Methylation of critical carboxyl groups in the vicinity of the sodium channel of guinea-pig atrium

J Mol Cell Cardiol 18, 99-108 (1986)

Methylation of Critical Carboxyl Groups in the Vicinity of the Sodium Channel of Guinea-pig Atrium S. E. Freeman, R. M. D a w s o n , M. P. Bladen and P. J. Gray Materials Research Laboratories, P.O. Box 50, Ascot Vale Vic. 3032, Australia (Received 9 October 1984, accepted in revisedform 14 March 1985) S. E. FREEMAN,R. M. DAWSON,M. P. BLADENANDP.J. GRAY.Methylation of Critical Carboxyl Groups in the Vicinity of the Sodium Channel of Guinea-pig Atrium. Journal of Molecularand CellularCardiology(1986) 18, 99-108. Methylation of a critical carboxyl group in guinea-pig left atrium with trimetboxonium ion leads to loss of excitability. The critical group(s) could be protected with a number of cationic drugs, so that on washout of the protecting drug and reaction products full excitability returned. Tetrodotoxin, edrophonium, cholinergic agonists and amantadine protected the preparation. During the recovery period readmissionof these drugs led to the same pharmacologicalresponse as during the control period, suggestingprotection of specific site(s). This thesis was confirmedin crossover experiments,in which the atrium was exposed to the methylating agent in the presence of one protecting drug, then exposed to another during recovery. The expected pharmacological responses were obtained. Assay of muscarinic receptors with [3H]-qninuclidinyl benzilate after methylation without protection or in the presence of tetrodotoxin or acetylcholine suggested that these receptors are not involved in the maintenance of excitability. It is postulated that the Na + channel and the K + channel are located in the same macromolecular membrane complex, and that the K + channel has a cbolinoreceptive sub-site. KEYWORDS: Guinea-pig atrium ; Na channel ; Tetrodotoxin ; Carboxyl group methylation ; Electrophysiology.

Introduction T r i m e t h o x o n i u m ion has been shown to react with carboxyl groups in the m e m b r a n e s of skeletal muscle leading to a loss of sensitivity to tetrodotoxin ( T T X ) a n d saxitoxin ( S T X ; [14]). C u r r e n t flow was reduced by this treatmeat, b u t the ionic specificity of the Na + c h a n n e l was not altered. Baker a n d R u b i n s o n [1] carried out similar experiments using trie t h o x o n i u m ion a n d crab nerve. Nerves treated in this way were also insensitive to T T X a n d STX. T r i m e t h o x o n i u m ion ( T M O ) has also been shown to inactivate acetylcholinesterase [13] a n d to modify a b i n d i n g subsite in the nicotinic acetylcholine r e c e p t o r [3]. These results are consistent with the ability of tri-alkyloxonium salts to alkylate weakly nucleophilic groups u n d e r mild aqueous conditions. W h e n used to modify proteins the reagent was found to react with a limited n u m b e r of carboxyl groups [12] suggesting some degree of specificity for the reaction. 0022-2828/86/010099+ 10 $03.00/0

P r e l i m i n a r y work in this laboratory [7, 8] showed that m e t h y l a t i o n of carboxyl groups in the guinea-pig left a t r i u m abolished excitability and contractility, Occasional small, n o n - p r o p a g a t i n g action potentials could be recorded from strands of pectinate muscle; they had no plateau. T h e loss of excitability was not due to the experimental conditions per se since sham experiments with the hydrolysis products of T M O did not affect excitability. T h e critical carboxyl group(s) can be protected with T T X , e d r o p h o n i u m , cholinergic agonists or a m a n t a d i n e . N o r m a l action potentials (AP) can be recorded after washout of the protecting drug a n d the reaction products of methylation. T h e ability of the protecting drug to modify the A P after m e t h y l a t i o n has been determined, T h e experimental protocol included experiments in which the preparation was protected with one drug, then exposed to a n o t h e r after methylation. These experiments are now reported in full. I n addition we have studied the b i n d i n g of the 9 1986 Academic Press Inc. (London) Limited

100

S.E. Freeman

muscarinic receptor ligand 1-quinuclidinyl [phenyl-4-3H] benzilate ([3H]QNB) to homogenates of the atrium after methylation in the presence or absence of protecting drugs. The results obtained have confirmed the previous suggestion [8] that the Na § channel in guinea-pig atrium must be located in close proximity to a cholinoceptive site. The binding studies with [-aH]QNB suggest that this cholinoceptive site may be distinct from the muscarinic receptor.

