T-Type Ca 2+ Channel Lowers the Threshold of ... - BioMedSearch
T-Type C a 2+ Channel Lowers the Threshold of Spike Generation in the Newt Olfactory Receptor Cell FUSAO KAWAI, TAKASHI
KURAHASHI,
and
AKIMICHI
KANEKO
From the D e p a r t m e n t o f Information Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444,Japan
ABSTRA CT Mechanisms underlying action potential generation in the newt olfactory receptor cell were investigated by using the whole-cell version of the patch-clamp technique. Isolated olfactory cells had a resting membrane potential of - 7 0 +- 9 mV. Injection of a depolarizing current step triggered action potentials under current clamp condition. The amplitude of the action potential was reduced by lowering external Na + concentration. After a complete removal of Na +, however, cells still showed action potentials which was abolished either by Ca 2+ removal or by an application of Ca z+ channel blocker (Co 2+ o r Ni2+), indicating an involvement of Ca 2+ current in spike generation of newt olfactory receptor cells. Under the voltage clamp condition, depolarization of the cell to - 4 0 mV from the holding voltage of - 1 0 0 mV induced a fast transient inward current, which consisted of Na + (INa) and T-type C a 2+ (ICa.T) currents. The amplitude of I(~.Twas about one fourth of that of IN~. Depolarization to more positive voltages also induced L-type Ca 2+ current (Ica.L)- ICa,L Was as small as a few pA in normal Ringer solution. The activating voltage of Ic~.xwas approximately 10 mV more negative than that of INa. Under current clamp, action potentials generated by a least effective depolarization was almost completely blocked by 0.1 mM Nf2+ (a specific T-type C a z+ channel blocker) even in the presence of Na +. These results suggest that Ic~,r contributes to action potential in the newt olfactory receptor cell and lowers the threshold of spike generation. KEY WORDS:
olfactory receptor cell • calcium channel • action potential • patch clamp • newt
INTRODUCTION
O d o r a n t binding to the receptor protein at the ciliary surface of the olfactory r e c e p t o r cell leads to the subseq u e n t enzymatic cascades (for review, see Bakalyar and Read, 1991; Breer a n d Boekhoff, 1992; Reed, 1992; R o n n e t t and Snyder, 1992) that finally leads to the o p e n i n g of three types of ionic channels; cAMP-gated cationic channels, CaZ+-gated C1 channels (for review, see Gold a n d Nakamura, 1987; Firestein, 1992; Kurahashi a n d Yau, 1994), and IP3-gated Ca 2+ channels (for review, see Reed, 1992; Restrepo et al., 1996; Ronnett and Snyder, 1992). This initial excitation causes a slow and graded voltage change; its amplitude is d e p e n d e n t on stimulus intensities (Trotier and MacLeod, 1983; Kurahashi, 1989a; Firestein et al., 1993). Graded receptor potential is then e n c o d e d into spike trains that transmit the olfactory information to the brain. At this step, the information relating to the stimulus intensity is e n c o d e d into the spike density. It has long b e e n believed that the action potential of olfactory cells is generated by the influx of Na+; u n d e r voltage clamp, the transient inward current has b e e n shown to be mediated by Na + channel (catfish: Miyamoto et al., 1992; coho salmon: Nevitt and Moody, 1992; xenopus: Schild, 1989; tiger salamander: Firestein and Werblin, 1987; Dr. Kurahashi's p r e s e n t address is School of Science, Osaka University, Toyonaka, Osaka 560, Japan. Address c o r r e s p o n d e n c e to Dr. Fusao Kawai, National Institute for Physiological Sciences, Myodaiji, Okazaki 444,Japan. Fax: 81-564-527913; E-mail: [email protected] 525
Dubin and Dionne, 1994; rat: T r o m b l e y and Westbrook, 1991). However, an earlier study by Trotier (1986) has pointed out that the salamander olfactory cell expresses a transient inward current in a Na+-free, tetrodotoxin-containing solution. F u r t h e r m o r e , in preliminary experiments by one of the present authors, Kurahashi (1989a) recorded action potentials using newt olfactory cells bathed in Na+-free m e d i u m . These observations raised a possibility that action potential is generated not only by Na + current, but also by o t h e r transient inward current in the a m p h i b i a n olfactory receptor cells. In the present study, we reexamined the mechanism of spike generation in newt olfactory receptor cells by analyzing m e m b r a n e currents under the whole-cell clamp condition in further detail. We f o u n d that the transient inward current activated by m e m b r a n e depolarization is a mixture of Na + a n d C a 2+ currents through two i n d e p e n d e n t ionic channels. The transient Ca 2+ c o m p o n e n t was shown to be carried through a T-type Ca 2+ channel. Activation range for the T-type C a 2+ current was approximately 10 mV m o r e negative than that of Na + current. This observation indicates that T-type Ca 2+ current determines the threshold of spike generation in the newt olfactory receptor cells. 1 1After the submission of o u r original manuscript, L i m a n a n d Corey (1996) reported that a T-type Ca 2+ c h a n n e l is expressed in the chemosenso~y n e u r o n s from the m o u s e vomeronasal organ. This observation raises a possibility that T-type Ca 2+ channels m i g h t be expressed n o t only in newt olfactory receptor cells b u t also in cells of the o t h e r species.
J. GEN. PHYSIOL. © The Rockefeller University Press • 0 0 2 2 - 1 2 9 5 / 9 6 / 1 2 / 5 2 5 / 1 1 $2.00 Volume 108 D e c e m b e r 1996 525-535
Indeed,
at threshold,
action potentials
N i 2+, a s e l e c t i v e b l o c k e r in the presence
o f N a +.
