Neuroscience Research, Suppl. 12 (1990) $51 $62 Elsevier Scientific Publishers Ireland Ltd.
MECHANO-ELECTRICAL RESPONSES
$51
TRANSDUCTION
AND
MUSCARINIC
CHOLINERGIC
IN THE C H I C K H A I R CELL
Harunori OHMORI
The N a t i o n a l Myodaiji,
I n s t i t u t e for P h y s i o l o g i c a l
Okazaki,
Sciences,
444, Japan
INTRODUCTION The s e n s o r y h a i r cell t r a n s d u c e s electrical
signal;
mechanical
information
such i n f o r m a t i o n as sound, acceleration,
into
and the
body axis in the g r a v i t y field of the earth are t r a n s d u c e d into the electrical central
signal
nervous
in
the
system
hair
for
cell
further
and
then
transmitted
information
to
processing.
the Some
i n f o r m a t i o n is also r e t u r n e d from the central nervous s y s t e m to the peripheral
h e a r i n g organ and to the v e s t i b u l a r o r g a n by the effer-
ent i n n e r v a t i o n
pathway.
The m e c h a n o - e l e c t r i c a l
p e r f o r m e d as a g a t i n g of the m e c h a n i c a l l y gated the h a i r cell.
In this
mechano-electrical
review
I will
discuss
transduction
is
ion channel w i t h i n
the
features
of
the
t r a n s d u c t i o n and will s u m m a r i z e the t r a n s d u c t i o n
m e c h a n i s m s t u d i e d in the chick hair cell. Hair cells r e c e i v e the c e n t r i f u g a l chlear bundle,
and the e l e c t r i c a l
i n n e r v a t i o n by the o l i v o c o -
s t i m u l a t i o n of the e f f e r e n t nerve
fiber is k n o w n to p r o d u c e a long lasting slow i n h i b i t o r y p o s t s y n a p tic p o t e n t i a l tylcholine
in the h a i r cell
induced
(5,12).
intracellular
second part of this review. concentration would
I will demonstrate
Ca c o n c e n t r a t i o n
change
an acein the
This increase of the i n t r a c e l l u l a r
lead to the h y p e r p o l a r i z a t i o n
Ca
of the m e m b r a n e
by the a c t i v a t i o n of Ca a c t i v a t e d K c o n d u c t a n c e and will e v e n t u a l l y suppress
the h a i r
cell
function
as a p r e s y n a p t i c
neural
element
(18).
M a t e r i a l s and M e t h o d s Hair treatment ciation
cells
were
isolated
from
f o l l o w e d by trituration. and
electrical
recording
a chick
inner
ear
Detailed methods have
been
by p a p a i n
of cell disso-
described
elsewhere
(13). ACh induced Ca r e s p o n s e s w e r e studied from hair cells isolated w i t h o u t to d i s p e r s e
proteolytic cells.
e n z y m e t r e a t m e n t w h e r e o n l y D N A s e was u s e d
Isolated hair cells were w h o l e
Presented at the 12th Taniguchi International Symposium on Visual Science, November 27-December 1, 1989 0168-0102/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
cell clamped
for
$52 current and voltage recordings. loaded with
fura-2
Ca c o n c e n t r a t i o n s .
Fluorescence
excitation wavelengths lated.
Detailed
In some experiments
for f l u o r e s c e n c e
images
of
were frame integrated
method
described p r e v i o u s l y
measurements
of
fluorescence
hair cells were of i n t r a c e l l u l a r
both
340
and
380
nm
and ratios were calcu-
measurements
have
been
(16,18).
(mY) A
peq
hc
20 mV
probe m o t i ~ / ~ ~ I
]
2 /Jr.
~
gr
I
100 msec
mV, o"l
B
O
(b ° o
-50 I I I I I
~ ~
t
%. ~ @'-'wO
f
t
/ I I I I o
loo
(mV) membrane potential
-L-50 Fig.l Mechano-electrical transduction (m-e.t.) p o t e n t i a l s . M e c h a n i c a l s t i m u l a t i o n was a p p l i e d to the hair b u n d l e by a glass rod of w h i c h m o t i o n is i l l u s t r a t e d in the b o t t o m of A. The inset shows the a r r a n g e m e n t of the hair cell (hc), a s t i m u l a t i n g glass rod (gr) and a recording patch electrode (pe). A, traces of m-e.t. p o t e n t i a l s u n d e r the w h o l e cell current clamp. B, a m p l i t u d s of me.t. p o t e n t i a l in A are p l o t t e d a g a i n s t the m e m b r a n e p o t e n t i a l s . F i l l e d c i r c l e s i n d i c a t e t r a c e s d e m o n s t r a t e d in A. Note that the a m p l i t u d e of d e p o l a r i z i n g m-e.t, p o t e n t i a l s is p l o t t e d downward. Amplitudes of d e p o l a r i z i n g m-e.t, potential decreased p r o g r e s s i v e l y at large n e g a t i v e m e m b r a n e p o t e n t i a l s w h e r e a n o m a l o u s r e c t i f i e r K conductance was activated.
$53
Results
Ion selectivity of the transduction channel. M e c h a n i c a l d i s p l a c e m e n t s of the hair bundle towards the taller stereocilium
generated
negative membrane
depolarizing
potentials
transduction
At p o s i t i v e m e m b r a n e p o t e n t i a l s
(i.e. at +96 mV),
cal d i s p l a c e m e n t
bundle
of
the
transduction potentials. duction
of the
hair
level of the r e v e r s a l of i n t r a c e l l u l a r
hair
Therefore,
cell
and
potentials
(a t r a c e r e c o r d e d at -43 mV,
hyperpolarizing
the m e c h a n o - e l e c t r i c a l a reversal
p o t e n t i a l was d e t e r m i n e d extracellular
the same m e c h a n i -
generated
demonstrated
medium.
at
Fig.l).
trans-
potential.
The
by the c o m p o s i t i o n
All
the
alkaline
ca-
tions and m o s t of the a l k a l i n e earth cations were p e r m e a b l e through the t r a n s d u c e r ence
to
Cs
follows:
channel,
ions
choline(0.33): Ca(4.65): cations
were
Li(1.39)
and the r e l a t i v e p e r m e a b i l i t i e s
determined
: Na(l.22)
TMA(0.20):
Sr(2.82):
from
the
reversal
: K(I.17).
TEA(0.17)
Ba(2.73):
potential
Rb(l.12)
for m o n o v a l e n t
Mn(2.50):
in r e f e r -
cations,
Mg(2.41)
as
: Cs(l.0)
for
:
and
divalent
(14).
From this
ion-selectivity
study,
the m - e . t ,
channel
gested to have a high field s t r e n g t h i o n - s e l e c t i v i t y site series XI, ability
of
4), w h i c h divalent
is s u g -
(Eisenman
is c o n s i s t e n t w i t h the r e l a t i v e l y high permecations
through
the
m-e.t,
channel.
If we
assume the d i a m e t e r of the least p e r m e a b l e organic cations TEA) as the size of pore,
the m-e.t,
(TMA, or
channel might be a pore of 6-7
diameter.
Single channel conductance of the mechano-electrical The mined
single
in the
channel
chick
steps d e t e c t e d under v o l t a g e
cell
in the w h o l e clamp
can be c a l c u l a t e d in a Cs m e d i u m
hair
conductance
(Fig.2).
