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Studiesinturtlerevealedthatelectricaltuningofhaircellscouldaccountforthatanimals

acousticfrequencyselectivity.Thiselectricaltuningappearedtodependonthecell
specificexpressionofBK(calciumsensitive,voltagegated)potassiumchannels.Thenext
challengewastotrytodeterminewhatmolecularfeaturesofBKchannelsmightaccount
forthisvariability.Thisprovedtobemorefeasibleinchickenears,sincetherewerecDNA
librariesofchickengenesavailable,andgrowingknowledgeofchickengenomics.

StructureandFunctionoftheAuditoryand
VestibularSystem 1

Increasingelaborationoftheauditoryendorganduringevolution.Increasinglengthofthe
cochleaassociatedwithhigherfrequencyhearing.Upperlimitinturtle~1kHz,inchicken
~5kHz,inhumans~20kHz.
Althoughthelengthoftheauditoryepitheliumisonly1mmlonginturtle(arepresentative
reptile)comparedto~30mmlonginamammal(human),nonethelesstheturtle
epitheliumistonotopicallyarranged,withhighestfrequenciesrepresentednearestthe
saccule,andlowerfrequenciesfurtheraway.

Thebird(chicken)auditoryorgan(basilarpapilla)isintermediateinformandfunction
betweenthatofturtleandmammals.About10,000haircellscoverthe5mmlongbasilar
membrane,encodingsoundsbetween50Hzand5kHz.Thehaircellsaredifferentially
innervation.Tallhaircells,likemammalianinnerhaircells,receivemostafferentcontacts.
Shorthaircells,likemammalianouterhaircells,receivemostefferentcontacts.

Thechickenbasilarpapilla,likethemammaliancochlea,isdifferentiallyinnervatedby
afferentandefferentneurons.Outerhaircellsinmammalsproduceactivemovements
thatenhancecochlearmotionandacousticsensitivity.Thisenhancementofcochlear
sensitivityandtuningissubjecttofeedbackregulationbyefferentcholinergicneuronsthat
inhibittheouterhaircells.
Cholinergicefferentneuronsalsoinhibitshort(outerlike)haircellsofthechickenbasilar
papilla givingrisetospeculationthatshorthaircellstoomayprovideamechanicalboost
toacousticsensitivityforthisanimal.

Chickencochlearafferentsaresharplytunedforacousticinputs.

Spontaneousactivityofchickencochlearafferentsexhibitspreferredintervals,strongly
suggestingthatchickenhaircells,likethoseofturtle,havespontaneousvoltage
oscillations,orringingthatindicateelectricaltuning.

A. Crosssectionofthechickenbasilarpapilla.
B. Surfaceviewofthepapilladuringanexperiment.
C. Schematicofthebasilarpapillaindicatingpositionsatwhichintracellularrecordings
weremadefromindividualtallorshorthaircells.

Likeinturtlehaircells,currentstepsexciteanelectricalresonanceintallhaircellsofthe
chicken.Bodytemp.forachickenis~40 C,sousefultowarmprepaboveroomtempto
getbetterideaofnativebehaviorofhaircells.NotethatfrequencyandQofringing
increaseswithtemperature.

Ringingfrequencyoftallhaircellsiscomparedtoacousticallyderivedtonotopicmapofthe
chickenbasilarpapilla.Correspondenceprettygoodforwarmertemperaturesandwith
moreeffectiveamplifier.

Voltageringinginshorthaircells.Generallynotasrobustasintallhaircells,butstill
present.

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Tonotopicmapofringingfrequencyfortallandshorthaircells.Atagivencrosssectional
positionthereisanoffsetinfrequency.Someanatomicalstudiessuggestthatthe
isofrequency linesmayactuallybecurvedacrossthepapilla(basedonorientationand
lengthofhairbundles) whichmayaccountforthisoffset.

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Wellnotdiscussmechanotransductiontoday,butnotefromthisfigurethatthe
mechanotransducer currentislargerinhigherfrequencyhaircells.Samepatternisseenin
turtles,andmammals.