MATERIALS
AND
were blocked
f o r T - t y p e C a 2+ c h a n n e l ,
Cells were c o n t i n u o u s l y s u p e r f u s e d (at a rate o f 0.9 m l / m i n ) with o n e o f t h e s o l u t i o n s listed in T a b l e I. T h e i n d i f f e r e n t e l e c t r o d e was a n Ag-AgC1 wire c o n n e c t e d to t h e c u l t u r e dish via a n a g a r o s e bridge. C u r r e n t a n d voltage signals f r o m t h e a m p l i f i e r were m o n itored o n a n oscilloscope ( T e k t r o n i x , Beaverton, OR) a n d a therm a l array r e c o r d e r (WR7900; G r a p h t e c , Fujisawa, J a p a n ) . Data were low-pass filtered (4-pole Bessel type) with a cut-off freq u e n c y o f 5 k H z a n d t h e n digitized at 10 kHz. All e x p e r i m e n t s were p e i - f o r m e d at r o o m t e m p e r a t u r e (23-25°C).
by
even
METHODS
Preparation Solitary r e c e p t o r cells were enzymatically dissociated f r o m t h e olfactory e p i t h e l i u m o f t h e newt, Cynopspyrrhogaster. Dissociation p r o t o c o l s were similar to t h o s e r e p o r t e d previously ( K u r a h a s h i , 1989b). In short, t h e a n i m a l was a n e s t h e t i z e d by c o o l i n g o n ice, d e c a p i t a t e d , a n d p i t h e d . T h e m u c o s a e e x c i s e d f r o m t h e olfactory cavity were i n c u b a t e d for 5 rain at 30°C in a s o l u t i o n (Table I solution 15) c o n t a i n i n g 0.1% c o l l a g e n a s e (Sigma C h e m i c a l Co., St. Louis, M O ) with n o a d d e d Ca z+ a n d M g ')+. T h e tissue was t h e n r i n s e d twice with a c o n t r o l s o l u t i o n (solution 1) a n d triturated. Isolated cells were p l a t e d o n t h e c o n c a n a v a l i n A - c o a t e d glass cover-slip. Cells were m a i n t a i n e d at 4°C ( u p to 10 h) b e f o r e use. In t h e p r e s e n t e x p e r i m e n t , we s e l e c t e d r e c e p t o r cells w h i c h lost t h e i r cilia to s t u d y t h e excitability o f t h e s o m a t i c m e m b r a n e .
Application of Drugs Several p h a r m a c o l o g i c a l a g e n t s were u s e d for m e m b r a n e c u r r e n t analyses: t e t r a e t h y l a m m o n i u m c h l o r i d e (TEA) a n d CsC1 for blocking K+ current, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid (SITS) for b l o c k i n g CI- c u r r e n t , COC12, NiCI~, a n d CdC12 for b l o c k i n g Ca 2+ c u r r e n t . D i h y d r o p y r i d i n e s (nifed i p i n e a n d Bay K 8644) a n d t0-conotoxin GVIA were also u s e d to identify t h e type o f t h e Ca 2+ c u r r e n t . Divalent cations a n d o t h e r p h a r m a c o l o g i c a l a g e n t s were dissolved in t h e s u p e r f u s a t e . T h e s e a g e n t s were b a t h a p p l i e d to t h e cell f r o m t h e p e f f u s i o n system. CoCI~, NiC12, a n d CdC12 were p u r c h a s e d f r o m Nacalai T e s q u e Inc. (Kyoto, J a p a n ) , T E A f r o m W a k o P u r e C h e m i c a l I n d u s t r i e s Ltd. (Osaka, J a p a n ) , a n d SITS, n i f e d i p i n e , Bay K 8644, a n d to-conotoxin GVIA f r o m S i g m a C h e m i c a l Co.
Recording Procedures M e m b r a n e c u r r e n t s were r e c o r d e d by a p a t c h c l a m p t e c h n i q u e in t h e w h o l e - c e l l - c l a m p c o n f i g u r a t i o n (Hamill et al., 1981). Pyrex t u b i n g (1.2 m m o.d.) was p u l l e d in two steps o n a p i p e t t e p u l l e r (PP-83; N a r i s h i g e Scientific I n s t r u m e n t s , Tokyo, J a p a n ) . T o m i n i m i z e stray c a p a c i t a n c e , t h e e x t e r n a l wall o f t h e p i p e t t e was c o a t e d with a n i n s u l a t i n g resin (Apiezon wax; A p i e z o n Products Ltd., L o n d o n , UK) u p to ~ 1 0 0 I*m away f r o m t h e tip. A residual c a p a c i t a n c e was c o m p e n s a t e d electrically. T h e r e c o r d i n g p i p e t t e was filled with K + s o l u t i o n (in m M ) : 119 KCI, 1 CaC12, 5 EGTA, 10 HEPES ( p H a d j u s t e d to 7.4 with K O H ) o r Cs + solution: 119 CsCI, 1 CaC12, 5 EGTA, 10 H E P E S ( p H a d j u s t e d to 7.4 with C s O H ) . Resistance o f t h e p i p e t t e was a b o u t 20 M ~ . For r e c o r d i n g , t h e c u l t u r e dish was m o u n t e d o n t h e stage o f a n i n v e r t e d m i c r o s c o p e with p h a s e c o n t r a s t optics ( D i a p h o t TMD-2; Nikon, T o k y o J a p a n ) . A stainless-steel r i n g was p u t into t h e d i s h to r e d u c e t h e d e a d s p a c e o f t h e r e c o r d i n g c h a m b e r to ~ 0 . 1 5 ml.
