4 IHCs). Between 230 and 130deviation from the single exponential fit to the initial

2 ms); four IHCs had no fast component (Figs. 5C mV, the mean activation time constants of the fast-


activating outward current records over the initial 2 to 0 mV. This P2X receptor conductance exhibited nodependence on IHC turn of origin ( p . 0.05, one-ms shortened from 0.9 6 0.1 ms to 0.3 6 0.03 ms in

turn 1 IHCs and from 1.4 6 0.3 ms to 0.6 6 0.04 ms in way ANOVA; Fig. 7). Membrane slope conductanceabout 275 mV for all cells increased significantly to aturn 4 IHCs (n 5 19; Fig. 5D). IHC slope conductance

about 0 mV did not vary across turns, as seen for the mean of 32.1 6 2.2 nS with 10 mM ATP and to 47.56 3.5 nS with 400 mM ATP. There was no significantvoltage-ramp analysis, and the turn 4 IHCs with slower

activation kinetics had the same conductance (meas- effect of ATP on IHC conductance about 0 mV (Fig.7B) because of inward rectification of the P2X receptorured at 40 ms) as the faster-activating IHC from all

four turns ( p . 0.05, Fig. 5E). conductance (Brake et al. 1994).The net ATP-activated conductance was derived by

subtraction of the I/V relationship with superfusionP2X receptor conductance in IHC with respect of Ca2+-free solution from the I/V during applicationto cell origin of ATP 1 Ca2+-free. As apparent from the absolute

conductances, there was no significant difference ( pDirect measurement of the P2X receptor conductance. 0.05, one-way ANOVA) in the IHC P2X receptorwas achieved by suppression of the KCa conductanceconductance about 275 mV with respect to cell originunder nominally Ca2+-free conditions, along with a(Fig. 7). There was no net P2X receptor conductancehigh level of intracellular Ca2+ buffering (10 mMabout 0 mV as a result of the inward rectificationBAPTA). Under these conditions, and using bath-(described above, Fig. 3B). Overall, the net P2X recep-applied ATP, there was no evidence for a P2Y receptor-tor conductance about 275 mV averaged 15.4 6 1.8mediated release of intracellular Ca2+ affecting mem-nS for 10 mM ATP and 31.0 6 3.2 nS for 400 mM ATP.brane conductance (Sugasawa et al. 1996). At a con-

centration of ATP (10 mM) close to the IHC P2Xreceptor conductance EC50 (Sugasawa et al. 1996), P2X

DISCUSSIONreceptor currents exhibited no dependence upon turnof origin ( p . 0.05, one-way ANOVA; Figs. 6A and C)and had an overall average of 2681 6 70 pA (n 5 48, The potential for variation in the expression of voltage-

and ligand-activated conductances to alter the IHCVh 5 260 mV). This lack of systematic variation withturn of origin was also apparent in the responses to membrane filter properties along the length of the

cochlea was investigated. The membrane filter shapesthe supramaximal concentration of ATP (400 mM; Figs.6B and C). The overall mean inward current response the AC and DC components of the receptor potential

which determine the temporal coding of neurotrans-to 400 mM ATP was 21319 6 121 pA (n 5 42). Subse-quent application of 1 mM ATP failed to elicit signifi- mitter release during sound stimulation (van Emst et

al. 1998; Cheatham and Dallos 1999). The principalcantly greater inward currents ( p . 0.05, paired t-test,n 5 5). ATP produced reversible depolarizing shifts finding of this study was the consistency of IHC mem-

brane capacitance and conductance throughout theof the Vz in a dose-dependent manner (to 238.9 60.9 mV with 10 mM ATP, n 5 48; to 232.3 6 0.8 mV cochlea. Approximately 40% of IHC basolateral K+

conductance was modulated by Ca2+. In addition, thewith 400 mM ATP, n 5 42; compared with the mean5 250.4 mV in Ca2+-free solution, stated above). apically located ATP-activated (P2X receptor) conduc-

tance may also influence the sound-induced receptorThe ATP-gated inward current was associated withan increased slope conductance at potentials negative potential. These data indicate that while the elements