Methods

Left atrial preparations were dissected from guinea-pigs weighing 200 to 350 g as described previously [10]. Transmembrane potentials were recorded with microelectrodes filled with 3 M KC1 and with a d.c. resistance of 10 to 15 MfL The AP was recorded as a function of time, and the first derivative, dV/dt, was recorded as a function of membrane voltage. The phaseplane trajectory which resulted was analyzed on the assumption that the atrial cell penetrated behaves as part of a fnnctional bundle which exhibits cable behaviour [10]. A procedure has been developed [9] whereby the inward ionic current (/i) can be derived by subtraction of the first and second derivatives of the AP: 1i :

(ldEV) C m k dt 2

dV ~-

where C m is the specific membrane capacitance and k is the rate constant of the capacitative current which flows at the 'foot' of the AP. The method enables us to display simultaneously the V,t signal of the AP, the phase plane trajectory and the inward ionic current as a function of membrane voltage. The oscilloscope trace was used to monitor each cell penetration. Data were then collected by an SBC 80/30 Single Board Computer (Intel Corporation, CA, USA) and an R T I - 1 2 0 0 Real Time Interface (Analog Devices, Inc., Norwood, MA, USA). The R T I 1200 provides a 12 bit analog to digital converter triggered by a crystal clock operating at 20 kHz. Data were stored on floppy discs using Remex disc drives (Ex-Cell-O Corporation, lrvine, CA, USA) and a Matrox FFD-1 floppy disc controller (Matrox Electronic Systems Ltd,

et aL

Quebec, Canada). The operating system used was CP/M (Digital Research, CA, USA). Following the closure of a trigger approximately 1600 data points were collected after the end of the next pulse from the tissue stimulator. At the completion of each experiment data were transmitted to a V A X 11/780 computer for analysis. This involves a least squares polynomial fitting procedure, and is described elsewhere [9]. Each potential recorded represents penetration of a different cell. It was usual to track across each atrial appendage to obtain a representative population. Preparations were stimulated at 2 Hz. Methylation of the atrium was carried out with trimethoxonium tetrafluoroborate (25 raM) made up immediately prior to use. It was dispensed into weighed ampoules in a dry box under an atmosphere of nitrogen. Each batch was checked for purity by nuclear magnetic resonance spectrometry. Methylation was carried out in a nutrient solution [5"] buffered with 10 m~a HEPES instead of bicarbonate and chilled in an ice bath. The reacting solution was maintained at p H 7'4 by the addition of 150 m~ N a O H from a Radiometer pH stat apparatus. Acid production was complete in 5 rain; the preparation was then returned to the organ bath at 37~ and washed with a bicarbonate buffered nutrient solution [5] to remove methanol and dimethyl ether produced during methylation. When a protecting drug was present excitability returned in a period varying from 10 to 60 min, and was maintained for approximately 3 h thereafter. This permitted collection of a second set of control data and a second exposure to a drug. [ 3 H ] Q N B binding assays were carried out as described previously [6]. Left and right atria (24 to 55 rag/atrium) were dissected from hearts bathed in nutrient solution at 37~ and were then homogenized in 10 ml of ice-cold Ringer's solution, using an ultra Turrax homogenizer on setting 6.5 with three homogenizations of 15 s each and 30 s pauses between bursts. The homogenates were filtered through nylon mesh to remove connective tissue and the filtrates were used for binding assays. [3H]QNB binding was performed as described previously ['4] except that (i) the buffer used was Ringer's solution (above; [5]); (ii) the concentration of

Carboxyl Group Methylation of Na Channel in Atrium [ 3 H ] Q N B varied from 0.03 to 0.8 riM, a n d (iii) the filters were not dried before a d d i t i o n ofscintillant. A m a n t a d i n e h y d r o c h l o r i d e was a gift o f C i b a - G e i g y Australia. E d r o p h o n i u m a n d unlabelled Q NB were gifts of R o c h e Products (Australia). 1-Quinuclidinyl [ p h e n y l - 4 - 3 H ] benzilate was o b t a i n e d from A m e r s h a m Australia at a specific activity of 31 Ci/mmol. T e t r o d o t o x i n was o b t a i n e d from F l u k a and t r i m e t h o x o n i u m tetrafluoroborate from Calbiochem-Behring. All other materials are freely available commercially.

Results M e t h y l a f i o n of surface carboxyl groups b y T M O c o m p l e t e l y abolished excitability. T h e e n d o c a r d i a l m e m b r a n e a p p e a r e d to be toughened by the process, as was evidenced by an increased rate of b r e a k a g e of rnicroelectrode tips. I t was possible to record 'resting' potentials of a b o u t - - 6 0 m V a m p l i t u d e . However, as r e p o r t e d previously [8] A P were rare, nonp r o p a g a t i n g a n d lacked a p l a t e a u phase. Prolonged washing did not restore excitability, so spontaneous d e m e t h y l a t i o n a p p e a r s not to occur.

