the supporting cells of the organ of corti are structurally and electrically coupled together by gap junctions 
  ( jahnke , 1975 ; gulley and reese , 1976 ; iurato et al . , 
  1976 ;
hama and saito , 1977 ; santos - sacchi and dallos , 
  1983 ; kikuchi et al . , 1995 ) .
such gap junctional coupling among the supporting cells provides for electrical 
  and metabolic uniformity ; cochlear homeostasis is believed to rely on intercellular coupling ( santos - sacchi , 
  1985 , 1986 , 1991 ; kikuchi et al . , 1995 ) .
gap junction channels are distinguished from other 
  ionic channels since the integration of two aligned 
  hemichannels from adjacent cells is required for normal function . in early work , hypertonic solutions , 
  which cause cell and tissue shrinkage , were found to 
  uncouple gap junctions in several different preparations ( barr et al . , 1965 , 1968 ; goodenough and gilula , 
  1974 ; loewenstein et al . , 1967 ) .
more recently , hypotonic treatments , which cause cell swelling , were determined to either increase ( kimelberg and kettenmann , 
  1990 ) or decrease ( ngezahayo and kolb , 1990 ) gap 
  junctional coupling .
these effects could have been due 
  to a variety of factors , including direct mechanical influences , changes in nonjunctional resistance , and 
  modulation of intracellular factors that are known to 
  uncouple cells . in the study of ngezahayo and kolb 
  ( 1990 ) , where junctional conductance was studied directly , the decrease in coupling
was abolished by 5 mm 
  egta in nominally ca - free internal solutions , and 
  was linked to the activity of pkc . in the present report
, 
  we used the whole - cell voltage clamp technique to examine the effects of turgor pressure on junctional coupling of isolated pairs or small groups of cochlear supporting cells . both input capacitance (
santos - sacchi , 
  1991 ; bigiani and roper , 1995 ) and transjunctional 
  conductance measures were used to gauge intercellular 
  communication .
we report that data obtained with 
  both techniques indicate that positive intracellular 
  pressure , which is known to induce membrane tension , 
  uncouples gap junctions of supporting cells in corti 's 
  organ .
detailed experimental methods can be found in previous reports 
  ( santos - sacchi , 1991 ; sato and santos - sacchi , 1994 ) . in brief , isolated supporting cells or cell aggregates were freshly obtained 
  from the organ of corti of the guinea pig cochlea by shaking for 
  515 min in nominally ca - free leibovitz medium containing 
  1  mg / ml trypsin . to reduce the voltage - dependent ionic currents from nonjunctional membrane during double voltage 
  clamp experiments
, cells were perfused with an ionic blocking solution containing ( mm ) : 100 nacl , 20 tea , 20 cscl , 1.25 cocl2 , 
  1.48 mgcl2 , 10 hepes , ph 7.2 , 300 mosm . in initial experiments , 
 
a modified leibovitz medium was used for measurement of input 
  capacitance ( cin ) with a single pipette voltage clamp containing 
  ( mm ) : 136.9 nacl , 5.37 kcl , 1.25 cacl2 , 1.48 mgcl2 , 10 hepes , 
  ph 7.2 , 300 mosm .
pipette solutions were composed of ( mm ) : 
  140 kcl , 10 egta or bapta , 2 mgcl2 , and 10 hepes , ph 7.2 . 
  for double voltage clamp recording ,
patch electrodes had initial resistances of 2.54 
  m , corresponding to 12 m in diameter .
series resistance ( rs ) 
  after whole cell configuration was estimated from the current in 
  response to 10-mv steps ( huang and santos - sacchi , 1993 ) . in single hensen cells , where rs could be unequivocally determined after whole cell configuration , the average value was 7.16  0.43 
  m ( mean  sem , n = 48 ) .
cells were typically held at 80 mv , 
  within the hensen cell 's linear current  voltage range ( santos - sacchi , 1991 ) .
currents were filtered at 10 khz with a four - pole 
  bessel filter ( axon instruments , foster city , ca ) .