,FIG. 5. Activation kinetics of IHC voltage-dependent currents. A. protocol (fitted between 3 and 40 ms). These traces had time constantsTurn 1 (basal) IHCs exhibit fast-activating outward currents at poten- ranging from 59.2 ms at 220 mV to 12.0 ms at 130 mV. This cell alsotials positive to 260 mV (voltage range: 2100 to 130 mV, 10 mV exhibited the fast-activating currents with kinetic properties similar toincrements, 40 ms, Vh 5 260 mV). The initial 3 ms of current traces those of the majority of IHC. The time constants for the initial 3 msfor 230 to 130 mV voltage steps are shown under the voltage proto- of current traces ranged from 3.2 ms at 0 mV to 1.7 ms at 130col. These current traces have been fitted over the initial 2 ms by a mV (shown immediately beneath the fits to the slower currents). D.recursive best-fit single exponential function. The time constants (t ) Analysis of the voltage dependence of the activation time constantsranged from 1.1 ms at 230 mV to 0.4 ms at 130 mV. B. Turn for IHC of different turns. The majority of the IHCs (turn 1 5 8, turn4 (apical) IHC exhibiting similar fast (f) activation kinetics. Single 2 5 8, turn 3 5 18, turn 4 5 14/23) had rapidly activating currentsexponential fits for the activation of the outward currents were deter- (symbols as for E ). A subpopulation of turn 4 IHCs (9/23) had slowermined as for A. The time constants varied from 0.6 ms to 0.4 ms. C. time constants; the activation kinetics of this subpopulation of IHCsTurn 4 IHCs from a subpopulation which exhibited an additional is shown in relation to rest of the dataset (inset). E. Analysis of theslower (s) activation component of the outward current response to current–voltage relationships (current amplitudes obtained at 40 ms)the depolarizing voltage steps. The single exponential fits for this for IHCs from turns 1–4, including subsets of turn 4 fast (f) and slowslower-activating current are shown immediately below the voltage (s) cells.


FIG. 7. IHC slope conductance measured during the sequentialsuperfusion of standard solution (steady state with BAPTA dialysis),Ca2+-free solution, 10 mM ATP in Ca2+-free solution, and 400 mMATP in Ca2+-free solution. A. Slope conductance measured about275 mV (G275mV). B. Slope conductance measured about 0 mV(G0mV). There was no systematic variation in G275mV and G0mV forgiven solutions across the turns of the cochlea ( p . 0.05, one-wayANOVA). Each bin represents the mean 6 SEM of 10-19 IHCs percochlear turn. * indicates significant difference ( p , 0.05, unpairedt-test for adjacent bars).

FIG. 6. IHC inward current responses to bath applied 10 and 400mM ATP in Ca2+-free solution. The net ATP-gated current (Vh 5 260

Raybould and Housley 1997). It has been proposedmV) was measured immediately prior to the voltage ramp used forthat this increase in GK,n contributes a tonotopic tun-measurement of slope conductance. A. The current responses to 10

mM ATP showed no systematic variation across the turns of the ing of the OHC membrane filter which is defined bycochlea (n 5 48, p . 0.05, one-way ANOVA). B. Current responses the time constant [tm 5 input resistance (R in) 3to 400 mM ATP were also independent of cell origin (n 5 42, p . membrane capacitance (Cm) (Hille 1992)]. Thus,0.05, one-way ANOVA). C. Combined net ATP-gated current data

reductions of both OHC R in and Cm toward the basefor each cochlear turn. Each bin represents the mean for 10-14 IHCsof the cochlea result in tm changing from 11 ms inper turn. Error bars indicate 6 SEM. * indicates significant difference

(p . 0.05, unpaired t-test for adjacent bars) the apex to 0.3 ms in turn 2 (Mammano and Ashmore1996). Therefore, the attenuation of the response tothe sound-modulated receptor potential has a cornerfrequency (2f 5 3 dB, f 5 1/2 p t) of approximatelydefining the IHC membrane filter are constant, local

activation of KCa or P2X conductance would generate 400 Hz for basal turn OHCs (R in 5 22 MV , Cm 5 18pF; Housley and Ashmore 1992). Preyer et al. (1996)significant increases in the corner frequency of the

forward transduction transfer function. similarly verify the tonotopic variation in OHC cornerfrequencies, showing an exponential decline fromThese data provide a comprehensive, quantitative

analysis of IHC conductance properties. Guinea pig approximately 550 Hz for cells in the basal turn ofthe cochlea to 35 Hz at the apex. In comparison,IHCs express a different complement of membrane

conductances from that of the adjacent OHCs. In IHCs at rest (negative to the activation threshold forthe K+ conductance, 255 mV) for any region of theOHCs there is a systematic variation in the principal

