AbstractThe patch clamp technique was used to record cAMP-dependent currents of the guinea pig cochlear hair cell plasma membrane. Data obtained indicate that the channels passing this current are moderately selective for monovalent cations and are effectively blocked by L-cis-diltiazem and reversibly blocked by 1 mM Mg2+ or Ca2+. The single-channel unit conductance estimated in the absence of divalent cations is about 16 pS. The results demonstrate that cyclic nucleotide-dependent channels of cochlear hair cells are virtually identical to the photoreceptor and olfactory ones

Kalamkarov, Grigorii R.

Kolesnikov, Stanislav S.

Rebrik, Tatyana I.

Tavartkiladze, George A.

Zhainazarov, Aslbek B.

Elsevier - Publisher Connector 

Volume 290, number 1,2, 167-170 FEBS 10189 
0 1991 Fcdcration of European Biochemical Societies 00145793/911%3.50 
ADONIS0014579391009007 
September 199 1 
Stanislav $. Kolesnikov’, Tatyana I. Webrik’, Aslbek B. Zhainazarov’, George A. Tavartkiladze3 and 
Grigorii R. Kalamkarov’*’ 
‘institute of Biological Physics. USSR Academy of Sciences, Pushchino, h4oscow Region 142292, USSR, ‘institute of Chemical 
Physics, USSR Academy of Sciences, Kosygin str.. 4, A4oscow II 7334, USSR and ‘All- Wnion Scientl#c Center fo  Audiology 
and tiealth, 1905 g. SW~, IOa 123022, Moscow, USSR 
Received 16 July 1991 
The patch clamp technique was used to record CAMP-dependent currents of the guinea pig cochlear hair cell plasma membrane. Data obtained 
indicate that the channels passing this current are moderately selective for monovalent cations and are effectively blocked by L-cis-diltiazem and 
reversibly blocked by 1 mM Mg2’ or Ca 2+ The single-channel unit conductance stimated in the absence of divalent cations is about 16 pS. The 
results demonstrate that cyclic nucleotide-dependent channels of cochlear hair cells are virtually identical to the photoreceptor and olfactory ones. 
Cochlear hair cell; CAMP-activated channel; Patch clamp technique 
1. INTRODUCTION 
I-Iair cells are the mechanoreceptors of the inner ear, 
Their transducing organells is the hair bundle, a cluster 
of interconnected microvilli up to 1Opm in length. Dis- 
placement of the hair bundle results in the generation 
of a transducer current of lo-250 pA on the hair cell 
plasma membrane [ 1,2]. Several types of ionic channels 
have been identified in the hair cell [3]. The prevailing 
view is that the transducer channel is mechanically 
gated when the hair bundle is deformed. 
and diluted with 200@ L-l 5. Inner and outer hair cells could be readily 
identified by their characteristic morphology [6]. Cells could be used 
for up to 3-4 hours after dissection. The second bulla could be kept, 
unopened, in moist tissue for subsequent use within about 5 h. Treat- 
ment with collagenase (Sigma, I mg/ml, 10 min) or pronase (Sigma, 
I m&ml, 15 min) and mechanical dissociation was also successfully 
used to obtain isolated cells. 
A conventional patch clamp technique was used to obtain gigaseal 
excised patches and to measure their electrical characteristics [7]. 
Here we report that excised patches of inner and 
outer hair cell membranes, from the organ of Corti of 
guinea pig, contain a Gonductance which is gated di- 
rectly by CAMP. It is non-selective to monovalent ca- 
tions and resembles the cyclic nucleotide-dependent 
conductance of photoreceptors and olfactory cells [4,5], 
but differs in that it is activated only by CAMP. 
Solution A was changed to solution B just after a gigaseal had been 
obtained. The patch pipette was usually filled with solution 8. The 
current signal was low-pas filtered (bandwidth 1500 Hz) before being 
digitized. A computer system was used to generate command voltage 
ramps and obtain I-V relationships. Solutions (mM): (A) NaCI, 150; 
CaC&, 1; MgCI,; IO; HEPES, 10; pH 7.5; (B) NaCI, 150; EGTA, 0.5; 
EDTA, 0.5; HEPES, 10; pH 7.5. In our experiments the CAMP-gated 
conductance was low when both sides of excised patches were bathed 
with physiological solution, while in low Ca”, Mg” solution with 
CAMP-gated conductance was much higher. Therefore to increase the 
CAMP-gated conductance (i.e. to ensure experimental accuracy) patch 
pipettes were filled with solution B containing less than 0.1 PM Ca” 
and Mg”. 
2. MATERIALS AND METHODS 3. RESULTS 
Isolated outer and inner hair cells were obtained from the organ of 
Corti of guinea pigs. Young adult guinea pigs of either sex (weight 
200-600 g) were killed by rapid cervical dislocation. Both bullae were 
quickly removed. One of the bullae was opened, the cochlea was 
removed and 4 turns of the organ of Corti were dissected from the 
cochlear spiral in L-15 cell culture medium (Flow laboratories; the 
major ions in the medium are (mM) Na’, 135; K’, 5; Ca*‘., 1.26; Mg2’, 