intracellular 
  pressure was modified either through the patch pipette with a syringe connected to the teflon tubing attached to the patch pipette holder or by changing osmolarity with  y - tube
pipette pressure was monitored via a t - connector to a pressure monitor ( world precision instruments , inc . ,
all experiments were video tape recorded and performed at 
  room temperature . since the input capacitance can be measured by a single pipette voltage clamp and
is correlated with junctional conductance ( santos - sacchi , 1991 ; bigiani and roper , 1995 ) , it can be 
  conveniently used to study gap junctional coupling under conditions of less cellular damage than the double voltage clamp technique .
input capacitance , in conjunction with input resistance 
  ( rin ) , was continually measured on line to monitor junctional 
  coupling .
cin and rin were determined from the transient charge 
  and steady state current , respectively , induced by small ( 10 
  mv ) test pulses with duration of 18 the clamp time constant at 
  the holding potential ; measures were made at 13 hz ( santos - sacchi , 1991 ) . 
 
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 \begin{equation*}c_{in}=\frac{q_{in}}{v_{test}}\end{equation*}\end{document}2\documentclass[10pt]{article } 
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 \begin{document } 
 \begin{equation*}r_{in}=\frac{v_{test}}{{\delta}i_{{\infty}}},\end{equation*}\end{document } 
  where 
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 \begin{document } 
 \begin{equation*}q_{in}={\int _ { 0}^{{\infty}}}i_{c}dt.\end{equation*}\end{document } 
  qin is the charge moved , vtest is the voltage of the test pulse , ic is 
  the capacitive current induced by the test pulse , and i is the 
  current difference between the steady state current induced by 
  the test pulse and the holding current at the holding potential . for the double voltage clamp ,
each cell in a cell pair was separately voltage clamped using 200a and 200b patch clamps ( axon 
  instruments ) .
both cells were clamped at the same holding potentials and a test pulse ( 10 mv , 10 ms ) superimposed only on 
  cell 1 .
the transjunctional current ( ij ) is equal to the current difference ( i2 ) in cell 2 caused by the test pulses applied to cell 1 . 
 
the transjunctional conductance ( gj ) can be calculated by : 
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 \begin{document } 
 \begin{equation*}g_{j}=\frac{-{\delta}i_{2}}{v_{test}},\end{equation*}\end{document } 
  where vtest is the test pulse voltage applied to cell 1 .
data collection and analysis were performed with an in - house developed 
  windows - based whole - cell voltage clamp program , jclamp ( http : 
  //www.med.yale.edu / surgery / otolar / santos / jclamp.html ) , using a digidata 1200 board ( axon instruments ) . in some experiments ,
gj was measured online at 24 hz and the corresponding 
  video images of recorded cells were digitally captured every 510  s 
  under software ( jclamp ) control .
the captured images were 
  printed at 1,700 and the plane cell areas calculated . to gauge 
  membrane stress , area strain ( a / a0 ) was calculated , where a is 
  the change of cell area after pressure or osmotic treatment and 
  a0 is the original cell area .
hensen cells can be easily distinguished from other inner ear supporting cells by their prominent lipid vacuoles .
the number of cells comprising isolates of hensen 
  cells can be determined under the light microscope , 
  and corresponds to the isolate 's cin since hensen cells 
  are well coupled electrically . although the size of 
  hensen cells is variable , the distributions of cin for one , 
  two , and three hensen cells , whose numbers were visually confirmed , were quite distinct ( fig .
the peaks of the isolate 
  distributions were clearly separated at 31.03  0.86 , 
  64.75  1.5 , and 103.9  3.05 pf , corresponding to 
  one , two , and three cell contributions , respectively . 
  the number of cells within isolates can also be confirmed using uncoupling agents , such as co2 , octanol , 
  or , as we now find , positive turgor pressure , to uncouple the cells .
when cells fully uncoupled , cin reached 
  single cell capacitance levels ( e.g. , figs . 2 and 3 ) .
the 
  correlation of cin with degree of cell coupling is illustrated by real measures of cin in a coupled two - cell electrical model ( fig .
cin of the electrical model was a 
  monotonic function of transjunctional resistance or 
  conductance , indicating the validity of cin as an indicator of cell coupling . 