K+ conductance (GK,n), with greatest expression in organ of Corti can now be modeled with R in 5 140MV around their resting potential and Cm 5 10 pF.the basal (high-frequency encoding) region (Housley

and Ashmore 1992; Mammano and Ashmore 1996; The present study clearly demonstrates the narrow


range of IHC conductance between 260 and 290 defined a fast-activating K+ current (IK,f) in the pres-ence of 4-aminopyridine (4AP), which was best-fittedmV irrespective of cell origin (Fig. 4A). Thus, the

average tm for a guinea pig IHC is approximately 1.4 by a second-order exponential with voltage-dependentand independent activation time constants. At roomms with a predicted corner frequency of approxi-

mately 115 Hz. At the point of K+ conductance activa- temperature, the voltage-dependent component var-ied between approximately 1.5 and 0.8 ms over thetion, the corner frequency has been estimated at

480–940 Hz in isolated IHCs (Kros and Crawford activation range, while the voltage-independent timeconstant was approximately 0.25 ms. A slower 4AP-1990). Our data suggest that this estimate is valid

throughout the cochlea. sensitive K+ current (IK,s) was also isolated using tetra-ethylammonium (TEA). The activation kinetics forOur measurements of IHC membrane time con-

stant and corner frequency also agree with the lower this conductance had two voltage-dependent time con-stants that ranged between approximately 1 and 10 msend of the estimate of a 0.9 ms time constant and a

178-1 Hz corner frequency for basal turn guinea pig and between 12 and 28 ms. While both IK,f and IK,s

would contribute to the activation kinetics of ourIHCs, based on high-impedance intracellular re-cordings in vivo (Sellick and Russell 1980). While no whole-cell currents, we have shown that IHC conduc-

tance (in the absence of K+ channel blockers) is well-data are available on the temperature sensitivity ofIHC conductance, our in vitro data closely matches fitted by a first-order exponential function with time

constants similar to the voltage-dependent IK,f compo-these in vivo measurements when corrected using ageneral Q10 value of 1.3 (Hille 1992). The fall-off in nent. In our study, the IHC activation kinetics across

turns varied between approximately 0.9 and 1.8 ms,basal (first) turn IHC AC receptor potentials fromapproximately 300 Hz (Palmer and Russell 1986) has close to the activation threshold for turns 1 and 2 IHCs

and turns 3 and 4 IHCs, respectively. Thus, the IHCsbeen ascribed to the low-pass characteristics of theIHC filter. An estimated time constant of 0.34 ms and from the more apical turns activated approximately

half as fast as the higher-frequency-encoding IHCs.a corner frequency of 472 Hz have previously beenmodeled for this filter for guinea pig IHCs from all However, in comparison with the subpopulation of

slowly activating IHCs from turn 4, these activationregions of the cochlea in vivo (Dallos 1984). The cur-rent study demonstrates that the estimates of the mem- kinetics are remarkably consistent (see Fig. 5D, inset).

Indeed, the data for the fast-activating IHCs from allbrane time constant will necessarily vary withrecording method and condition of the IHCs, which four turns lie within the range of activation time con-

stants for IK,f described by Kros and Crawford (1990).in turn will affect the activation level of the KCa chan-nels. Our estimate is likely to be at the lower end of The slowly activating outward current of the subpopu-

lation of turn 4 IHCs had voltage-dependent time con-the experimental range because of the Ca2+ bufferingthat we achieved in our whole-cell recordings. Never- stants considerably slower than those reported for IK,s

(Kros and Crawford 1990) and slower than those acti-theless, the key element of the present analysis is thedemonstration of the constancy of the IHC low-pass vation time constants of the TEA-sensitive KCa conduc-

tance in OHCs (30 ms at 230 mV; Housley andmembrane filter throughout the cochlea. This is incontrast with all other cochlear hair cell systems Ashmore 1992).