Correction of the Resting Potential Olfactory r e c e p t o r cells s h o w a very h i g h i n p u t resistance ( > 2 GI]; see also Trotier, 1986), so a small leakage c u r r e n t c a u s e s a n e r r o r in t h e r e c o r d e d m e m b r a n e potential. In t h e p r e s e n t study, t h e r e f o r e , t h e r e s t i n g p o t e n t i a l was c o r r e c t e d for by u s i n g t h e following e q u a t i o n s : l / R i n p .-=- 1/Rmem+
( v.~- V,oa,)/~,,~
+
1/R~e,I,
Vap/~oa, = 0,
w h e r e Psnp is t h e i n p u t resistance, R;,le,,1 t h e m e m b r a n e resistance, a n d P~,.,i t h e seal resistance. Vap a n d Vreal a r e t h e a p p a r e n t a n d t h e real r e s t i n g potential, respectively. H e r e , we a s s u m e d t h a t t h e
TABLE
I
Superfusates 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15
NaC1
KC1
CaC12
MgC12
CholineC1
control 65 Na + 10 Ca 2+
110 65
0 Na + 10 Ca 2+
--
3.7 3.7 3.7 3.7
3 10 10 --
1 l l 1
. -65 15
--
3.7
--
1
80
--
35
--
3.7
--
1
65
10
35
--
3.7
--
1
65
10
35
65 Na + 0 Ca 2+ 0 Na + 0 Ca2+ 0 Na + 10 Baz+ 0 Na + 10 Ba 2+, 0.1 Ni 2+ 65 Na + 10 Ca 2+, 0.1 Cd 2+ 0 Na + 10 Ca "-'+,0.1 Cd 2+ 65 Na + 10 Ca 2+, 1 Co 2+ 65 Na ÷ 0 Ca 2+, 1 Co ~+ 0 Na + 110 Choline + 0 Na + 110 Choline +, 0.1 Ni 2+ 0.1 Ni ~+ 0 Ca 2+, 0Mg 2+
65
65 --
65 65
.
TEA-C1
.
. 35
--
CdC12
NiCI~
CoClz
HEPES
Glucose
--
--
--
2 2
15 15 15 15 15
.
.
--
35
--
--
--
2
--
35
--
--
--
2
--
--
--
2
--
--
--
2
--
0.1
--
2
3.7
10
1
--
--
35
0.1
--
--
2
3.7
10
1
65
--
35
0.1
--
--
2
3.7 3.7
10 --
1 1
--.
35 35
---
---
--
--
3.7
3
1
-15 110
--
3.7
3
1
110
3.7 3.7
3
1
.
110 110
BaC12
.
.
.
. .
.
. .
. --
.
. --
. .
.
1 1
.
2 2 2
0.1
--
2
0.1
--
2 2
15
15 15 15 15 15 15 15 15 15
pH was adjusted to 7.4 with KOH. All solutions contained phenol red (0.01 mg/ml). Nifedipine, Bay K 8644, c0-conotoxin GVIA, and SITS were tested by adding them to solution 6. 526
T-typeCa2+ Channel in Olfactory Receptor Cells
seal resistance at on-cell mode was equal to the resistance of the seal under whole-cell conditions. Vreaiand l~nem w e r e estimated from /~,lp, R~eal,and VT~precorded in the experiment. For example, when/~np = 2 GgI, Rseal : 10 Gft, and V,p = - 6 0 mV, V~,l and/~ ..... are calculated as:
Control
0 [-
I Na free
-20
Vr,.,i = - 75 mV, R....... = 2.5 G ~ . The resting potentials to be described below are the corrected values. RESULTS
Na free + Ni 0.1raM
> E 4o
-60
Action Potentials Recorded under Various Ionic Conditions T h e i s o l a t e d n e w t o l f a c t o r y r e c e p t o r cells h a d a r e s t i n g m e m b r a n e p o t e n t i a l o f - 7 0 +- 9 m V ( m e a n -+ SD, n = 18). T r a n s i e n t a n d r e g e n e r a t i v e a c t i o n p o t e n t i a l s w e r e i n i t i a t e d w h e n cells w e r e s t i m u l a t e d by a d e p o l a r i z i n g c u r r e n t . Since N a + was b e l i e v e d to b e a d o m i n a n t c h a r g e c a r r i e r f o r s p i k e g e n e r a t i o n in t h e o l f a c t o r y cells (catfish: M i y a m o t o et al., 1992; x e n o p u s : Schild, 1989; t i g e r s a l a m a n d e r : F i r e s t e i n a n d W e r b l i n , 1987; D u b i n a n d D i o n n e , 1994; rat: T r o m b l e y a n d W e s t b r o o k , 1991), we first e x a m i n e d t h e c o n t r i b u t i o n o f e x t e r n a l N a + to spike i n i t i a t i o n . I n t h e e x p e r i m e n t o f Fig. 1, 10-pA current injection induced three repetitive action potentials u n d e r t h e n o r m a l c o n d i t i o n . W h e n c h o l i n e + was s u b s t i t u t e d f o r N a +, t h e r i s i n g p h a s e o f t h e a c t i o n p o t e n t i a l b e c a m e slower a n d t h e a m p l i t u d e o f t h e a c t i o n p o t e n t i a l b e c a m e smaller. However, t h e cell still s h o w e d a t r a n s i e n t a c t i o n p o t e n t i a l t h a t l o o k e d s i m i l a r to t h a t g e n e r a t e d in c o n t r o l s o l u t i o n . S i m i l a r results w e r e obs e r v e d in 11 cells o u t o f 12 e x a m i n e d . F u r t h e r m o r e , as s h o w n with a t h i c k line in Fig. 1, add i t i o n o f 100 ixM Ni 2+ s u p p r e s s e d t h e r e m a i n i n g a c t i o n p o t e n t i a l in Na+-free s o l u t i o n c o m p l e t e l y . Ni 2+ is k n o w n to b e a selective b l o c k e r f o r t h e T-type Ca z+ c h a n n e l s in v a r i o u s e x c i t a b l e m e m b r a n e s (Tsien et al., 1988; also see b e l o w ) , a n d t h e r e f o r e it is h i g h l y likely t h a t T-type C a 2+ c u r r e n t is i n v o l v e d in spike g e n e r a t i o n o f t h e n e w t o l f a c t o r y r e c e p t o r cells. B e c a u s e T-type C a 2+ c u r r e n t is k n