(50 pS,
f r o m the d i s c r e t e cell
recorded
The a b s o l u t e
transducer.
at 30 °C) w a s d e t e r amplitude
transduction
permeability
from this 50 pS single m-e.t,
channel c o n d u c t a n c e
S i n c e Ca ions are 4.65
times m o r e p e r m e a b l e than Cs ions through the channel, 30 ~M Ca c o n c e n t r a t i o n in the e n d o l y m p h
will
channel raise
coefficient
(14); the single channel p e r m e a b i l i t y c o e f f i c i e n t of
Cs ions at -50 m v is 7.04 x 10 -14 cm3/sec.
m-e.t,
current currents
at -50 mV, w i l l
the
carry
if we assume
(i), i0 m s e c o p e n i n g of the
3.8 x 10 -22 mol Ca ions,
i n t r a c e l l u l a r Ca c o n c e n t r a t i o n
within
a
and
volume
$54
• , so
=v
Cs/Cs
A
500 - 50
"
300
mV
- -
b, ,.Q
25 pA
c,
100
-
-
/ l
I
l
i0
o
l
1
20
1
|00
msec
30
(pA)
Fig.2 D i s c r e t e amplitudes of m-e.t, currents. A, an a m p l i t u d e h i s t o g r a m c a l c u l a t e d f r o m t r a c e s in B. B, m - e . t , c u r r e n t s w e r e generated at +50mV in Cs based e x t r a c e l l u l a r m e d i u m w h i l e r e c o r d i n g with C s / E G T A b a s e d i n t r a c e l l u l a r medium. S t e a d y h a i r b u n d l e d i s p l a c e m e n t s g e n e r a t e d s t e p w i s e m - e . t , c u r r e n t s . By s u p e r i m p o s i n g these current traces in B-c, several current levels were detected. The a m p l i t u d e of these current levels are reflected as the peak in the histogram. These current levels were located a p p r o x i m a t e l y at 2.5 pA separation, and were likely g e n e r a t e d by a channel of 50 pS conductance. (modified from Figs.liB, 12 of Ohmori (1985)).
equivalent to a single s t e r e o c i l i u m close to the #M level. calculation might the channel
still u n d e r e s t i m a t e
This
the e x a c t Ca i n f l u x
since the p o s i t i v e l y p o l a r i z e d
endolymphatic
through
potential
would facilitate the Ca influx
(3). The i n t r a c e l l u l a r Ca concentra-
tion could be raised close to
#M levels, by the m e c h a n o - e l e c t r i c a l
transduction, this
at
least
in the v i c i n i t y
of the m-e.t,
level of Ca c o n c e n t r a t i o n w o u l d be sufficient
channel
and
to trigger m a n y
Ca-dependent b i o l o g i c a l reactions. Holton
and H u d s p e t h
(9) e s t i m a t e d
the s i n g l e m - e . t ,
channel
$55 current
and
the
conductance
nique a p p l i e d to a series frog s a c c u l a r hair cell m a t e of 13 pS pS);
by the
ensemble
noise
of m-e.t,
currents
recorded
in the e p i t h e l i a l
(at 10 °C) was m u c h
small value m i g h t between
chick
coefficient
Their esti-
smaller than my observation
and
(9),
but m o r e
c o m m o n p r o b l e m in the n o i s e a n a l y s i s ,
resulted
lowpass
filtering
the
current
fluctuation
of
bullfrog
the
signal,
power
of
particularly
(25 pS).
likely
the
This
channel
reflects
a
from the e x t e n s i v e
underestimation in
(50
case even at 37
of 1.3 was a s s u m e d
reflect a d i f f e r e n t nature of the m-e.t,
(14)
tech-
in the bull-
preparation.
the c o n d u c t a n c e was about half of the chick's
°C when a t e m p e r a t u r e
analysis
higher
of
the
frequency
region.
Gating sensitivity of the m-e.t,
channel.
Figure 3A d e m o n s t r a t e s a series of current traces g e n e r a t e d by mechanical
stimulation applied
tion to the c u t i c u l a r plate. generate
a transduction
This d i s p l a c e m e n t
5 ~m above the hair bundle's
inser-
The smallest d i s p l a c e m e n t w h i c h
current
corresponds
was
100 ~
in
this
hair
could cell.
to 0.1 d e g r e e a n g u l a r d i s p l a c e m e n t
of the hair bundle. F i g . 3 B plots the n o r m a l i z e d a m p l i t u d e of the t r a n s d u c t i o n currents a g a i n s t several
the a n g u l a r d i s p l a c e m e n t
experiments
hair b u n d l e
made
on h a i r
of hair bundle,
cells w i t h
(15). W h e n d i s p l a c e m e n t s
summarizing
different
were applied
lengths
of
to h a i r b u n d l e s
shorter than 7.5 ~m the d i s p l a c e m e n t was applied at 5 ~m above the hair b u n d l e ' s ~m w h e n
insertion
applied
the i n s e t
to the
pictures
to the c u t i c u l a r plate, tall
hair bundle
in Fig.3).
The
taller
transduction
and at 5 or at i0 than
12.5
current
was plotted a g a i n s t the a n g u l a r d i s p l a c e m e n t a f t e r scaling, three types
of e x p e r i m e n t s
The a n g u l a r
displacement
showed a common relationship
of h a i r b u n d l e
is t h e r e f o r e
~m
(see
amplitude and all
(Fig.3B).
the
primary
factor in the g a t i n g of t r a n s d u c t i o n channel.
Site of the mechano-electrical The
site
controversial
of
transduction
issue.
small f i e l d p o t e n t i a l were
channel
in h a i r
cell
is
still
a
By m e a s u r i n g the e x t r a c e l l u l a r field p o t e n t i a l
changes w h i l e v i b r a t i n g
which
transduction channel.
the h a i r bundle,
changes
Hudspeth
a b o u t the d i s t a l
r e v e r s i b l y e l i m i n a t e d by
(ii) d e t e c t e d
a
end of h a i r b u n d l e
streptomycin.
Hudspeth
has
$56
A
B
@59*m
O 001
(#m)
0.02
t%
~
E
0 0 ~A-0%0-0
1.0
(D
"o :D
A
0.03
0.05
O
007
~
o.o
E
0.1
short hair
0
tail hair
o/"
.....
A
>
5 pA /~100
q
n"
msec O~
-10
< 7.5 pm
-5 t
A
~
A
O~ )~
5
10
I
t
~ 12.5 pm 15
Angular d i s p l a c e m e n t
o_____o._____~__----o---"°"
(degree)
Fig.3 Mechano-electrical t r a n s d u c t i o n c u r r e n t and the n o r m a l ized a n g u l a r d i s p l a c e m e n t vs. response relationship. A, m e c h a n i c a l d i s p l a c e m e n t was a p p l i e d to the hair bundle at 5 ~m above the hair b u n d l e ' s i n s e r t i o n to the c u t i c u l a r p l a t e . B, d i s p l a c e m e n t vs. response relationship after normalization. The s h o r t h a i r b u n d l e hair cell (_& 7.5 ~m, open circles) was s t i m u l a t e d at 5 /an, and the tall h a i r b u n d l e h a i r cell (_~ 12.5 /an) was s t i m u l a t e d e i t h e r at 5 /an (open t r i a n g l e s ) or at i0 /an (filled c i r c l e s ) . The i n s e t pictures indicate these stimulation heights . (modified from Figs.4,5,6,7, of Ohmori (1987)).
proposed
that
individual structure
the
which
connects
mechano-electrical review
transduction
stereocilium
channel
somewhat adjacent
transduction
is
at
associated stereocilia has
been
the
distal
with (17).
described
the His in
end
of
tip-link idea a
of
recent
(i0).