2; and amino acids, adjusted to pH 7.4), The isol-ited coil was trans. 
ferred to a 100 PM aliquot to which was added 100 ~1 of L-15 with 
1 mgml trypsin. The dissection step could be completed within 3 min. 
After 20 min the droplet was gently triturated to dissociate the cells 
Correspondence &dress: G.R. Kalamkarov, Institute of Biological 
Physics, USSR Academy of Sciences, Pushchino, Moscow Region 
142292, USSR. 
We have examined the conductance of the isolated 
hair cell plasma membrane using the patch-clamp tech- 
nique [?‘I. After gigaseal formation, the membrane patch 
was excised in the ‘inside-out’ configuration so the cyto- 
plasmic surface was exposed to the bath solution. The 
application of CAMP resulted in a reversible increase in 
patch conductance (Fig. 2) which occurred in the ab- 
sence of nucleotide triphosphates and thus cannot have 
resulted from CAMP-stimulated protein phosphoryla- 
tion. This was observed in 18 out of 37 outer hair cell 
patches and 9 out of 22 inner hair cell patches. The 
subsequent addition of 1 mM MgZC or Ca” ions in the 
presence of CAMP reversibly blocked the conductance 
?ubiished by Elsevier Science Publishers B. V, 167 
Fig. 1. Photomicrograph of dissociated inner (a) and outer (b) hair cell 
showing the position of the patch pipette during gigascal formation. 
The photomicrograph was taken 4 hours after isolation of the cell. 
(Scale bar = IO pm) 
(Fig. 2). Further, I-cis-diltiazem, which effectively 
blocks the cyclic nucleotide gated conductance in pho- 
toreceptor and olfactory receptor cells [4,5], in 0.5 mM 
concentration blocked the CAMP-gated conductance of 
hair cells as well (not shown in the figure). 
The outer segments of retinal rods and cones are 
known to be highly specific for cGMP, while olfactory 
cell conductance is activated by both CAMP and cGMP. 
We therefore investigated the sensitivity of the hair cell 
conductance to cGMP. In our experiments cGMP at 1 
mM concentration did not affect the conductance (Fig. 
2) although 30 ,uM of cAMP application was sufficient 
to significantly increase conductance in the same patch 
(Fig. 2). 
The conductance of the excised patch varied with the 
concentration of agonist (Fig. 3a). We characterized 
conductance by measuring the current-voltage relation- 
ss aopl CAMP I 88 
ships of the patches. In Fig. 3b, the normalized conduct- 
ance of a patch from an inner hair cell is plotted against 
cAMP concentration. The conductance shows a second- 
ary decrease at a high concentration of agonist, similar 
to the conductance of photoreceptor and olfactory cells 
[4,5]. Similar results were obtained in an identical exper- 
iment on an outer hair cell. 
The selectivity of the CAMP-gated channels was 
characterized by the shift of the reversal potential of the 
CAMP-dependent current seen upon equimolar substi- 
tution of NaCl by other monovalent metal chlorides in 
the bathing solution. Two patches, in which the con- 
ductance was sufficiently high and stable to provide 
appropriate accuracy of measurements yielded the fol- 
lowing reversal potential shifts (mV): -0.3-CO.5; 
5.4+0.8; 7.3C 1, for Cs+, I.2 and Wb’, respectively. This 
suggests the following order of permeabilities: 
Cs’ > Na’ > Li’ > Rb’ 
The shift induced by substituting NaCl for KC1 was not 
measured exactly because of the high density of potas- 
sium channels which are not gated by CAMP in the cell. 
But this value did not exceed 10 mV and therefore the 
K+ permeability of the CAMP-gated channels seems to 
be of the same order as for other monovalent cations. 
We failed in our attempts to record the single channel 
current. Therefore, the amplitude and kinetic parame- 
ters of the channels were estimated by analysis of the 
fluctuations of the CAMP-gated current. The power 
spectrum for the current, fitted with two least-square 
Lorenzians (Fig. 4), indicated cut-off frequencies of 170 
I-Iz and 1000 kI-Iz, corresponding to an intrinsic time of 
channel fluctuation in the order of 1 ms. The single- 
~OJJM CAMP I 30 JJM CAMP * 9.5 mbj 
M@$ 1 SB 1 100 JJM cAMP 1 1mMcGMP I100 pMbAMP 1 SB 
Fig. 2. Chart recordinf of the action of CAMP, h?g2+ ions and cGMP on the conductance of an isolated patch of the hair cell plasma membrane. 
The bars indicate changes in bathing solution. Mg”, cCMP and CAMP were added to solution B (SB). A@” strongly suppressed the CAMP-gated 
conductance. The same results were obtained with Ca” (not shown), 10 mV, 15 Hz pulses were applied to measure the patch conductance. 
Volume 290, number 1.2 FEBS LETTERS September 199 I 
a r~~lts in a decrease of the unit event almplitude, the 
4-M single channel conductance in the normal divalent ca- 
tion concentration seems to be not higher than 1 pS. 
4. DISCUSSION 
Our data indicate that the properties of cyclic nucleo- 