o w n to show a t r a n s i e n t t i m e c o u r s e , it is s u i t a b l e f o r g e n e r a t i n g t r a n s i e n t a c t i o n p o t e n t i a l s . T h e r e f o r e , we r e e x a m i n e d v o l t a g e - g a t e d c u r r e n t s o f n e w t o l f a c t o r y rec e p t o r cells in d e t a i l u n d e r t h e v o l t a g e c l a m p . Transient Inward Current and Sustained Outward Current Under the voltage clamp condition, depolarizing step p u l s e s i n d u c e d time- a n d v o l t a g e - d e p e n d e n t c u r r e n t s (Fig. 2). M e m b r a n e d e p o l a r i z a t i o n to voltages b e t w e e n - 9 0 a n d + 2 0 m V f r o m a h o l d i n g v o l t a g e (Vh) o f - - 1 0 0 m V i n d u c e d a r a p i d l y ( < 1 5 ms) d e c a y i n g initial i n w a r d c u r r e n t a n d a late o u t w a r d c u r r e n t . T h e t r a n s i e n t inw a r d c u r r e n t s h o w e d a p e a k value at a b o u t - 2 0 m V (Fig. 2 B). It has b e e n s h o w n t h a t t h e o u t w a r d c u r r e n t consists o f two o r t h r e e types o f K + c u r r e n t s (IK(ca) a n d IK(v), 527
KAWAI ET AL.
-80 I 0
,
I 200
,
I 400
~
I 600
msec
FIGURE 1. Action potentials evoked by injection of 10 pA depolarizing current for 250 ms recorded under current clamp condition. Continuous line: recording in control (Table I, solution 1) solution. Dotted line: recording in Na+-free solution (solution 12). Thick line: recording in Na+-free solution containing 0.1 mM Ni2+ (solution 13). Recording pipette was filled with K+ solution.
a n d possibly IK(A) ) in t h e n e w t ( K u r a h a s h i , 1989a) a n d in o t h e r a n i m a l s p e c i e s (catfish: M i y a m o t o et al., 1992; t i g e r s a l a m a n d e r : F i r e s t e i n a n d W e r b l i n , 1987; m u d p u p p y : D u b i n a n d D i o n n e , 1994; rat: T r o m b l e y a n d W e s t b r o o k , 1991; m o u s e : M a u e a n d D i o n n e , 1987). T h e s e K + c u r r e n t s a r e k n o w n to b e b l o c k e d by a d d i n g T E A to t h e b a t h o r by l o a d i n g cells with Cs + (see below). In t h e f o l l o w i n g e x p e r i m e n t s , K + c u r r e n t s w e r e m i n i m i z e d by t h e s e t r e a t m e n t s . Inward Currents Consist of Three Components Fig. 3 A shows a m i x t u r e o f i n w a r d c u r r e n t s o b s e r v e d in a cell l o a d e d with Cs + a n d e x p o s e d to 35 m M TEA. Dep o l a r i z i n g steps i n d u c e d a fast t r a n s i e n t i n w a r d c u r r e n t f o l l o w e d by a small s u s t a i n e d i n w a r d c u r r e n t . T h e p e a k a m p l i t u d e o f t h e t r a n s i e n t c o m p o n e n t was ~ 3 0 0 pA. W h e n N a + was r e p l a c e d by c h o l i n e +, t h e a m p l i t u d e o f t h e i n w a r d c u r r e n t was r e d u c e d to ~ 1 0 0 pA. T h e amp l i t u d e o f this c u r r e n t c o m p o n e n t was also d e p e n d e n t o n C a 2+ c o n c e n t r a t i o n ; in 3 m M Ca 2+ t h e p e a k a m p l i t u d e was ~ 6 0 pA. T h e N a + c o m p o n e n t c o u l d b e isol a t e d by s u b t r a c t i n g t h e c u r r e n t u n d e r t h e Na+-free condition from that under the control condition, and was p l o t t e d by a d o t t e d line. T h e N a + c u r r e n t (INa) reached a peak ~3 ms after the onset of the stimulus a n d i n a c t i v a t e d in ~ 4 ms. T h e i n w a r d c u r r e n t r e c o r d e d in Na+-free c o n d i t i o n was a l m o s t c o m p l e t e l y e l i m i n a t e d by f u r t h e r r e m o v i n g C a 2+ f r o m t h e b a t h i n g s o l u t i o n . This r e s u l t suggests t h a t t h e c u r r e n t e v o k e d in Na+-free s o l u t i o n was carr i e d solely by Ca 2+. T h e Ca 2+ c u r r e n t s (Ica) c o n s i s t e d o f
A
600/f
800
B
600
~
400
20mV
~ 200
//
10m V 1f1--
~
0
m
400
V i0m
KURAHASHI,
and
AKIMICHI
KANEKO
From the D e p a r t m e n t o f Information Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444,Japan