The
site
of Ca i n f l u x
during
mechano-electrical
transduction
$57 was s t u d i e d by a f l u o r e s c e n c e are s i g n i f i c a n t duction,
Ca influx through the m-e.t,
and the Ca s e n s i t i v e dye fura-2
the cell by the e s t e r tion
of
imaging technique
Ca
ions
and
mechano-electrical
form of the dye, changes
of
the
(16).
Since there
channel d u r i n g
trans-
(6) is e a s i l y loaded
into
the i n t r a c e l l u l a r d i s t r i b u Ga
concentration
during
t r a n s d u c t i o n have been imaged by the c o m b i n a t i o n
of f l u o r e s c e n c e m i c r o s c o p e and image p r o c e s s i n g c o m p u t e r system.
A
C
w/+
B
w/÷ Mn
1.0
!
1,2
2
,
3
3,4 i
4 i
S
5,6
6
7
7,8
8
Fig.4 L o c a l i z a t i o n of f l u o r e s c e n c e c h a n g e s d u r i n g m e c h a n o e l e c t r i c a l transduction. Hair bundle was s i n u s o i d a l l y s t i m u l a t e d at 1 kHz, first in a 25 mM Ca m e d i u m (A) and then in a 2 mM Mn, 23 mM Ca m e d i u m (B) in a single hair cell. F l u o r e s c e n c e ratio i n t e n s i t i e s w e r e d e m o n s t r a t e d in a t h r e e d i m e n s i o n a l w a y in A a n d in B. C, f l u o r e s c e n c e r a t i o s in s e v e r a l p l a n e s l o n g i t u d i n a l l y c r o s s i n g the hair bundle and the cell b o d y are d e m o n s t r a t e d in a Ca m e d i u m (left column, t r a c e s 1,3,5,7), and in a Mn m e d i u m (right column, t r a c e s 2 , 4 , 6 , 8 ) . T h e s e two m e a s u r e m e n t s are s u p e r i m p o s e d in the c e n t e r column. (from Fig.9, of Ohmori (1988)).
$58 F i g . 4 A d e m o n s t r a t e s ratio i n t e n s i t y d i s t r i b u t i o n over the hair cell c a l c u l a t e d
f r o m an e x p e r i m e n t
hair b u n d l e was
stimulated
peak emerges about plate.
Fig.4C,
by
25 m M Ca s a l i n e .
sinusoidal
the insertion of hair bundle
and a p e a k
cuticular plate.
is o b s e r v e d
longitudinally
into the c u t i c u l a r
cross
at the h a i r b u n d l e ' s
the h a i r bun-
insertion
to the stimu-
raised all over the cell body
the ratio i n t e n s i t y has been
(see Fig.8 of
(16)). This is p r o b a b l y
b e c a u s e of the a c t i v a t i o n
of v o l t a g e g a t e d Ca c h a n n e l s
the b a s o l a t e r a l
membrane
and
channels.
such
Under
by
the
circumstances
the cell b o d y towards
influx
close
Ca c h a n n e l s
channel (6).
Fig.4B
of
(14)
and
measurement
the h a i r
and
4C,
of Ca
of the Ca c h a n n e l
are d i f f i c u l t
cell were
is
right
in Mn m e d i u m
known
to q u e n c h
column,
cations the
demonstrate
(2 mM Mn,
d e p r e s s i o n of the f l u o r e s c e n c e insertion
fluorescence
to
the
in the c e n t e r
column
in Fig.4C,
the f l u o r e s c e n c e
through
induced
the m - e . t ,
electrical
the
channel.
changes
by
It was
channels
when the hair frequency.
By
superimposing
peak
(Ca-peak)
observed
A the
and
the
loca-
here h a v e b e e n
the
influx
concluded
are m o s t
of
these
that
likely
displacement
that the g a t i n g k i n e t i c s of
the
hair
absolute d i s p l a c e m e n t of the hair bundle
bundle
(15).
cations
the m e c h a n o -
located
This conclusion accords with
understanding
angular
of
the Ca c o n c e n t r a t i o n and the Mn c o n c e n t r a -
base of the hair bundle.
on the
the
fluorescence
have a p p e a r e d e x a c t l y at the same
directly
transduction
physiological
plate.
the r a t i o
tion. T h e r e f o r e ,
changes
to Mn 2+.
through
in the Ca m e d i u m and in the Mn m e d i u m
(Mn-peak)
tion
differ-
ratio a p p e a r e d a b o u t the h a i r bun-
ratio d e p r e s s i o n
confirmed as r e f l e c t i n g
The
and the m-
fluorescence
23 mM Ca saline),
cuticular
ratio profiles
Ca
from
for the moment.
bundle was s i n u s o i d a l l y stimulated at 1 kHz v i b r a t i o n dle's
these ions
impermeable
Mn 2+ is one of the m o s t p e r m e a b l e
fura-2
through
(14) and p h a r m a c o l o g i c a l
e n t i a t i o n of t h e s e two ion c h a n n e l s
m-e.t,
l o c a t e d on
the hair bundle could not be negligible.
channel are r e l a t i v e l y
Conversely,
Ca
the d i f f u s i o n
ion s e l e c t i v i t y and the p h a r m a c o l o g y
However,
the
a sharp
However, when a larger a m p l i t u d e m e c h a n i c a l
lation was a p p l i e d to the hair bundle,
e.t.
When
waveform,
left column, d e m o n s t r a t e s the f l u o r e s c e n c e i n t e n s i t y
ratios in s e v e r a l p l a n e s w h i c h dle,
in
1 kHz
but
at
the
the e l e c t r o is d e p e n d e n t not
on
the
$59
A 0.9 O3
-~ O
0.8 100 pM Ach
O3
0.7
10 pM atropine
I
100
I
I
200
300
I'"
400
I
f
800
900
I
1000 (sec)
B o
0.8 ~r
100 pM Ach 2.5 Ca
2.5 Ca
0.7
0
r
!
I
I
100
200
300
I
400
500
(sec)
Fig.5 A C h g e n e r a t e d i n t r a c e l l u l a r Ca r e s p o n s e s in h a i r cells. One h u n d r e d ~M ACh was p r e s s u r e puff applied to hair cells. A, ACh induced Ca r e s p o n s e s w e r e r e v e r s i b l y suppressed by the e x t r a c e l l u larly a p p l i e d 10 ~M a t r o p i n e . B, Ca r e s p o n s e was g e n e r a t e d by ACh in a Ca f r e e e x t r a c e l l u l a r m e d i u m . ( f r o m F i g . 6 of S h i g e m o t o & Ohmori (1990)).
Muscarinic cholinergic receptor mechanism might mediate
the effer-
ent innervation to the hair cell. Efferent and
fibres m a k e a x o s o m a t i c contacts with outer hair cells
axodendritic
contacts
mainly
inner h a i r c e l l s of m a m m a l s efferent nerves most primary
generates
afferent
at t h e s e
terminals,
onto
afferent
Electrical
nerve
fibres
(5,12).
innervation
as the m o s t however
nerve
fibres
stimulation
inhibitory effects
receive excitatory efferent has b e e n c o n s i d e r e d
(19).
in h a i r cells Some
(7).