 
 
  
positive turgor pressure induced either by osmolarity 
  changes or directly via the patch pipette decreased cin 
  of cell pairs or three - cell groups ( figs . 2 and 3 ) , but did 
  not reduce single cell capacitance ( fig .
this indicates that positive turgor pressure uncouples gap junctions between adjacent hensen cells . 
   in fig .
2 a , bath application of hypo - osmotic solution 
  ( 150 mosm ) caused a hensen cell pair to swell ( insets ) 
  and decreased cin of the pair to single cell levels .
the 
  uncoupling induced by increased turgor pressure is reversible since return to normal osmolarity solution often restored initial cin values ; subsequent reperfusion 
  with hypo - osmotic solution remained effective as an uncoupling stimulus ( fig .
2 b ) . in single cells , while the 
  same hypo - osmotic treatment caused cell swelling , cin 
  remained stable ( fig . 4 b ) .
3 illustrates the uncoupling effect of cell turgor 
  pressure change induced by patch pipette pressure . as 
  turgor pressure
was directly increased to 1.2 kpa via 
  the patch pipette , cin decreased to almost single cell 
  levels ( after an initial delay possibly due to pipette plugging ) , and immediately began to return when the pressure was released ( fig .
the cells could be permanently uncoupled during the application of prolonged , 
  continuous positive pressure ( fig .
the uncoupling effect of positive turgor pressure was found in 40 
  of 42 cell pairs , or three - cell groups . as with osmolarity 
  change , direct application of positive turgor pressure 
  via the patch pipette
also did not decrease the measured capacitance in single hensen cells despite cell 
  swelling ( fig .
4 a , insets ) . although cin can be easily measured by single pipette 
  voltage clamp to gauge the degree of cell coupling , 
  transjunctional conductance can not be measured directly since transjunctional voltage and current are unknown .
additionally , a quantitative estimate of degree 
  of coupling based on cin is not easily established since 
  cin is a nonlinear function of transjunctional conductance ( see fig
to further investigate the uncoupling effect of positive turgor pressure 
  on gap junctions in hensen cells , the transjunctional 
  conductance was directly assessed with a double voltage 
  clamp technique , and corresponding changes of the 
  cell plane surface areas ( a / a0 ) ( i.e. , an indicator of 
  membrane strain ) were simultaneously measured .
cell areas increased in concert with decreases of 
  transjunctional conductance as positive turgor pressure 
  was delivered to the cells .
the changes in cell area were 
  observable before gap junctional uncoupling and occurred faster than gj decay ( figs . 5 and 6 ) . however , 
  unlike pressure changes induced by pipette pressure , 
  hypo - osmotic shocks produced changes in gj and cell 
  areas that were quite fast . with extracellular perfusion 
  of a 150-mosm solution ,
6 b , and the average value was 5.1  
  1.86 s ( n = 6 ) . in fig
. 6 b , the rise time constant of 
  membrane strain was 4.43 s. the average rise time constant of membrane strain is estimated to be close to or 
  less than that of the average gj decay since in most 
  cases the swelling fully occurred within the 510-s video 
  capture rate . in most , but not all , cases ,
it was noted 
  that after membrane tension stabilized , transjunctional 
  conductance likewise stabilized ( fig .
the correlated 
  and reciprocal changes in gj and membrane strain 
  ( a / a0 ) were reversible and repeatable ( fig . 6 a ) , 
  strongly indicating that gj decreases were relative to increases of membrane strain ; i.e. , membrane tension .
it 
  should be noted that the latency to gj change after a/ 
  a0 change is possibly due to the absence of significant 
  membrane stress during the initial cell inflation , which 
  clearly ( based on the magnitude of cell enlargement ) 
  was accompanied by membrane unfolding . 