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Work from several laboratories showed that the responsible potassium channel was
the large-conductance (BK), calcium-sensitive, voltage-gated potassium channel.
This channel is encoded by the slo gene (named for the Drosophila mutant from
which it was first cloned), now known as KCNMA1.
This channel has six transmembrane segments that characterize most voltagegated ion channels. Plus some variants have an additional N-terminus
transmembrane segment (S0). Different from other voltage-gated potassium
channels, the slo channel has an extended C-terminus tail that may be entirely
cytoplasmic, although that has not been proven. There are potentially 4 more
transmembrane segments, based on sequence homology. The most significant
feature of the sequence is that there are numerous splice sites and which
alternative exons can occur. This potentially gives rise to an enormous number of
combinatorial variants on the basic channel structure. Does this potential genetic
heterogeneity underlie the kinetic variability seen in hair cells?

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Navaratnam etalandotherlabsusedRTPCRtoobtainandsequencemRNAwithstrong
homologytoBKchannelsknownfromfruitfly andcalledslo aftertheslowpokemutantfly.
BKmRNAhasatleast7splicesitesatwhichalternativeexonscanbeaddedtothe
sequence.

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ThegeneraltopologyoftheBKchannelisthoughttoinclude6Kchannelstandard
transmembranesegmentsthatformthechannelperse.InadditionBKchannelshaveextra
aminoterminalsequence,andextendedCterminal,portionsofwhicharethoughttobe
cytoplasmicandtocontaincalciumbindingsites.

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RTPCRwasconductedondissectedquartersofthechickenbasilarpapilla.Thisrevealed
someregionalheterogeneityinexpressionofalternativeexons.

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RTPCRwasconductedonindividualhaircellsisolatedfromselectedquartersofthebasilar
papilla.EachlaneshowsPCRproductsaroundsinglesplicesites.Notethatindividualcells
canexpressmorethanonsplicevariant!RighthandcolumnshowscompetitivePCRfor
actintocontrolfordifferencesinstartingmaterialorreactionefficiency.

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Twovariantsoccurringinthechickenbasilarpapillawerecombinedwithafulllength
minimalcDNA.ThesealternativecDNAs wereexpressedinHEK(humanembryonic
kidney)celllines,astandardsystemforionchannelstudies.The61aa versionwas
comparedtotheminimalchannelwithoutthatexon.

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VoltagecommandswereusedtoactivateBKchannelsinpatchesexcisedfromHEKcells.
BK channelsarehighlyexpressedsoexcisedinsideoutpatchescanbeusedtorecord
macroscopicBKcurrents.Sincethecytoplasmicsideofthepatchfacesthebath,calcium
concentrationcanbeexperimentallycontrolled,enablingstudyofvoltage andcalcium
dependenceofeachchanneltype.AsshowninA.andB.,theminimalchannelhadfaster
gatingthanthe+61exonvariant.The+61variantalsowasmoresensitivetovoltageand
calcium.

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Inmanycelltypes,theBKchannelalsoincludesabetasubunitthatstrongly modulates
gating.Thisalsooccurredforthechickenhaircellsplicevariants.Thebetasubunitslowed
gatingkinetics,andshiftedthevoltagesensitivityofbothvariants.

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Calciumsensitivityandgatingkineticsforthe2splicevariants(withandwithout61aa
exon)aloneorexpressedincombinationwithbetasubunit.The4channeltypeshave
consistentlydifferentkinetics,covering~50foldrange.

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Insituhybridizationofthebetasubunitinthequailbasilarpapilla.Expressionishighestin
thelowfrequencyregion.Thispatternisconsistentwiththeexpectationthatco
expressionwithbetaproducesBKchannelswithslowkinetics,andsosupportinglow
frequencyelectricaltuning.

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BKmRNAsplicingalsooccursinturtlebasilarpapilla.

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BKsplicevariantsaredifferentiallydistributedalongturtlebasilarpapilla.
Turtlebetasubunitsunknownsonotstudied.
minimaltranscriptpresentthroughoutthepapilla,variousalternateexonstendedtoshow
upinlowerfrequencyquadrants.
However,the+61exon(encodingmoreslowlygatingBKchannelsinheterologous
expressionstudies)wasfoundinthehighestfrequencyquadrantoftheturtlebp contrary
toprediction

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ResultsfromRTPCRofratcochleafoundmostlytheminimaltranscript,alternateexons
withverylowprobability.InsituhybridizationforthecoreregionofBKalphasubunit
(NOTsplicevariantspecific)foundexpressioninhaircells(andothercells)butnogradient
alongthelengthofthecochlea.ISHforbetasubunitfoundexpressioninhaircells(and
othercells)butnogradientalongthecochlearlength.
TheauthorsconcludedthatBKchannelsinmammaliancochleaprobablynottunedalong
thetonotopicaxisandsoelectricaltuningofhaircellsunlikely.Directrecordingfromhair
cellshasyettorevealvoltageringing.BKchannelkineticshavenotbeenstudied.