ACh
has
not
yet
and in
afferent
fibres
Acetylcholine
likely neurotransmitter been
in
of t h e s e
(ACh)
candidate
unequivocally
d e t e r m i n e d to be the n e u r o t r a n s m i t t e r of this synapse. Shigemoto
& Ohmori
loaded i s o l a t e d
(18)
applied
ACh
rise of the i n t r a c e l l u l a r Ca concentration. intracellular ances
and
Ca
and
ATP
h a i r cell or c l u s t e r of h a i r cells
concentration
hyperpolarized
the
activated membrane.
to
the
fura-2
and d e t e c t e d
a
This i n c r e a s e d level of Ca a c t i v a t e d Fig.5
K conduct-
demonstrates
the
$60 increase
of
the
extracellulariy atropine
intracellular applied
(Fig.5~,
sponse was
application),
but
Muscarine
responses
similar
ent
terminal
induced
the
in
the
intracellular
curare
1 mM,
Ca response
pressure
from
puff
medium
the
receptor
at
by re-
intracellular
released
muscarinic
the
(Ca
extracellular
induced
ACh
by
was b l o c k e d
by
at
Ca-free
Therefore,
to a c t i v a t e
and
control
carbamylcholine
to ACh.
induced
This Ca r e s p o n s e
application)
30% of the
and
seems
and to induce
bath
to
was
(Fig.5B).
concentration
100 ~M ACh.
i0 ~M,
suppressed
Ca
least
Ca
effer-
mechanism
in chick
hair
cells. ATP
is
known
cholinergic responses lular
to
be
terminals
in chick
hair
lar m e d i u m
ATP
the inward
current
(18).
was
that
from
of Ca ions
in
various
generates of
Ca
intracel-
the ACh
induced
from the e x t r a c e l l u -
from the i n t r a c e l l u l a r
in the v o l t a g e
followed
ACh ATP
the mode
different
Ca ions
current
with
shown
However,
was
an influx
released
inward
together
have
increase
induced
and also
ATP g e n e r a t e s
We
cells
Ca c o n c e n t r a t i o n
Ca response.
released
(2).
clamped
by a slowly rising
stores.
hair
cell
outward
and
current.
DISCUSSION Inferences
on
the
gating
The d i s p l a c e m e n t
mechanism
tion of the m e m b r a n e
structure
al s t e r e o c i l i u m
the
into
ing or r e l a x a t i o n there.
From
cation
through
diameter the
of
m-e.t,
these
vs.
displacement
to
deformation diameter
presence any
relationship,
the
the
m-e.t,
cuticular
could work
were
TEA
diameter
smallest
current
plate
its d i a m e t e r
at
arguement
linkages present
was
This the
as
as the m e c h a n i c a l
This
of
does
the
If the
the pore
From
the
hair
bundle
0.i
degree
the
hair
displacement base
0.2 gate not
to the t r a n s d u c e r between
permeable
5 ~m a b o v e
(15).
deformation
located
(14).
diameter.
a p p l i e d at
stretch-
channels
largest
or
the 7 ~
channel.
linkages
as
of the i n d i v i d u -
a membrane
the
TMA
6 to
of any c y t o s k e l e t a l
structural
a
if we a s s u m e
the
was
taken
have
2 ~ membrane
membrane
study,
i00 ~ w h e n
to the
the
stereocilium
7 ~
or
Such
to gate the m-e.t,
were
generated
lead to the d i s t o r -
the insertion
channel
response
channel.
should
plate.
selectivity
might
insertion
corresponds
be able
cations
that
about
cuticular
m-e.t,
displacement
bundle's
vidual
ion
the
channel
displacement
angular
might
the
of m-e.t,
of the hair bundle
~m.
of
indi-
This
directly assume channel.
channel
and
2 on the If the
$61 cytoskelton, more
the s m a l l m e m b r a n e d e f o r m a t i o n
directly
be
condensed
onto
the
estimated
channel
protein
a mediator
of
above
could
and
would
facilitate the channel gating. Is m u s c a r i n i c synapse
cholinergic
onto
the h a i r cell
Although
efferent
?
by ACh and ATP in these hair cells,
t h a t m u s t be e x p l a i n e d
least one of these m o l e c u l e s candidate
the
it is a c o n s i s t e n t o b s e r v a t i o n to induce i n t r a c e l l u -
lar Ca r e s p o n s e s problems
receptor
of
the
efferent
before
there are two
t h e s e two m o l e c u l e s
or at
to be c o n s i d e r e d as a n e u r o t r a n s m i t t e r
synapse.
(i) The
intracellular
Ca
ions
themselves have been shown to release n e u r o t r a n s m i t t e r in the o t h e r synapses
(8). However,
the Ca a c t i v a t e d
K channel
is l i k e l y local-
ized in close v i c i n i t y to the i n h i b i t o r y e f f e r e n t synapse w h e r e the muscarinic
ACh
concentration
receptor
is
is increased,
channel were
frequently
cell a t t a c h e d
patch
activated
and
the
since activities
observed
pipette
with
while
the c h a n n e l
(2)
The
second
problem
course of the i n t r a c e l l u l a r Ca r e s p o n s e
Ca
of the Ca a c t i v a t e d
the p r e s e n c e
of A C h
activity
when ACh was a p p l i e d o u t s i d e the patch m e m b r a n e al c o m m u n i c a t i o n ) .
intracellular
was
(Shigemoto,
is
while
rare
person-
a prolonged
(Fig.3C),
K
in the
time
the hyper-
p o l a r i z a t i o n induced by e f f e r e n t nerve a c t i v i t y was in the o r d e r of 100 msec
(12).
Neurotransmitters
v i c i n i t y of p o s t s y n a p t i c
are r e l e a s e d as a packet
receptor
lose its a c t i v i t y in in vivo condition. tion
and
required
rapid
breakdown
to s h o r t e n
of
the
the o v e r a l l
a l s o be e s s e n t i a l
Ca r e s p o n s e
thW
to
particular
Such c o n c e n t r a t e d a p p l i c a -
transmitter time
range and m i g h t
from the
and t h e n h y d r o l y z e d and r a p i d l y
course
molecule to the
in l o c a l i z i n g
site
close
to
might
be
physiological
the i n t r a c e l l u l a r
the
Ca
activated
K
channels.
ACKNOWLEDGMENTS This w o r k was s u p p o r t e d by grants M i n i s t r y of Education,
(62870005)
Science and C u l t u r e to H.O.
from the J a p a n e s e
$62 REFERENCES
i.
Bosher SK, Warren RL (1968) Proc Royal Society BI71:
2.
Burnstock
3.
Davis,H.
G (1984) J Physiol (1965).In:Symposium
Harbor Laboratory NY,vol.30,
227-244
313:1-35 on Quantal Biology,
of Quantitative
Biology.
Cold Spring
ColdSpring
Harbor,
pp 181-190
4.
Eisenman G (1962) Biophys J 2 : 2 5 9 - 3 2 3
5.
Galambos
6.
Grynkiewicz
7.
Highstein
SM, Baker R (1985) J Neurophysiol
8.
Higashida
H (1988) J Physiol
R (1956) J Neurophysiol
19:424-437
G, Poenie M, Tsien RY (1985) J Biol Chem 260:
3440-3450 54: 370-384
397:209-222
9.
Holton T, Hudspeth AJ (1986) J Physiol
i0.
Howard J, Roberts WM, Hudspeth AJ (1983) Ann Rev Biophys
ii.