 
  
uncoupling of hensen cell gap junctions by membrane stress was not inhibited by using pipette solutions 
  containing 50 m h-7 ( dihydrochloride ; calbiochem 
  corp . , la jolla , ca ) , a broad - based serine / threonine 
  kinase inhibitor ( boulis and davis , 1990 ) ( figs .
these data imply that the uncoupling effect of 
  positive turgor pressure on inner ear gap junctions is 
  independent of protein kinases , and that the effect is 
  different from previous observations that cell volume 
  changes induced uncoupling of gap junctions via the 
  pkc pathway ( ngezahayo and kolb , 1990 ) .
nevertheless , cell swelling induced by hypo - osmotic shocks has 
  been linked to increases of another uncoupling agent , 
  intracellular ca ( hoffmann and simonsen , 1989 ; suzuki et al . ,
however , uncoupling by ca , which 
  occurs at millimolar intracellular concentrations in 
  hensen cells ( sato and santos - sacchi , 1994 ) , can be 
  ruled out since pipette solutions contained 10 mm 
  bapta , a fast highly selective calcium chelating reagent , and extracellular and intracellular solutions 
  were nominally ca free .
considering all evidence , the 
  observed uncoupling effect of positive turgor pressure 
  on inner ear gap junctions , which is fast ( within seconds ) , correlated with changes of membrane strain , 
  and independent of protein kinases and ca , is likely 
  to occur via direct mechanical effects on the plasmalemma ; i.e. , membrane tension . the effect of membrane tension on gap junctional 
  conductance
was further studied by increasing turgor 
  pressure in cell 1 and measuring ij in cell 2 at different 
  membrane potentials ( fig .
gap junctional conductance in hensen cells at a holding potential of 80 mv 
  was 52.9  12.1 ns ( n = 51 ) .
as the turgor pressure in 
  cell 1 was increased , ij decreased ( fig . 7 a ) .
the junctional conductance at different membrane potentials 
  reduced in parallel when the turgor pressure was increased . in those cell pairs where turgor pressure alterations were successfully applied without losing the cells , 
  gj at 80 mv holding potential decreased 38.3  9.5% 
  from 50.5  14 ns ( n = 9 ) at a turgor pressure of 1.41 
   0.05 kpa .
7 . in this case , as the cells were depolarized , gj decreased ( fig .
7 b ) . other vm dependencies of transjunctional conductance were also found , including vm 
  insensitivity and an increase with depolarization .
we provide evidence , based on input capacitance and 
  double voltage clamp measures , that junctional coupling is sensitive to positive turgor pressure - induced 
  membrane tension .
turgor pressure has been used to 
  induce membrane tension in a wide variety of cells , including the outer hair cell ( ohc ) , where it has been 
  shown that motility and motility - related gating current 
  characteristics are directly altered ( iwasa , 1993 ; gale 
  and ashmore , 1994 ; kakehata and santos - sacchi , 1995 ) . 
  membrane tension ( possibly acting via cytoskeletal interactions )
is also known to gate stretch - activated ionic 
  channels ( yang and sachs , 1989 ) , which have been observed in outer hair cells ( ding et al . , 1991 ; iwasa et al . , 
 
it is possible that membrane tension also alters 
  gating characteristics of supporting cell gap junctions . 
 
we show , however , that unlike stretch channels , inner 
  ear gap junctional conductance decreases with membrane stress .
recently , it has been postulated that gap 
  junction channels possess two distinct gating mechanisms , namely , a voltage gating mechanism and a 
  chemical gating mechanism ( bukauskas et al . , 1995 ; 
  bukauskas and peracchia , 1997 ;
chemical uncoupling agents , such as co2 , 
  h , and ca , may act on sensor elements from the cytoplasmic side . supporting cell coupling
has been 
  shown to be sensitive to a variety of chemical uncoupling agents ( santos - sacchi , 1985 ; 1991 ) , and we now 
  report that supporting cell coupling is voltage dependent as well .
the existence of voltage - dependent gap 
  junctional conductance may account in part for previous reports of temperature - induced depolarization on 
  supporting cell coupling ratios ( santos - sacchi , 1986 ) . 