tide-activated channels in hair cell, olfactory cell and 
-4- fllV 
photoreceptor cell patches are very similar in respect o 
conductance, ion selectivity, blockage by Ca2+ and Mg2+ 
-30 30 and sensitivity to diltiazem. Likewise, the cAMP-con- 
ductance of hair cell decreased at high agonist concen- 
trations as has been observed in photoreceptor and ol- 
factory patches [4,S]. In rod and cone photoreceptors, 
b 
photoresponses are known to be generated by the clo- 
sure of channels directly gated by cGMP [9,10]. Direct 
gating of conductance by cyclic nucleotides has also 
been observed in ti;e plasma membrane of olfactory 
cells [4,1 l] and has been suggested to play an important 
role in the olfactory transduction. 
The demonstration of a cyclic nucleotide-gated con- 
ductance in outer and inner hair cells raises the possibil- 
ity that mechanotransduction might be mediated by cy- 
Fig. 3. Concentration dependence of the CAMP-gated conductance clic nwcleotides in a similar manner as phototransduc- 
measured with divalcnt-action-free solution on both sides of the mem- tion and olfactory transduction. To what extent do 
brane: a I-V CUI’VCS measured in the presence of different concentra- 
tions of CAMP (concentration (,!&I) indicated lo the right of each 
these characteristics agree with those which are thought 
trace): b. normalized conductance plotted as a function of CAMP to control the hair cell transducer current? At least two 
concentration. The Hill coefficient estimated from tbe linear part of characteristics are available for comparison: (1) selec- 
the curve (O-100 PM) is 1.2. tivity, and (2) unit conductance. (1) The reversal poten- 
tial of the transducer current of isolated hair cell is close 
channel unit conductance estimated in the absence of to zero [1,12]. It means that the channels are non-selec- 
divalent cations from the variance-to-mean ratio of the tive which is consistent with the properties of the 
CAMP-gated current [8], was 16 pS (Ca”‘, Mg”’ free cAMP-gated conductance described here. (2) The 
solution, holdiag potential -40 mV). Divalent cations single-channel conductance estimated in ref. [13] from 
decreased the CAMP-gated conductance by more than the variance of the transducer current in the presence of 
an order of magnitude. Suggesting that the blockage divalent cations is 12 pS. This value does not agree with 
the very small unit conductance of our cAMP-gated 
1 _ channel in a physiological concentration of divalent ca- 
tions. On the other hand, the estimation in ref. [13] is 
in principle susceptible to several sources of error. It 
was based on whole-cell recording, and thus it was 
necessary to block other conductances by some inhi- 
0.1 _ bitors; e.g., if the blockage of Ca2+-activated potassium 
channels by Cs’ ions was incomplete, their contribution 
to the current variance could be substantial. 
It might be argued that if the CAMP-gated channels 
O.OL are really connected with transduction, they should be 
I I I , Hz located in the microvilli rather than in the cell body. Our 
PO 102 IO3 lo4 attempts to obtain gigaseal recordings from the micro- 
Fig. 4. Power density spectrum of the CAMP-gated fluctuations from 
villi were unfortunately unsuccessful. However in olfac- 
a patch of outer hair cell plasma membrane at holding potentin! -40 tory cells, where the cilia are considered to be the site 
mV. The curve is a Lore&an function of transduction [ 14,151 the cyclic nucleotide-gated con- 
0.35 0.05 
ductance is found also in the dendrite and cell body [4]. 
sy> = 
I + (r/170)2 + I + ~1000)~ 
A role in transduction would require a mechanism by 
fitted by the least-squares method. The difference spectrum of patch 
which mechanical movement could change the concen- 
current fluctuations in the presence and absence of CAMP was calcu- tration of CAMP. But there is presently no evidence for 
kited to obtain the CAMP-gated current spectrum. the existence of an adenylate cyclase or phosphodies- 
169 
Voiume 29U, number i,Z 
terase stimulated by hair bundle displacement. It is also 
possible that there are several parallel molecular mech- 
anisms providing the transducer current and cAMP- 
gated channels are involved only in one of them. Thus 
the involvement of cyclic nucleotides in mechano- 
transduction would not exclude the possibility that the 
displacement of microvilli could also directly gate chan- 
nels in the plasma membrane [3]. 
Acknowledgenrents: We would like to thank Dr K. Dormer, Prof. A. 
Byzov and Prof. T. Reuter for critically reading the manuscript. 
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A cyclic-AMP-gated conductance in cochlear hair cells 

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A cyclic-AMP-gated conductance in cochlear hair cells

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