Hudspeth AJ
12
Klinke R, Galley N (1974) Physiol Rev 54: 316-357
Bio Chem
375: 195-227
17:99-124 (1982) J Neurosci
2: 1-10
13
Ohmori H (1984) J physiol
350:561-581
14
Ohmori H (1985) J Physiol
359:189-217
15
Ohmori H (1987) J Physiol
387:589-609
16
Ohmori H (1988) J Physiol
399:
17
Pickles JO, Comis SD, Osborne MP (1984) Hearing Res 15: 103-112
115-137
18
Shigemoto
T, Ohmori H (1990) J Physiol
19
Spoendlin
H (1984)
In:Comparative
eds. L.Bolis,R.D.Keynes Press, pp163-188
420:127-148
Physiology of Sensory Systems
& S.H.P. Maddrell,
Cambridge
University
MECHANO-ELECTRICAL RESPONSES
$51
TRANSDUCTION
AND
MUSCARINIC
CHOLINERGIC
IN THE C H I C K H A I R CELL
Harunori OHMORI
The N a t i o n a l Myodaiji,
I n s t i t u t e for P h y s i o l o g i c a l
Okazaki,
Sciences,
444, Japan
INTRODUCTION The s e n s o r y h a i r cell t r a n s d u c e s electrical
signal;
mechanical
information
such i n f o r m a t i o n as sound, acceleration,
into
and the
body axis in the g r a v i t y field of the earth are t r a n s d u c e d into the electrical central
signal
nervous
in
the
system
hair
for
cell
further
and
then
transmitted
information
to
processing.
the Some
i n f o r m a t i o n is also r e t u r n e d from the central nervous s y s t e m to the peripheral
h e a r i n g organ and to the v e s t i b u l a r o r g a n by the effer-
ent i n n e r v a t i o n
pathway.
The m e c h a n o - e l e c t r i c a l
p e r f o r m e d as a g a t i n g of the m e c h a n i c a l l y gated the h a i r cell.
In this
mechano-electrical
review
I will
discuss
transduction
is
ion channel w i t h i n
the
features
of
the
t r a n s d u c t i o n and will s u m m a r i z e the t r a n s d u c t i o n
m e c h a n i s m s t u d i e d in the chick hair cell. Hair cells r e c e i v e the c e n t r i f u g a l chlear bundle,
and the e l e c t r i c a l
i n n e r v a t i o n by the o l i v o c o -
s t i m u l a t i o n of the e f f e r e n t nerve
fiber is k n o w n to p r o d u c e a long lasting slow i n h i b i t o r y p o s t s y n a p tic p o t e n t i a l tylcholine
in the h a i r cell
induced
(5,12).
intracellular
second part of this review. concentration would
I will demonstrate
Ca c o n c e n t r a t i o n
change
an acein the
This increase of the i n t r a c e l l u l a r
lead to the h y p e r p o l a r i z a t i o n
Ca
of the m e m b r a n e
by the a c t i v a t i o n of Ca a c t i v a t e d K c o n d u c t a n c e and will e v e n t u a l l y suppress
the h a i r
cell
function
as a p r e s y n a p t i c
neural
element
(18).
M a t e r i a l s and M e t h o d s Hair treatment ciation
cells
were
isolated
from
f o l l o w e d by trituration. and
electrical
recording
a chick
inner
ear
Detailed methods have
been
by p a p a i n
of cell disso-
described
elsewhere
(13). ACh induced Ca r e s p o n s e s w e r e studied from hair cells isolated w i t h o u t to d i s p e r s e
proteolytic cells.
e n z y m e t r e a t m e n t w h e r e o n l y D N A s e was u s e d
Isolated hair cells were w h o l e
Presented at the 12th Taniguchi International Symposium on Visual Science, November 27-December 1, 1989 0168-0102/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
cell clamped
for
$52 current and voltage recordings. loaded with
fura-2
Ca c o n c e n t r a t i o n s .
Fluorescence
excitation wavelengths lated.
Detailed
In some experiments
for f l u o r e s c e n c e
images
of
were frame integrated
method
described p r e v i o u s l y
measurements
of
fluorescence
hair cells were of i n t r a c e l l u l a r
both
340
and
380
nm
and ratios were calcu-
measurements
have
been
(16,18).
(mY) A
peq
hc
20 mV
probe m o t i ~ / ~ ~ I
]
2 /Jr.
~
gr
I
100 msec
mV, o"l
B
O
(b ° o
-50 I I I I I
~ ~
t
%. ~ @'-'wO
f
t
/ I I I I o
loo
(mV) membrane potential
-L-50 Fig.l Mechano-electrical transduction (m-e.t.) p o t e n t i a l s . M e c h a n i c a l s t i m u l a t i o n was a p p l i e d to the hair b u n d l e by a glass rod of w h i c h m o t i o n is i l l u s t r a t e d in the b o t t o m of A. The inset shows the a r r a n g e m e n t of the hair cell (hc), a s t i m u l a t i n g glass rod (gr) and a recording patch electrode (pe). A, traces of m-e.t. p o t e n t i a l s u n d e r the w h o l e cell current clamp. B, a m p l i t u d s of me.t. p o t e n t i a l in A are p l o t t e d a g a i n s t the m e m b r a n e p o t e n t i a l s . F i l l e d c i r c l e s i n d i c a t e t r a c e s d e m o n s t r a t e d in A. Note that the a m p l i t u d e of d e p o l a r i z i n g m-e.t, p o t e n t i a l s is p l o t t e d downward. Amplitudes of d e p o l a r i z i n g m-e.t, potential decreased p r o g r e s s i v e l y at large n e g a t i v e m e m b r a n e p o t e n t i a l s w h e r e a n o m a l o u s r e c t i f i e r K conductance was activated.
$53
Results
Ion selectivity of the transduction channel. M e c h a n i c a l d i s p l a c e m e n t s of the hair bundle towards the taller stereocilium
generated
negative membrane
depolarizing
potentials
transduction
At p o s i t i v e m e m b r a n e p o t e n t i a l s
(i.e. at +96 mV),
cal d i s p l a c e m e n t
bundle
of
the
transduction potentials. duction
of the
hair
level of the r e v e r s a l of i n t r a c e l l u l a r
hair
Therefore,
cell
and
potentials
(a t r a c e r e c o r d e d at -43 mV,
hyperpolarizing
the m e c h a n o - e l e c t r i c a l a reversal
p o t e n t i a l was d e t e r m i n e d extracellular
the same m e c h a n i -
generated
demonstrated
medium.
at
Fig.l).
trans-
potential.
The
by the c o m p o s i t i o n
All
the
alkaline
ca-
tions and m o s t of the a l k a l i n e earth cations were p e r m e a b l e through the t r a n s d u c e r ence
to
Cs
follows:
channel,
ions
choline(0.33): Ca(4.65): cations
were
Li(1.39)
and the r e l a t i v e p e r m e a b i l i t i e s
determined
: Na(l.22)
TMA(0.20):
Sr(2.82):
from
the
reversal
: K(I.17).
TEA(0.17)
Ba(2.73):
potential
Rb(l.12)
for m o n o v a l e n t
Mn(2.50):
in r e f e r -
cations,
Mg(2.41)
as
: Cs(l.0)
for
:
and
divalent
(14).
From this
ion-selectivity
study,
the m - e . t ,
channel
gested to have a high field s t r e n g t h i o n - s e l e c t i v i t y site series XI, ability
of
4), w h i c h divalent
is s u g -
(Eisenman
is c o n s i s t e n t w i t h the r e l a t i v e l y high permecations
through
the
m-e.t,
channel.