 
interestingly , junctional voltage dependence is unaffected by concomitant tension - induced junctional conductance change , possibly indicating that an independent tension gating mechanism may exist .
gap junctions consist of two aligned transmembrane 
  hemichannels ( connexons ) , one from each cell ( revel 
  et al . , 1984 ;
goodenough et al . , 1988 ; bennett et al . , 
  1991 ) .
each of these hemichannels is formed by six 
  connexin subunits ( kumar and gilula , 1996 ; perkins et 
  al . , 1997 ) .
our data indicate that membrane stress acts 
  on inner ear gap junctions in a manner independent of 
  ca , ph , and protein kinases .
the rapid and reversible nature of the uncoupling also indicates that the 
  mechanism is not due to some sort of mechanical destruction of the channels . while there may be other unknown links between membrane stress and junctional 
  conductance ,
it is conceivable that tension may gate 
  gap junction channels by a conformational change in 
  connexon structure , possibly causing only the stressed 
  membrane 's hemichannel to close .
gap junction connexins represent a family of homologous proteins that have differing voltage gating characteristics ( harris et al . , 1981 ; spray et al . , 1981 ; bennett et al . , 1991
cx26 was 
  found in gap junctions of the rat ( kikuchi et al . , 1995 ) 
  and gerbil ( forge et al . , 1997 )
more recently , cx26 , cx30 , cx32 , and cx43 have been localized to supporting cell regions of the rat cochlea ( lautermann et al . , 1997 ) .
such diversity of connexins within 
  the organ may provide for a variety of junctional communication characteristics ; for example , rectifying junctional conductance .
indeed , in addition to our direct 
  observation that voltage - dependent junctional communication exists in the supporting cells , we have preliminary evidence that junctional rectification occurs . directional flow of ions mediated by rectified gap junctions may be crucial for normal cochlea homeostasis 
  ( see below ) . since the mid 1980 's , gap junctional coupling
however , input capacitance and resistance reflect the degree of electrical coupling and can be conveniently 
  measured using a single voltage clamp ( santos - sacchi , 
  1991 ; sato and santos - sacchi , 1994 ; bigiani and roper , 
  1995 ) .
. 1 b , 
  inset ) , and assuming that the individual cells have the 
  same input impedance , the following equations are obtained ( bigiani and roper , 1995 ) , 
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 \begin{equation*}c_{in}=\frac{(2r_{m}r_{j}+2r^{2}_{m}+r^{2}_{j})r^{2}_{m}}{(2r_{m}r_{s}+r^{2}_{m}+r_{s}r_{j}+r_{m}r_{j})^{2}}c_{m},\end{equation*}\end{document}6\documentclass[10pt]{article } 
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 \begin{document } 
 \begin{equation*}r_{in}=\frac{r_{s}r_{j}+2r_{s}r_{m}+r_{j}r_{m}+r^{2}_{m}}{r_{j}+2r_{m}},\end{equation*}\end{document } 
  where rs and rm are electrode series resistance and 
  nonjunctional membrane resistance , respectively , and 
  cm is single cell capacitance ( see fig
since 
  rm is not readily available from recordings , we can solve 
  eqs . 5 and 6 to remove rm .
rj can be finally expressed : 
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 \begin{equation*}r_{j}= \left{\mid } \frac{c_{in}r^{2}_{in}+4c_{m}r_{s}r_{in}-2c_{m}r^{2}_{s}-2c_{m}r^{2}_{in}}{\sqrt{2c^{2}_{m}r_{s}r_{in}+c_{in}c_{m}r^{2}_{in}-c^{2}_{m}r^{2}_{s}-c^{2}_{m}r^{2}_{in } } } \right{\mid } .\end{equation*}\end{document } 
  cin , rin , and rs are readily obtained from recordings . 
 
8 illustrates the measurement of these parameters 
  during an uncoupling event , and the bottom panel 
  shows the estimated gj based on those data .
it 
  should be noted that rs changes can also produce 
  changes in cin and rin .
for example , to obtain the observed maximum change in cin , an order of magnitude 
  increase of rs would be required in this case . in our experiments ,
changes solely in rs required to produce a 
  comparable change in cin were not observed .
series resistance remained constant , being 7.79  0.49 m ( n = 
  7 ) for cell pairs that were well coupled and 6.34  1.13 
  m after those same cells were uncoupled with positive 
  pipette pressure . 