ABSTRA CT Mechanisms underlying action potential generation in the newt olfactory receptor cell were investigated by using the whole-cell version of the patch-clamp technique. Isolated olfactory cells had a resting membrane potential of - 7 0 +- 9 mV. Injection of a depolarizing current step triggered action potentials under current clamp condition. The amplitude of the action potential was reduced by lowering external Na + concentration. After a complete removal of Na +, however, cells still showed action potentials which was abolished either by Ca 2+ removal or by an application of Ca z+ channel blocker (Co 2+ o r Ni2+), indicating an involvement of Ca 2+ current in spike generation of newt olfactory receptor cells. Under the voltage clamp condition, depolarization of the cell to - 4 0 mV from the holding voltage of - 1 0 0 mV induced a fast transient inward current, which consisted of Na + (INa) and T-type C a 2+ (ICa.T) currents. The amplitude of I(~.Twas about one fourth of that of IN~. Depolarization to more positive voltages also induced L-type Ca 2+ current (Ica.L)- ICa,L Was as small as a few pA in normal Ringer solution. The activating voltage of Ic~.xwas approximately 10 mV more negative than that of INa. Under current clamp, action potentials generated by a least effective depolarization was almost completely blocked by 0.1 mM Nf2+ (a specific T-type C a z+ channel blocker) even in the presence of Na +. These results suggest that Ic~,r contributes to action potential in the newt olfactory receptor cell and lowers the threshold of spike generation. KEY WORDS:
olfactory receptor cell • calcium channel • action potential • patch clamp • newt
INTRODUCTION
O d o r a n t binding to the receptor protein at the ciliary surface of the olfactory r e c e p t o r cell leads to the subseq u e n t enzymatic cascades (for review, see Bakalyar and Read, 1991; Breer a n d Boekhoff, 1992; Reed, 1992; R o n n e t t and Snyder, 1992) that finally leads to the o p e n i n g of three types of ionic channels; cAMP-gated cationic channels, CaZ+-gated C1 channels (for review, see Gold a n d Nakamura, 1987; Firestein, 1992; Kurahashi a n d Yau, 1994), and IP3-gated Ca 2+ channels (for review, see Reed, 1992; Restrepo et al., 1996; Ronnett and Snyder, 1992). This initial excitation causes a slow and graded voltage change; its amplitude is d e p e n d e n t on stimulus intensities (Trotier and MacLeod, 1983; Kurahashi, 1989a; Firestein et al., 1993). Graded receptor potential is then e n c o d e d into spike trains that transmit the olfactory information to the brain. At this step, the information relating to the stimulus intensity is e n c o d e d into the spike density. It has long b e e n believed that the action potential of olfactory cells is generated by the influx of Na+; u n d e r voltage clamp, the transient inward current has b e e n shown to be mediated by Na + channel (catfish: Miyamoto et al., 1992; coho salmon: Nevitt and Moody, 1992; xenopus: Schild, 1989; tiger salamander: Firestein and Werblin, 1987; Dr. Kurahashi's p r e s e n t address is School of Science, Osaka University, Toyonaka, Osaka 560, Japan. Address c o r r e s p o n d e n c e to Dr. Fusao Kawai, National Institute for Physiological Sciences, Myodaiji, Okazaki 444,Japan. Fax: 81-564-527913; E-mail: [email protected] 525
Dubin and Dionne, 1994; rat: T r o m b l e y and Westbrook, 1991). However, an earlier study by Trotier (1986) has pointed out that the salamander olfactory cell expresses a transient inward current in a Na+-free, tetrodotoxin-containing solution. F u r t h e r m o r e , in preliminary experiments by one of the present authors, Kurahashi (1989a) recorded action potentials using newt olfactory cells bathed in Na+-free m e d i u m . These observations raised a possibility that action potential is generated not only by Na + current, but also by o t h e r transient inward current in the a m p h i b i a n olfactory receptor cells. In the present study, we reexamined the mechanism of spike generation in newt olfactory receptor cells by analyzing m e m b r a n e currents under the whole-cell clamp condition in further detail. We f o u n d that the transient inward current activated by m e m b r a n e depolarization is a mixture of Na + a n d C a 2+ currents through two i n d e p e n d e n t ionic channels. The transient Ca 2+ c o m p o n e n t was shown to be carried through a T-type Ca 2+ channel. Activation range for the T-type C a 2+ current was approximately 10 mV m o r e negative than that of Na + current. This observation indicates that T-type Ca 2+ current determines the threshold of spike generation in the newt olfactory receptor cells. 1 1After the submission of o u r original manuscript, L i m a n a n d Corey (1996) reported that a T-type Ca 2+ c h a n n e l is expressed in the chemosenso~y n e u r o n s from the m o u s e vomeronasal organ. This observation raises a possibility that T-type Ca 2+ channels m i g h t be expressed n o t only in newt olfactory receptor cells b u t also in cells of the o t h e r species.
J. GEN. PHYSIOL. © The Rockefeller University Press • 0 0 2 2 - 1 2 9 5 / 9 6 / 1 2 / 5 2 5 / 1 1 $2.00 Volume 108 D e c e m b e r 1996 525-535
Indeed,
at threshold,
action potentials
N i 2+, a s e l e c t i v e b l o c k e r in the presence
o f N a +.