If we
assume the d i a m e t e r of the least p e r m e a b l e organic cations TEA) as the size of pore,
the m-e.t,
(TMA, or
channel might be a pore of 6-7
diameter.
Single channel conductance of the mechano-electrical The mined
single
in the
channel
chick
steps d e t e c t e d under v o l t a g e
cell
in the w h o l e clamp
can be c a l c u l a t e d in a Cs m e d i u m
hair
conductance
(Fig.2).
(50 pS,
f r o m the d i s c r e t e cell
recorded
The a b s o l u t e
transducer.
at 30 °C) w a s d e t e r amplitude
transduction
permeability
from this 50 pS single m-e.t,
channel c o n d u c t a n c e
S i n c e Ca ions are 4.65
times m o r e p e r m e a b l e than Cs ions through the channel, 30 ~M Ca c o n c e n t r a t i o n in the e n d o l y m p h
will
channel raise
coefficient
(14); the single channel p e r m e a b i l i t y c o e f f i c i e n t of
Cs ions at -50 m v is 7.04 x 10 -14 cm3/sec.
m-e.t,
current currents
at -50 mV, w i l l
the
carry
if we assume
(i), i0 m s e c o p e n i n g of the
3.8 x 10 -22 mol Ca ions,
i n t r a c e l l u l a r Ca c o n c e n t r a t i o n
within
a
and
volume
$54
• , so
=v
Cs/Cs
A
500 - 50
"
300
mV
- -
b, ,.Q
25 pA
c,
100
-
-
/ l
I
l
i0
o
l
1
20
1
|00
msec
30
(pA)
Fig.2 D i s c r e t e amplitudes of m-e.t, currents. A, an a m p l i t u d e h i s t o g r a m c a l c u l a t e d f r o m t r a c e s in B. B, m - e . t , c u r r e n t s w e r e generated at +50mV in Cs based e x t r a c e l l u l a r m e d i u m w h i l e r e c o r d i n g with C s / E G T A b a s e d i n t r a c e l l u l a r medium. S t e a d y h a i r b u n d l e d i s p l a c e m e n t s g e n e r a t e d s t e p w i s e m - e . t , c u r r e n t s . By s u p e r i m p o s i n g these current traces in B-c, several current levels were detected. The a m p l i t u d e of these current levels are reflected as the peak in the histogram. These current levels were located a p p r o x i m a t e l y at 2.5 pA separation, and were likely g e n e r a t e d by a channel of 50 pS conductance. (modified from Figs.liB, 12 of Ohmori (1985)).
equivalent to a single s t e r e o c i l i u m close to the #M level. calculation might the channel
still u n d e r e s t i m a t e
This
the e x a c t Ca i n f l u x
since the p o s i t i v e l y p o l a r i z e d
endolymphatic
through
potential
would facilitate the Ca influx
(3). The i n t r a c e l l u l a r Ca concentra-
tion could be raised close to
#M levels, by the m e c h a n o - e l e c t r i c a l
transduction, this
at
least
in the v i c i n i t y
of the m-e.t,
level of Ca c o n c e n t r a t i o n w o u l d be sufficient
channel
and
to trigger m a n y
Ca-dependent b i o l o g i c a l reactions. Holton
and H u d s p e t h
(9) e s t i m a t e d
the s i n g l e m - e . t ,
channel
$55 current
and
the
conductance
nique a p p l i e d to a series frog s a c c u l a r hair cell m a t e of 13 pS pS);
by the
ensemble
noise
of m-e.t,
currents
recorded
in the e p i t h e l i a l
(at 10 °C) was m u c h
small value m i g h t between
chick
coefficient
Their esti-
smaller than my observation
and
(9),
but m o r e
c o m m o n p r o b l e m in the n o i s e a n a l y s i s ,
resulted
lowpass
filtering
the
current
fluctuation
of
bullfrog
the
signal,
power
of
particularly
(25 pS).
likely
the
This
channel
reflects
a
from the e x t e n s i v e
underestimation in
(50
case even at 37
of 1.3 was a s s u m e d
reflect a d i f f e r e n t nature of the m-e.t,
(14)
tech-
in the bull-
preparation.
the c o n d u c t a n c e was about half of the chick's
°C when a t e m p e r a t u r e
analysis
higher
of
the
frequency
region.
Gating sensitivity of the m-e.t,
channel.
Figure 3A d e m o n s t r a t e s a series of current traces g e n e r a t e d by mechanical
stimulation applied
tion to the c u t i c u l a r plate. generate
a transduction
This d i s p l a c e m e n t
5 ~m above the hair bundle's
inser-
The smallest d i s p l a c e m e n t w h i c h
current
corresponds
was
100 ~
in
this
hair
could cell.
to 0.1 d e g r e e a n g u l a r d i s p l a c e m e n t
of the hair bundle. F i g . 3 B plots the n o r m a l i z e d a m p l i t u d e of the t r a n s d u c t i o n currents a g a i n s t several
the a n g u l a r d i s p l a c e m e n t
experiments
hair b u n d l e
made
on h a i r
of hair bundle,
cells w i t h
(15). W h e n d i s p l a c e m e n t s
summarizing
different
were applied
lengths
of
to h a i r b u n d l e s
shorter than 7.5 ~m the d i s p l a c e m e n t was applied at 5 ~m above the hair b u n d l e ' s ~m w h e n
insertion
applied
the i n s e t
to the
pictures
to the c u t i c u l a r plate, tall
hair bundle
in Fig.3).
The
taller
transduction
and at 5 or at i0 than
12.5
current
was plotted a g a i n s t the a n g u l a r d i s p l a c e m e n t a f t e r scaling, three types
of e x p e r i m e n t s
The a n g u l a r
displacement
showed a common relationship
of h a i r b u n d l e
is t h e r e f o r e
~m
(see
amplitude and all
(Fig.3B).
the
primary
factor in the g a t i n g of t r a n s d u c t i o n channel.
Site of the mechano-electrical The
site
controversial
of
transduction
issue.
small f i e l d p o t e n t i a l were
channel
in h a i r
cell
is
still
a
By m e a s u r i n g the e x t r a c e l l u l a r field p o t e n t i a l
changes w h i l e v i b r a t i n g
which
transduction channel.
the h a i r bundle,
changes
Hudspeth
a b o u t the d i s t a l
r e v e r s i b l y e l i m i n a t e d by
(ii) d e t e c t e d
a
end of h a i r b u n d l e
streptomycin.
Hudspeth
has
$56
A
B
@59*m
O 001
(#m)
0.02
t%
~
E
0 0 ~A-0%0-0
1.0
(D
"o :D
A
0.03
0.05
O
007
~
o.o
E
0.1
short hair
0
tail hair
o/"
.....