  
finally , how might the turgor pressure dependence 
  of junctional coupling in the organ of corti affect cochlear function ? in vivo ,
the organ of corti , comprising 
  hair cells and supporting cells , is bathed in two different media , high k endolymph apically and low k perilymph basally . since the receptor current through hair 
  cells is predominantly carried by k ,
an accumulation 
  of k within the perilymphatic space along the basolateral region of the hair cells is unavoidable
. this would 
  potentially depolarize hair cells with disastrous consequences for both forward and reverse sensory transduction . in the mammal ,
forward transduction ( gating of 
  stereociliar transduction channels ) relies on the large 
  driving force present across the hair cell 's apical plasma 
  membrane .
voltage gradients across the apical membranes of inner and outer hair cells ( i.e. , endolymphatic potential minus membrane potential ) range 
  from 125 to 150 mv , and drive the k - based receptor 
  currents .
reduction of this gradient ( e.g. , by membrane depolarization ) will reduce the magnitude of receptor potentials and synaptic output .
reverse transduction is a phenomenon that is restricted to the outer 
  hair cell and is believed to provide for the enhanced 
  high frequency selectivity and sensitivity enjoyed by 
  mammals .
ohcs , which are additionally mechanically 
  active , possess lateral membrane motors that are driven 
  by voltage ( santos - sacchi and dilger , 1988 ) ; the cell 's 
  mechanical response provides feedback into the basilar 
  membrane , thereby enhancing the stimulus to the primary receptor cells , the inner hair cells ( for review see 
  ruggero and santos - sacchi , 1997 ) . not only will depolarization of the ohc alter the driving force for the 
  mechanical response , but the function relating mechanical response to voltage will be shifted along the 
  voltage axis as well , resulting in an altered gain for the 
   cochlear amplifier  ( santos - sacchi et al . , 1998 ) .
a nutritive and k sinking role for gap junctions 
  in the avascular organ of corti has been proposed 
  ( santos - sacchi , 1985 , 1991 ; santos - sacchi and dallos , 
  1983 ) .
inner ear supporting cells have been shown to 
   share  plasmalemmal voltage - dependent conductances 
  due to the high degree of cell coupling ( santos - sacchi , 
  1991 ) .
the magnitude and stability of their resting potentials is pronounced ( close to 100 mv ) , and likely 
  depends on cell coupling since isolated cell resting input conductance is only 1 ns . at the normal resting 
  potential of this cellular syncytium ,
an inward rectifier 
  appears continuously activated and may result in k removal from perilymphatic spaces .
it should be noted 
  that the large perilymphatic fluid spaces may provide 
  little support in sinking k or directing its movement , 
  since hair cell regions that are likely to experience k 
  elevations are not directly exposed to those spaces .
inner hair cells are closely surrounded by supporting 
  cells , and the region of the ohcs that possesses voltage - dependent conductances ( e.g. , outward k ) is restricted to the basal pole of the cell ( santos - sacchi et 
  al . , 1997 ) , which is surrounded by a deiters cell cup . 
 
( 1995 ) provided morphological 
  evidence detailing epithelial and connective tissue gap 
  junctional systems within the cochlea that may complete the mechanism responsible for recycling k from 
  the perilymphatic space near hair cells to the k - rich 
  endolymph via the stria vascularis .
the maintenance of 
  normal fluid space architecture within the inner ear requires fine osmotic control ; imbalances can lead to serious auditory and vestibular problems ( e.g. , meniere 's 
  disease ) . while at present we do not know the normal 
  physiological significance of tension - dependent gap 
  junctional communication , it is likely that fluid balance 
  disorders in the inner ear will affect gap junctional 
  communication , thus compromising sensory function 
  by indirectly modifying hair cell activity .
the cell numbers ( 1 , 2 , or 3 ) in the isolates were determined 
  under the light microscope , and the cin was obtained at the holding potential 80 mv .
each bar represents the number of isolates 
  within a bin width of 10 pf .
the lines plotted over each histogram 
  represent the fitted gaussian distribution for the three isolate 
  groups .
( b ) cin was measured for a coupled two - cell electrical 
  model as transjunctional resistance ( rj ) was changed .
rs , 4.7 m ; 
  rm , 500 m ; cm , 33 pf .
( a ) when an extracellular 150 mosm solution was perfused ( indicated by the horizontal bar ) the cell pair swelled ( see 
  insets ) .
rs , 6.5 m. ( insets ) captured cell images before and during the perfusion of the hypo - osmotic solution .