MATERIALS
AND
were blocked
f o r T - t y p e C a 2+ c h a n n e l ,
Cells were c o n t i n u o u s l y s u p e r f u s e d (at a rate o f 0.9 m l / m i n ) with o n e o f t h e s o l u t i o n s listed in T a b l e I. T h e i n d i f f e r e n t e l e c t r o d e was a n Ag-AgC1 wire c o n n e c t e d to t h e c u l t u r e dish via a n a g a r o s e bridge. C u r r e n t a n d voltage signals f r o m t h e a m p l i f i e r were m o n itored o n a n oscilloscope ( T e k t r o n i x , Beaverton, OR) a n d a therm a l array r e c o r d e r (WR7900; G r a p h t e c , Fujisawa, J a p a n ) . Data were low-pass filtered (4-pole Bessel type) with a cut-off freq u e n c y o f 5 k H z a n d t h e n digitized at 10 kHz. All e x p e r i m e n t s were p e i - f o r m e d at r o o m t e m p e r a t u r e (23-25°C).
by
even
METHODS
Preparation Solitary r e c e p t o r cells were enzymatically dissociated f r o m t h e olfactory e p i t h e l i u m o f t h e newt, Cynopspyrrhogaster. Dissociation p r o t o c o l s were similar to t h o s e r e p o r t e d previously ( K u r a h a s h i , 1989b). In short, t h e a n i m a l was a n e s t h e t i z e d by c o o l i n g o n ice, d e c a p i t a t e d , a n d p i t h e d . T h e m u c o s a e e x c i s e d f r o m t h e olfactory cavity were i n c u b a t e d for 5 rain at 30°C in a s o l u t i o n (Table I solution 15) c o n t a i n i n g 0.1% c o l l a g e n a s e (Sigma C h e m i c a l Co., St. Louis, M O ) with n o a d d e d Ca z+ a n d M g ')+. T h e tissue was t h e n r i n s e d twice with a c o n t r o l s o l u t i o n (solution 1) a n d triturated. Isolated cells were p l a t e d o n t h e c o n c a n a v a l i n A - c o a t e d glass cover-slip. Cells were m a i n t a i n e d at 4°C ( u p to 10 h) b e f o r e use. In t h e p r e s e n t e x p e r i m e n t , we s e l e c t e d r e c e p t o r cells w h i c h lost t h e i r cilia to s t u d y t h e excitability o f t h e s o m a t i c m e m b r a n e .
Application of Drugs Several p h a r m a c o l o g i c a l a g e n t s were u s e d for m e m b r a n e c u r r e n t analyses: t e t r a e t h y l a m m o n i u m c h l o r i d e (TEA) a n d CsC1 for blocking K+ current, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid (SITS) for b l o c k i n g CI- c u r r e n t , COC12, NiCI~, a n d CdC12 for b l o c k i n g Ca 2+ c u r r e n t . D i h y d r o p y r i d i n e s (nifed i p i n e a n d Bay K 8644) a n d t0-conotoxin GVIA were also u s e d to identify t h e type o f t h e Ca 2+ c u r r e n t . Divalent cations a n d o t h e r p h a r m a c o l o g i c a l a g e n t s were dissolved in t h e s u p e r f u s a t e . T h e s e a g e n t s were b a t h a p p l i e d to t h e cell f r o m t h e p e f f u s i o n system. CoCI~, NiC12, a n d CdC12 were p u r c h a s e d f r o m Nacalai T e s q u e Inc. (Kyoto, J a p a n ) , T E A f r o m W a k o P u r e C h e m i c a l I n d u s t r i e s Ltd. (Osaka, J a p a n ) , a n d SITS, n i f e d i p i n e , Bay K 8644, a n d to-conotoxin GVIA f r o m S i g m a C h e m i c a l Co.
Recording Procedures M e m b r a n e c u r r e n t s were r e c o r d e d by a p a t c h c l a m p t e c h n i q u e in t h e w h o l e - c e l l - c l a m p c o n f i g u r a t i o n (Hamill et al., 1981). Pyrex t u b i n g (1.2 m m o.d.) was p u l l e d in two steps o n a p i p e t t e p u l l e r (PP-83; N a r i s h i g e Scientific I n s t r u m e n t s , Tokyo, J a p a n ) . T o m i n i m i z e stray c a p a c i t a n c e , t h e e x t e r n a l wall o f t h e p i p e t t e was c o a t e d with a n i n s u l a t i n g resin (Apiezon wax; A p i e z o n Products Ltd., L o n d o n , UK) u p to ~ 1 0 0 I*m away f r o m t h e tip. A residual c a p a c i t a n c e was c o m p e n s a t e d electrically. T h e r e c o r d i n g p i p e t t e was filled with K + s o l u t i o n (in m M ) : 119 KCI, 1 CaC12, 5 EGTA, 10 HEPES ( p H a d j u s t e d to 7.4 with K O H ) o r Cs + solution: 119 CsCI, 1 CaC12, 5 EGTA, 10 H E P E S ( p H a d j u s t e d to 7.4 with C s O H ) . Resistance o f t h e p i p e t t e was a b o u t 20 M ~ . For r e c o r d i n g , t h e c u l t u r e dish was m o u n t e d o n t h e stage o f a n i n v e r t e d m i c r o s c o p e with p h a s e c o n t r a s t optics ( D i a p h o t TMD-2; Nikon, T o k y o J a p a n ) . A stainless-steel r i n g was p u t into t h e d i s h to r e d u c e t h e d e a d s p a c e o f t h e r e c o r d i n g c h a m b e r to ~ 0 . 1 5 ml.