A
>
5 pA /~100
q
n"
msec O~
-10
< 7.5 pm
-5 t
A
~
A
O~ )~
5
10
I
t
~ 12.5 pm 15
Angular d i s p l a c e m e n t
o_____o._____~__----o---"°"
(degree)
Fig.3 Mechano-electrical t r a n s d u c t i o n c u r r e n t and the n o r m a l ized a n g u l a r d i s p l a c e m e n t vs. response relationship. A, m e c h a n i c a l d i s p l a c e m e n t was a p p l i e d to the hair bundle at 5 ~m above the hair b u n d l e ' s i n s e r t i o n to the c u t i c u l a r p l a t e . B, d i s p l a c e m e n t vs. response relationship after normalization. The s h o r t h a i r b u n d l e hair cell (_& 7.5 ~m, open circles) was s t i m u l a t e d at 5 /an, and the tall h a i r b u n d l e h a i r cell (_~ 12.5 /an) was s t i m u l a t e d e i t h e r at 5 /an (open t r i a n g l e s ) or at i0 /an (filled c i r c l e s ) . The i n s e t pictures indicate these stimulation heights . (modified from Figs.4,5,6,7, of Ohmori (1987)).
proposed
that
individual structure
the
which
connects
mechano-electrical review
transduction
stereocilium
channel
somewhat adjacent
transduction
is
at
associated stereocilia has
been
the
distal
with (17).
described
the His in
end
of
tip-link idea a
of
recent
(i0).
The
site
of Ca i n f l u x
during
mechano-electrical
transduction
$57 was s t u d i e d by a f l u o r e s c e n c e are s i g n i f i c a n t duction,
Ca influx through the m-e.t,
and the Ca s e n s i t i v e dye fura-2
the cell by the e s t e r tion
of
imaging technique
Ca
ions
and
mechano-electrical
form of the dye, changes
of
the
(16).
Since there
channel d u r i n g
trans-
(6) is e a s i l y loaded
into
the i n t r a c e l l u l a r d i s t r i b u Ga
concentration
during
t r a n s d u c t i o n have been imaged by the c o m b i n a t i o n
of f l u o r e s c e n c e m i c r o s c o p e and image p r o c e s s i n g c o m p u t e r system.
A
C
w/+
B
w/÷ Mn
1.0
!
1,2
2
,
3
3,4 i
4 i
S
5,6
6
7
7,8
8
Fig.4 L o c a l i z a t i o n of f l u o r e s c e n c e c h a n g e s d u r i n g m e c h a n o e l e c t r i c a l transduction. Hair bundle was s i n u s o i d a l l y s t i m u l a t e d at 1 kHz, first in a 25 mM Ca m e d i u m (A) and then in a 2 mM Mn, 23 mM Ca m e d i u m (B) in a single hair cell. F l u o r e s c e n c e ratio i n t e n s i t i e s w e r e d e m o n s t r a t e d in a t h r e e d i m e n s i o n a l w a y in A a n d in B. C, f l u o r e s c e n c e r a t i o s in s e v e r a l p l a n e s l o n g i t u d i n a l l y c r o s s i n g the hair bundle and the cell b o d y are d e m o n s t r a t e d in a Ca m e d i u m (left column, t r a c e s 1,3,5,7), and in a Mn m e d i u m (right column, t r a c e s 2 , 4 , 6 , 8 ) . T h e s e two m e a s u r e m e n t s are s u p e r i m p o s e d in the c e n t e r column. (from Fig.9, of Ohmori (1988)).
$58 F i g . 4 A d e m o n s t r a t e s ratio i n t e n s i t y d i s t r i b u t i o n over the hair cell c a l c u l a t e d
f r o m an e x p e r i m e n t
hair b u n d l e was
stimulated
peak emerges about plate.
Fig.4C,
by
25 m M Ca s a l i n e .
sinusoidal
the insertion of hair bundle
and a p e a k
cuticular plate.
is o b s e r v e d
longitudinally
into the c u t i c u l a r
cross
at the h a i r b u n d l e ' s
the h a i r bun-
insertion
to the stimu-
raised all over the cell body
the ratio i n t e n s i t y has been
(see Fig.8 of
(16)). This is p r o b a b l y
b e c a u s e of the a c t i v a t i o n
of v o l t a g e g a t e d Ca c h a n n e l s
the b a s o l a t e r a l
membrane
and
channels.
such
Under
by
the
circumstances
the cell b o d y towards
influx
close
Ca c h a n n e l s
channel (6).
Fig.4B
of
(14)
and
measurement
the h a i r
and
4C,
of Ca
of the Ca c h a n n e l
are d i f f i c u l t
cell were
is
right
in Mn m e d i u m
known
to q u e n c h
column,
cations the
demonstrate
(2 mM Mn,
d e p r e s s i o n of the f l u o r e s c e n c e insertion
fluorescence
to
the
in the c e n t e r
column
in Fig.4C,
the f l u o r e s c e n c e
through
induced
the m - e . t ,
electrical
the
channel.
changes
by
It was
channels
when the hair frequency.
By
superimposing
peak
(Ca-peak)
observed
A the
and
the
loca-
here h a v e b e e n
the
influx
concluded
are m o s t
of
these
that
likely
displacement
that the g a t i n g k i n e t i c s of
the
hair
absolute d i s p l a c e m e n t of the hair bundle
bundle
(15).
cations
the m e c h a n o -
located
This conclusion accords with
understanding
angular
of
the Ca c o n c e n t r a t i o n and the Mn c o n c e n t r a -
base of the hair bundle.
on the
the
fluorescence
have a p p e a r e d e x a c t l y at the same
directly
transduction
physiological
plate.
the r a t i o
tion. T h e r e f o r e ,
changes
to Mn 2+.
through
in the Ca m e d i u m and in the Mn m e d i u m
(Mn-peak)
tion
differ-
ratio a p p e a r e d a b o u t the h a i r bun-
ratio d e p r e s s i o n
confirmed as r e f l e c t i n g
The
and the m-
fluorescence
23 mM Ca saline),
cuticular
ratio profiles
Ca
from
for the moment.
bundle was s i n u s o i d a l l y stimulated at 1 kHz v i b r a t i o n dle's
these ions
impermeable
Mn 2+ is one of the m o s t p e r m e a b l e
fura-2
through
(14) and p h a r m a c o l o g i c a l
e n t i a t i o n of t h e s e two ion c h a n n e l s
m-e.t,
l o c a t e d on
the hair bundle could not be negligible.
channel are r e l a t i v e l y
Conversely,
Ca
the d i f f u s i o n
ion s e l e c t i v i t y and the p h a r m a c o l o g y
However,
the
a sharp
However, when a larger a m p l i t u d e m e c h a n i c a l
lation was a p p l i e d to the hair bundle,
e.t.
When
waveform,
left column, d e m o n s t r a t e s the f l u o r e s c e n c e i n t e n s i t y
ratios in s e v e r a l p l a n e s w h i c h dle,
in
1 kHz
but
at
the
the e l e c t r o is d e p e n d e n t not
on
the
$59
A 0.9 O3
-~ O
0.8 100 pM Ach
O3
0.7
10 pM atropine
I
100
I
I
200
300
I'"
400
I
f
800
900
I
1000 (sec)
B o
0.8 ~r
100 pM Ach 2.5 Ca
2.5 Ca
0.7
0
r
!
I
I
100
200
300
I
400
500
(sec)
Fig.5 A C h g e n e r a t e d i n t r a c e l l u l a r Ca r e s p o n s e s in h a i r cells. One h u n d r e d ~M ACh was p r e s s u r e puff applied to hair cells. A, ACh induced Ca r e s p o n s e s w e r e r e v e r s i b l y suppressed by the e x t r a c e l l u larly a p p l i e d 10 ~M a t r o p i n e . B, Ca r e s p o n s e was g e n e r a t e d by ACh in a Ca f r e e e x t r a c e l l u l a r m e d i u m . ( f r o m F i g . 6 of S h i g e m o t o & Ohmori (1990)).