( b ) decreases of cin due to hypo - osmotic challenge were reversible and repeatable . in this case , the cells finally 
  fully uncoupled and cin remained at the single cell capacitance 
  level .
rs , 3.8 m. hensen cells uncouple as turgor pressure is directly increased via the patch pipette .
( a ) 
  in this example , increased pipette pressure 
  caused cin to decrease to single cell levels after an 
  initial delay ( possibly due to pipette plugging ) , 
  but returned to the original level soon after pipette pressure was released .
rs , 9.76 m. ( b ) a 
  hensen cell pair uncoupled after prolonged application of pressure .
rs , 8.5 m. changes of turgor pressure caused either directly via the patch pipette ( a ) or by perfusion of hypo - osmotic solutions ( b ) do not 
  decrease the measured capacitance in a single hensen cell .
rs : ( a ) 8.4 m , ( b ) 4.4 m. positive turgor pressure - induced reduction in transjunctional conductance is correlated with increases in membrane 
  strain ( a / a0 ) .
transjunctional 
  conductance was measured with 
  a double voltage clamp technique , and membrane strains 
  were calculated from captured 
  images ( see methods ) .
( a ) positive 
  pipette pressure induced an increase in membrane strain and 
  concomitantly reduced gj .
( b ) 
  perfusion of 150 mosm solution 
  also induced an increase in membrane strain and concomitantly 
  reduced gj .
( a ) hypo - osmotic solutions induced reversible , concomitant changes in transjunctional conductance and membrane 
  strain .
( b ) the 
  time course of the uncoupling effect caused by hypo - osmotic 
  shock is compared with the increase of membrane strain .
the beginning of treatment is indicated by an arrow and continued during the observed period .
turgor pressure does not affect membrane potential 
  ( vm or vi - o ) dependence of gap junctions in hensen cells .
( a ) voltage stimulus protocols for each cell and current trace recorded in 
  cell 2 are plotted ( only five traces are shown for clarity ) .
each cell 
  in a hensen cell pair was separately voltage clamped at the same 
  holding potential of 80 mv .
voltage steps from 140 to 70 mv 
  for 100 ms in 10-mv increments were simultaneously delivered 
  into both cells except for 10 mv , 10-ms test pulses superimposed 
  on cell 1 only .
three current traces from cell 2 at different pressures were 
  zeroed and superimposed in the middle .
rs at 0 kpa , 12.5 m ; at 1.2 kpa , 12.7 
  m ; at 2 kpa , 12.2 m. transjunctional conductance is estimated by cin and 
  rin in single pipette voltage clamp .
rs , 8.73 m. ( bottom ) gj is estimated 
  from cin and rin measurements from the cell pair data based on 
  the coupled two - cell model .
cm was set as half of cin , 27.5 pf , at 
  zero applied pressure , and rs was 8.5 m. the change in estimated gj corresponds well to changes in cin .
the magnitude of gj 
  is larger than the largest obtained under double voltage clamp 
  ( 500 ns ) , and may be due to the fact that cell impedances are 
  not actually identical as required in the model .
 the effects of turgor pressure - induced membrane tension on junctional coupling of hensen cell isolates from the inner ear were evaluated by input capacitance or transjunctional conductance measurement techniques . 
 turgor pressure was altered by changing either pipette pressure or the osmolarities of extracellular solutions . 
 both positive pipette pressure and extracellular applications of hypotonic solutions , which caused cell size 
  to concomitantly increase , uncoupled the cells as indicated by reduced input capacitance and transjunctional conductance . 
 these changes were , in many cases , reversible and repeatable . 
 intracellular application of 50 m h-7 , a 
  broad - based protein kinase inhibitor , and 10 mm bapta did not block the uncoupling effect of positive turgor 
  pressure on inner ear gap junctions . 
 the transjunctional conductance at a holding potential of 80 mv was 53.6  
  5.8 ns ( mean  sem , n = 9 ) and decreased 40% at a turgor pressure of 1.41  0.05 kpa . 
 considering the coincident kinetics of cell deformation and uncoupling , we speculate that mechanical forces work directly on gap 
  junctions of the inner ear . 
 these results suggest that pathologies that induce imbalances in cochlear osmotic pressure regulation may compromise normal cochlear homeostasis . 