Correction of the Resting Potential Olfactory r e c e p t o r cells s h o w a very h i g h i n p u t resistance ( > 2 GI]; see also Trotier, 1986), so a small leakage c u r r e n t c a u s e s a n e r r o r in t h e r e c o r d e d m e m b r a n e potential. In t h e p r e s e n t study, t h e r e f o r e , t h e r e s t i n g p o t e n t i a l was c o r r e c t e d for by u s i n g t h e following e q u a t i o n s : l / R i n p .-=- 1/Rmem+
( v.~- V,oa,)/~,,~
+
1/R~e,I,
Vap/~oa, = 0,
w h e r e Psnp is t h e i n p u t resistance, R;,le,,1 t h e m e m b r a n e resistance, a n d P~,.,i t h e seal resistance. Vap a n d Vreal a r e t h e a p p a r e n t a n d t h e real r e s t i n g potential, respectively. H e r e , we a s s u m e d t h a t t h e
TABLE
I
Superfusates 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15
NaC1
KC1
CaC12
MgC12
CholineC1
control 65 Na + 10 Ca 2+
110 65
0 Na + 10 Ca 2+
--
3.7 3.7 3.7 3.7
3 10 10 --
1 l l 1
. -65 15
--
3.7
--
1
80
--
35
--
3.7
--
1
65
10
35
--
3.7
--
1
65
10
35
65 Na + 0 Ca 2+ 0 Na + 0 Ca2+ 0 Na + 10 Baz+ 0 Na + 10 Ba 2+, 0.1 Ni 2+ 65 Na + 10 Ca 2+, 0.1 Cd 2+ 0 Na + 10 Ca "-'+,0.1 Cd 2+ 65 Na + 10 Ca 2+, 1 Co 2+ 65 Na ÷ 0 Ca 2+, 1 Co ~+ 0 Na + 110 Choline + 0 Na + 110 Choline +, 0.1 Ni 2+ 0.1 Ni ~+ 0 Ca 2+, 0Mg 2+
65
65 --
65 65
.
TEA-C1
.
. 35
--
CdC12
NiCI~
CoClz
HEPES
Glucose
--
--
--
2 2
15 15 15 15 15
.
.
--
35
--
--
--
2
--
35
--
--
--
2
--
--
--
2
--
--
--
2
--
0.1
--
2
3.7
10
1
--
--
35
0.1
--
--
2
3.7
10
1
65
--
35
0.1
--
--
2
3.7 3.7
10 --
1 1
--.
35 35
---
---
--
--
3.7
3
1
-15 110
--
3.7
3
1
110
3.7 3.7
3
1
.
110 110
BaC12
.
.
.
. .
.
. .
. --
.
. --
. .
.
1 1
.
2 2 2
0.1
--
2
0.1
--
2 2
15
15 15 15 15 15 15 15 15 15
pH was adjusted to 7.4 with KOH. All solutions contained phenol red (0.01 mg/ml). Nifedipine, Bay K 8644, c0-conotoxin GVIA, and SITS were tested by adding them to solution 6. 526
T-typeCa2+ Channel in Olfactory Receptor Cells
seal resistance at on-cell mode was equal to the resistance of the seal under whole-cell conditions. Vreaiand l~nem w e r e estimated from /~,lp, R~eal,and VT~precorded in the experiment. For example, when/~np = 2 GgI, Rseal : 10 Gft, and V,p = - 6 0 mV, V~,l and/~ ..... are calculated as:
Control
0 [-
I Na free
-20
Vr,.,i = - 75 mV, R....... = 2.5 G ~ . The resting potentials to be described below are the corrected values. RESULTS
Na free + Ni 0.1raM
> E 4o
-60
Action Potentials Recorded under Various Ionic Conditions T h e i s o l a t e d n e w t o l f a c t o r y r e c e p t o r cells h a d a r e s t i n g m e m b r a n e p o t e n t i a l o f - 7 0 +- 9 m V ( m e a n -+ SD, n = 18). T r a n s i e n t a n d r e g e n e r a t i v e a c t i o n p o t e n t i a l s w e r e i n i t i a t e d w h e n cells w e r e s t i m u l a t e d by a d e p o l a r i z i n g c u r r e n t . Since N a + was b e l i e v e d to b e a d o m i n a n t c h a r g e c a r r i e r f o r s p i k e g e n e r a t i o n in t h e o l f a c t o r y cells (catfish: M i y a m o t o et al., 1992; x e n o p u s : Schild, 1989; t i g e r s a l a m a n d e r : F i r e s t e i n a n d W e r b l i n , 1987; D u b i n a n d D i o n n e , 1994; rat: T r o m b l e y a n d W e s t b r o o k , 1991), we first e x a m i n e d t h e c o n t r i b u t i o n o f e x t e r n a l N a + to spike i n i t i a t i o n . I n t h e e x p e r i m e n t o f Fig. 1, 10-pA current injection induced three repetitive action potentials u n d e r t h e n o r m a l c o n d i t i o n . W h e n c h o l i n e + was s u b s t i t u t e d f o r N a +, t h e r i s i n g p h a s e o f t h e a c t i o n p o t e n t i a l b e c a m e slower a n d t h e a m p l i t u d e o f t h e a c t i o n p o t e n t i a l b e c a m e smaller. However, t h e cell still s h o w e d a t r a n s i e n t a c t i o n p o t e n t i a l t h a t l o o k e d s i m i l a r to t h a t g e n e r a t e d in c o n t r o l s o l u t i o n . S i m i l a r results w e r e obs e r v e d in 11 cells o u t o f 12 e x a m i n e d . F u r t h e r m o r e , as s h o w n with a t h i c k line in Fig. 1, add i t i o n o f 100 ixM Ni 2+ s u p p r e s s e d t h e r e m a i n i n g a c t i o n p o t e n t i a l in Na+-free s o l u t i o n c o m p l e t e l y . Ni 2+ is k n