Muscarinic cholinergic receptor mechanism might mediate
the effer-
ent innervation to the hair cell. Efferent and
fibres m a k e a x o s o m a t i c contacts with outer hair cells
axodendritic
contacts
mainly
inner h a i r c e l l s of m a m m a l s efferent nerves most primary
generates
afferent
at t h e s e
terminals,
onto
afferent
Electrical
nerve
fibres
(5,12).
innervation
as the m o s t however
nerve
fibres
stimulation
inhibitory effects
receive excitatory efferent has b e e n c o n s i d e r e d
(19).
in h a i r cells Some
(7).
ACh
has
not
yet
and in
afferent
fibres
Acetylcholine
likely neurotransmitter been
in
of t h e s e
(ACh)
candidate
unequivocally
d e t e r m i n e d to be the n e u r o t r a n s m i t t e r of this synapse. Shigemoto
& Ohmori
loaded i s o l a t e d
(18)
applied
ACh
rise of the i n t r a c e l l u l a r Ca concentration. intracellular ances
and
Ca
and
ATP
h a i r cell or c l u s t e r of h a i r cells
concentration
hyperpolarized
the
activated membrane.
to
the
fura-2
and d e t e c t e d
a
This i n c r e a s e d level of Ca a c t i v a t e d Fig.5
K conduct-
demonstrates
the
$60 increase
of
the
extracellulariy atropine
intracellular applied
(Fig.5~,
sponse was
application),
but
Muscarine
responses
similar
ent
terminal
induced
the
in
the
intracellular
curare
1 mM,
Ca response
pressure
from
puff
medium
the
receptor
at
by re-
intracellular
released
muscarinic
the
(Ca
extracellular
induced
ACh
by
was b l o c k e d
by
at
Ca-free
Therefore,
to a c t i v a t e
and
control
carbamylcholine
to ACh.
induced
This Ca r e s p o n s e
application)
30% of the
and
seems
and to induce
bath
to
was
(Fig.5B).
concentration
100 ~M ACh.
i0 ~M,
suppressed
Ca
least
Ca
effer-
mechanism
in chick
hair
cells. ATP
is
known
cholinergic responses lular
to
be
terminals
in chick
hair
lar m e d i u m
ATP
the inward
current
(18).
was
that
from
of Ca ions
in
various
generates of
Ca
intracel-
the ACh
induced
from the e x t r a c e l l u -
from the i n t r a c e l l u l a r
in the v o l t a g e
followed
ACh ATP
the mode
different
Ca ions
current
with
shown
However,
was
an influx
released
inward
together
have
increase
induced
and also
ATP g e n e r a t e s
We
cells
Ca c o n c e n t r a t i o n
Ca response.
released
(2).
clamped
by a slowly rising
stores.
hair
cell
outward
and
current.
DISCUSSION Inferences
on
the
gating
The d i s p l a c e m e n t
mechanism
tion of the m e m b r a n e
structure
al s t e r e o c i l i u m
the
into
ing or r e l a x a t i o n there.
From
cation
through
diameter the
of
m-e.t,
these
vs.
displacement
to
deformation diameter
presence any
relationship,
the
the
m-e.t,
cuticular
could work
were
TEA
diameter
smallest
current
plate
its d i a m e t e r
at
arguement
linkages present
was
This the
as
as the m e c h a n i c a l
This
of
does
the
If the
the pore
From
the
hair
bundle
0.i
degree
the
hair
displacement base
0.2 gate not
to the t r a n s d u c e r between
permeable
5 ~m a b o v e
(15).
deformation
located
(14).
diameter.
a p p l i e d at
stretch-
channels
largest
or
the 7 ~
channel.
linkages
as
of the i n d i v i d u -
a membrane
the
TMA
6 to
of any c y t o s k e l e t a l
structural
a
if we a s s u m e
the
was
taken
have
2 ~ membrane
membrane
study,
i00 ~ w h e n
to the
the
stereocilium
7 ~
or
Such
to gate the m-e.t,
were
generated
lead to the d i s t o r -
the insertion
channel
response
channel.
should
plate.
selectivity
might
insertion
corresponds
be able
cations
that
about
cuticular
m-e.t,
displacement
bundle's
vidual
ion
the
channel
displacement
angular
might
the
of m-e.t,
of the hair bundle
~m.
of
indi-
This
directly assume channel.
channel
and
2 on the If the
$61 cytoskelton, more
the s m a l l m e m b r a n e d e f o r m a t i o n
directly
be
condensed
onto
the
estimated
channel
protein
a mediator
of
above
could
and
would
facilitate the channel gating. Is m u s c a r i n i c synapse
cholinergic
onto
the h a i r cell
Although
efferent
?
by ACh and ATP in these hair cells,
t h a t m u s t be e x p l a i n e d
least one of these m o l e c u l e s candidate
the
it is a c o n s i s t e n t o b s e r v a t i o n to induce i n t r a c e l l u -
lar Ca r e s p o n s e s problems
receptor
of
the
efferent
before
there are two
t h e s e two m o l e c u l e s
or at
to be c o n s i d e r e d as a n e u r o t r a n s m i t t e r
synapse.
(i) The
intracellular
Ca
ions
themselves have been shown to release n e u r o t r a n s m i t t e r in the o t h e r synapses
(8). However,
the Ca a c t i v a t e d
K channel
is l i k e l y local-
ized in close v i c i n i t y to the i n h i b i t o r y e f f e r e n t synapse w h e r e the muscarinic
ACh
concentration
receptor
is
is increased,
channel were
frequently
cell a t t a c h e d
patch
activated
and
the
since activities
observed
pipette
with
while
the c h a n n e l
(2)
The
second
problem
course of the i n t r a c e l l u l a r Ca r e s p o n s e
Ca
of the Ca a c t i v a t e d
the p r e s e n c e
of A C h
activity
when ACh was a p p l i e d o u t s i d e the patch m e m b r a n e al c o m m u n i c a t i o n ) .
intracellular
was
(Shigemoto,
is
while
rare
person-
a prolonged
(Fig.3C),
K
in the
time
the hyper-
p o l a r i z a t i o n induced by e f f e r e n t nerve a c t i v i t y was in the o r d e r of 100 msec
(12).
Neurotransmitters
v i c i n i t y of p o s t s y n a p t i c
are r e l e a s e d as a packet
receptor
lose its a c t i v i t y in in vivo condition. tion
and
required
rapid
breakdown
to s h o r t e n
of
the
the o v e r a l l
a l s o be e s s e n t i a l
Ca r e s p o n s e
thW
to
particular
Such c o n c e n t r a t e d a p p l i c a -
transmitter time
range and m i g h t
from the
and t h e n h y d r o l y z e d and r a p i d l y
course
molecule to the
in l o c a l i z i n g
site
close
to
might
be
physiological
the i n t r a c e l l u l a r
the
Ca
activated
K
channels.
ACKNOWLEDGMENTS This w o r k was s u p p o r t e d by grants M i n i s t r y of Education,
(62870005)
Science and C u l t u r e to H.O.
from the J a p a n e s e
$62 REFERENCES
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Burnstock
3.
Davis,H.
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4.
Eisenman G (1962) Biophys J 2 : 2 5 9 - 3 2 3
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Galambos
6.
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7.
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H (1988) J Physiol
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Hudspeth AJ
12
Klinke R, Galley N (1974) Physiol Rev 54: 316-357
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Ohmori H (1984) J physiol
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17
Pickles JO, Comis SD, Osborne MP (1984) Hearing Res 15: 103-112
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H (1984)
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& S.H.P. Maddrell,
Cambridge
University