In mouse IHC, expression of IK,f coincides with thestudied.Our findings are also supported by recent in vivo onset of hearing function (Kros et al. 1998) and its

pharmacology links this conductance to KCa channelrecordings which show the development of phase lagsin IHC receptor potentials relative to basilar mem- activity. In addition, excised patch recordings of KCa

channels from adult guinea pig IHCs show characteris-brane motion as stimulus frequency increases are inde-pendent of IHC origin (Cheatham and Dallos 1999). tics consistent with the IK,f classification (Appenrodt

and Kros 1997). Thus, the present study shows theThis roll-off in phase-related encoding of sound, attrib-utable to the membrane filter, is likely to underlie the potential physiological significance of Ca2+ signaling

to modulate the major IHC membrane conductance.limited dynamic range of phase-locked auditory nervefiring, at least in the guinea pig (Palmer and Russell In contrast to the mammalian cochlea, lower verte-

brates, such as birds, turtles, and frogs, do not separate1986). Thus, the IHC membrane filter may be mod-eled as a function which receives pretuned input from frequencies efficiently along their auditory papilla and

principally rely on intrinsic electrical tuning of thethe cochlear amplifier and changes dynamically withalterations in basolateral K+ channel activation (mod- sensory hair cells (Fettiplace and Fuchs 1999). This

electrical filter arises from an interplay between volt-eled by van Emst et al. 1998).Using pharmacological channel blockers, Kros and age-gated Ca2+ channels and KCa channels (Hudspeth

and Lewis 1988) and a tonotopic variation in KCa chan-Crawford (1990) dissected two separate K+ currentsfrom turns 3 and 4 guinea pig IHCs, which exhibited nel conductance and kinetics (Art et al. 1993; Art and

Fettiplace 1987; Wu et al. 1995; Fettiplace and Fuchswidely differing activation time constants. They


1999). Isolated guinea pig IHC current clamp experi- brane time constant and raise the corner frequencyof the membrane filter.ments (Kros and Crawford 1990) failed to detect the

In conclusion, the present study provides a compre-electrical resonance intrinsic to such systems. Whilehensive analysis of the membrane properties of IHCIHC KCa channel activation by Ca2+ influx throughalong the guinea pig cochlea. A feature of these sen-voltage-gated Ca2+ channels has been reported (Dulonsory hair cells is their remarkable uniformity in mem-et al. 1995), our demonstration of the constancy inbrane capacitance, in the kinetics and amplitude ofIHC K+ channel properties precludes the variation inthe voltage-activated conductances, and in the relatedion channels that form the basis of electrical tuningexpression of the ATP-activated P2X receptor conduc-in non-mammalian vertebrates.tance. These observations suggest that transduction ofOur quantitative analysis of the P2X receptor con-sound information by IHC is constant along theductance provides further information on the regula-cochlea and that frequency and intensity coding bytion of IHC membrane filter properties along theneurotransmitter release arises from elements up-cochlea. The present study shows that the proportionstream of the membrane filter.of IHC P2X receptor conductance to voltage-depen-

dent slope conductance measured about 275 mV isapproximately 3:1. This is similar to the ratio previously

ACKNOWLEDGMENTSdetermined for OHCs (Raybould and Housley 1997).We estimate that IHCs possess approximately 1500

This research was funded with the assistance of the HealthATP-gated ion channels, irrespective of origin. This isResearch Council of New Zealand, the New Zealand Lotterybased on a 31 nS IHC P2X receptor conductance andGrants Board, the Deafness Research Foundation of Newthe 21 pS unitary conductance reported for ATP-gatedZealand, and the Wallath Trust. Denise Greenwood is

ion channels assembled from P2X2 receptor subunits thanked for technical support.(Evans 1996). In comparison, the number of ATP-gated ion channels on OHCs increases from approxi-mately 3000 in the apex to 8000 in the basal turn REFERENCES(Raybould and Housley 1997). This gradient is in par-allel with the tonotopic increase in OHC GK,n . There- APPENRODT P, KROS CJ. Single-channel recordings of large conduc-fore, it seems likely that regulation of P2X receptor tance Ca2+ activated K+ channels from guinea-pig inner hair cells.

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