o w n to b e a selective b l o c k e r f o r t h e T-type Ca z+ c h a n n e l s in v a r i o u s e x c i t a b l e m e m b r a n e s (Tsien et al., 1988; also see b e l o w ) , a n d t h e r e f o r e it is h i g h l y likely t h a t T-type C a 2+ c u r r e n t is i n v o l v e d in spike g e n e r a t i o n o f t h e n e w t o l f a c t o r y r e c e p t o r cells. B e c a u s e T-type C a 2+ c u r r e n t is k n o w n to show a t r a n s i e n t t i m e c o u r s e , it is s u i t a b l e f o r g e n e r a t i n g t r a n s i e n t a c t i o n p o t e n t i a l s . T h e r e f o r e , we r e e x a m i n e d v o l t a g e - g a t e d c u r r e n t s o f n e w t o l f a c t o r y rec e p t o r cells in d e t a i l u n d e r t h e v o l t a g e c l a m p . Transient Inward Current and Sustained Outward Current Under the voltage clamp condition, depolarizing step p u l s e s i n d u c e d time- a n d v o l t a g e - d e p e n d e n t c u r r e n t s (Fig. 2). M e m b r a n e d e p o l a r i z a t i o n to voltages b e t w e e n - 9 0 a n d + 2 0 m V f r o m a h o l d i n g v o l t a g e (Vh) o f - - 1 0 0 m V i n d u c e d a r a p i d l y ( < 1 5 ms) d e c a y i n g initial i n w a r d c u r r e n t a n d a late o u t w a r d c u r r e n t . T h e t r a n s i e n t inw a r d c u r r e n t s h o w e d a p e a k value at a b o u t - 2 0 m V (Fig. 2 B). It has b e e n s h o w n t h a t t h e o u t w a r d c u r r e n t consists o f two o r t h r e e types o f K + c u r r e n t s (IK(ca) a n d IK(v), 527
KAWAI ET AL.
-80 I 0
,
I 200
,
I 400
~
I 600
msec
FIGURE 1. Action potentials evoked by injection of 10 pA depolarizing current for 250 ms recorded under current clamp condition. Continuous line: recording in control (Table I, solution 1) solution. Dotted line: recording in Na+-free solution (solution 12). Thick line: recording in Na+-free solution containing 0.1 mM Ni2+ (solution 13). Recording pipette was filled with K+ solution.
a n d possibly IK(A) ) in t h e n e w t ( K u r a h a s h i , 1989a) a n d in o t h e r a n i m a l s p e c i e s (catfish: M i y a m o t o et al., 1992; t i g e r s a l a m a n d e r : F i r e s t e i n a n d W e r b l i n , 1987; m u d p u p p y : D u b i n a n d D i o n n e , 1994; rat: T r o m b l e y a n d W e s t b r o o k , 1991; m o u s e : M a u e a n d D i o n n e , 1987). T h e s e K + c u r r e n t s a r e k n o w n to b e b l o c k e d by a d d i n g T E A to t h e b a t h o r by l o a d i n g cells with Cs + (see below). In t h e f o l l o w i n g e x p e r i m e n t s , K + c u r r e n t s w e r e m i n i m i z e d by t h e s e t r e a t m e n t s . Inward Currents Consist of Three Components Fig. 3 A shows a m i x t u r e o f i n w a r d c u r r e n t s o b s e r v e d in a cell l o a d e d with Cs + a n d e x p o s e d to 35 m M TEA. Dep o l a r i z i n g steps i n d u c e d a fast t r a n s i e n t i n w a r d c u r r e n t f o l l o w e d by a small s u s t a i n e d i n w a r d c u r r e n t . T h e p e a k a m p l i t u d e o f t h e t r a n s i e n t c o m p o n e n t was ~ 3 0 0 pA. W h e n N a + was r e p l a c e d by c h o l i n e +, t h e a m p l i t u d e o f t h e i n w a r d c u r r e n t was r e d u c e d to ~ 1 0 0 pA. T h e amp l i t u d e o f this c u r r e n t c o m p o n e n t was also d e p e n d e n t o n C a 2+ c o n c e n t r a t i o n ; in 3 m M Ca 2+ t h e p e a k a m p l i t u d e was ~ 6 0 pA. T h e N a + c o m p o n e n t c o u l d b e isol a t e d by s u b t r a c t i n g t h e c u r r e n t u n d e r t h e Na+-free condition from that under the control condition, and was p l o t t e d by a d o t t e d line. T h e N a + c u r r e n t (INa) reached a peak ~3 ms after the onset of the stimulus a n d i n a c t i v a t e d in ~ 4 ms. T h e i n w a r d c u r r e n t r e c o r d e d in Na+-free c o n d i t i o n was a l m o s t c o m p l e t e l y e l i m i n a t e d by f u r t h e r r e m o v i n g C a 2+ f r o m t h e b a t h i n g s o l u t i o n . This r e s u l t suggests t h a t t h e c u r r e n t e v o k e d in Na+-free s o l u t i o n was carr i e d solely by Ca 2+. T h e Ca 2+ c u r r e n t s (Ica) c o n s i s t e d o f
A
600/f
800
B
600
~
400
20mV
~ 200
//
10m V 1f1--
~
0
m
400
V i0m