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Cellulardifferentiation/specializationalongthetonotopicaxisofthecochlea.
Haircellshape iseitherflasklike(e.g.IHC)orcolumnar(e.g.OHC),regardlessofspecies.
Classificationbyshapeoftenmimicsclassificationbyinnervationpatterns.Interestingly,
thesize(primarilylength)ofthehaircellisassociatedwithitslocationalongthefrequency
(tonotopic)axisofthesensoryepithelium.Therearefunctionalconsequencesofthis
variationinsize.
Stereociliumlength alsovarieswiththelocationofthehaircellalongthecochlea.
Resonanceofastructurevariesproportionatelywithsquarerootofstiffness.Fora
cantileveredbeam(likethestereocilia),stiffnessisinverselyproportionaltosomepowerof
thebeamslength.Thus,asthelengthofthestereociliumincreasesitsresonantfrequency
decreases.Inthisslide,weshowthechangeinstereociliumlengthforIHCandOHChair
bundlesinhumans(thetalleststereociliaarereported).Aswemovefromthebaseofthe
cochleatotheapex(HFtoLF),thelengthofthestereociliaincrease.Thehypothetical
resonantfrequencyforthehairbundlewouldthendecrease.Isthisamechanismfor
passiveresonanceofthehairbundletoassistintuning?Itturnsoutthatthereisa
correlationinthelizard,butpassiveresonancedoesnotseemtobepresentinhairbundles
ofotherspecies(althoughsomeactiveprocessesmayindeedbepresentnextlecture).
Butresonantfrequenciesbasedonstereociliarylengtharemanytimeshigherthanthe
cochlearplacecode.

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Whataboutthechannel?Canwelearnanythingaboutthetransductionapparatusby
lookingatcurrentflowthroughopenchannels?
TonotopicVariationsinMechanotransducerChannelAmplitude(A)Singlechannelrecords
fromtwohaircellsin0.05mMCa2+forhairbundledisplacementsof150nm.Thecells
werelocatedatfractionaldistances,d,of0.22and0.67fromthelowfrequencyendofthe
cochlea.Holdingpoten al,80mV.(B)Singlechannelamplitudesrecordedat80mVin
haircellsatdifferentrelativedistancesalongthecochleafromthelowfrequencyend.
Measurementsateachlocationcamefromdifferentturtles.(o)2.8mMCa2+;()0.05mM
Ca2+.Therighthandordinategivesthechannelconductanceassumingareversalpotential
of0mV.StraightlinesareleastsquaresfitsusingtheLevenbergMarquardtalgorithm.Note
thatthechannelconductanceincreasesforhaircellstunedtohighfrequencies.
(RicciandFettiplace)

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Structure and Function of the Auditory


and Vestibular System - 1

Outerhaircellsintheratandgerbilcochleahavelargertransducercurrentsasone
progressesfromlowtohighfrequencypositions.AND,basolateralvoltagegated
potassiumconductancesalsoarelargerinhigherfrequencyouterhaircells.

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Structure and Function of the Auditory


and Vestibular System - 1

Smallersurfaceareameansthatinputcapacitanceissmallerinhigherfrequencyhaircells.
Increasingconductance(transducerandbasolateral)meansthatinputresistanceisloweris
higherfrequencyhaircells.Themembranetimeconstantistheproductofmembrane
resistancetimescapacitance,
m =RmCm
sothistermissmallerinhigherfrequencyhaircells.
Consequently,thecornerfrequencyofalowpassfilter(whereoutputfallstohalfthe
input)
Thecornerfrequencyofalowpassfilter,
fcorner =1/2m
representingthehaircellshiftstohigherfrequenciesinouterhaircellsfromhighfrequency
positionsalongthecochleastonotopicaxis.

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