Protection studies T e t r o d o t o x i n (5 /2M) was able to protect the critical carboxyl group(s) d u r i n g methylation, so that on washout of reaction products a n d T T X n o r m a l A P were recorded. T h e experi-

101

m e n t a l protocol was designed to o b t a i n control a n d T T X - t r e a t e d potentials prior to a n d after methylation. I t m a y be seen from T a b l e 1 that control potentials were similar before a n d after methylation, a n d also t h a t T T X (5 #M) p r o d u c e d a similar reduction i n i n w a r d c u r r e n t before a n d after m e t h y l a t i o n in the presence of T T X (see also ref. [9]). T h e p r i n c i p a l effects of this dose of T T X were to reduce the m a x i m u m rate of rise Of the spike, the m a x i m u m ionic current, a n d the rate constant k, which represents the discharge o f c a p a c i t a t i v e current. T h e r e was no effect on the threshold voltage of the p r o p a g a t e d AP. These d a t a indicate that T T X at 5/~M must bind to a sufficient n u m b e r o f c a r b o x y l groups at or n e a r the N a + channel to m a i n t a i n the integrity of the A P after washout of d r u g plus reaction products. T h e close similarity in A P configuration before a n d after rnethylation suggests that the architecture of the N a + channel has been m a i n t a i n e d unchanged. W e noted previously [8] t h a t the anticholinesterase d r u g e d r o p h o n i u m was also able to protect the excitability of the a t r i u m d u r i n g methylation. These d a t a are shown in full in T a b l e 2. A comparison of control I d a t a with control I I d a t a o b t a i n e d after methylat• in the presence of 50 /tM e d r o p h o n i u m indicates t h a t there was some reduction in most p a r a m e t e r s of the AP. This was not however a constant finding; in other experiments ( d a t a not shown) e d r o p h o n i u m was found to protect A P p a r a m e t e r s completely.

TABLE i. The effect oftetrodotoxin on atrial potentials before and after methylation of the atrium with T M O in the presence of T T X

Control I T T X 5/~ra Control II (after T M O ) T T X 5 ,UM (after T M O ) Probability lv. 3 2v. 4

Spike height

Maximum diastolic potential

Overshoot

(mY)

(mY)

(mY)

89.7 _ 0.7 (109) 84.94- 1.1 (57) 92.0• 1.2 (8t) 82.74-2.1 (35)

72.3 + 0.6 (109) 73.4• (57) 73.8+ 1.0 (81) 74.5• (35)

0.07 0.3

0.2 0.5

Maximum r a t e of rise (V/s)

17.4 • 0.5 128 + 3.2 (109) (107) 11.5• 63__+2.2 (57) (46) 18.2_-t-0.6 140• (81) (79) 8.2• 744-3.3 (35) (30) 0.3 0.02

0,06 0,01

T i m e to 50% repolarization (ms)

34 __0.7 (107) 394-0.5 (56) 334-0.9 (80) 36• (35) 0,6 0.01

Rate constant k (m/s)

Voltage of maximum current (mY)

Maximum ionic current (mA/cm 2)

5.4 • 0.2 (107) 2.64-0.1 (53) 5.1 4-0.2 (80) 2.94-0.2 (33)

--20• 0.8 (106) --22_+1.5 (35) --15+0.9 (78) --24• (28)

0.33• 0.01 (106) 0.204-0.01 (35) 0.37• (78) 0.21_+0.01 (28)

0.001 0.4

0.01 0.6

0,3 0,2

Pooled data from four atria. In this and subsequent tables values are shown _ S.E.M. The n u m b e r of cells penetrated is shown in parentheses. P values refer to significance of difference of means using Student's ~-test. Preparations were stimulated at 2 Hz.

102

S . E . F r e e m a n et al.

TABLE 2. The effect ofedrophonium on atrial potentials before and after methylation of the atrium with TMO in the presence ofedrophonium Spike height (mV)

Maximum diastolic potential (mV)

Overshoot (mV)

Maximum rate of rise (V/s)

Time to 50% repolarization (m/s)

Rate constant k (m/s)

Voltage of maximum current (mV)

Maximum loruc current (mA/cm 2)

Control I

88.3_+0.9 (100)

70.5_+0.6 (98)

17.6_+0.6 (98)

155-+4.2 (100)

37.2-+0.8 (100)

4.4-+0.1 (99)

-7.1-+0.9 (98)

0.38_+0.01 (100)

Edrophonium 50 /aM

89.2 -+ 1.2 (63)

68.6 -+ 0.8 (60)

21.2 -+ 0.8 (60)

177 -+ 7.8 (63)

39.3 -+ 0.6 (63)

4.6 -+ 0.1 (63)

0.3 -4-_1.3 (60)

0.43 _+ 0.02 (63)

Control II (after TMO)

84.1 • 0.9 (108)

66.7_+0.6 (100)

17.2_+0.7 (100)

130-+3.6 (I09)

28.8-+0.5 (10g)

3.5-+0.1 (101)

--3,7-+1.0 (99)

Edrophonium (after TMO)

84.4 + 1.5 (52)

67.9 _+ 0.9 (50)

15.6 _+ 0.7 (50)

105 -+ 6.5 (52)

40.7 _+ 1.1 (52)

3.3 • 0.1 (51)

- 13.9 -4-_1.8 (50)

0.24 • 0.02 (52)

0,001 0.02

0.001 0.6

0.7 0.001

0.001 0.001

0,001 0,3

0.001 0.001

0.02 0.001

0,001 0,001

Probability 1 v. 3 2 v. 4

0.31• (109)

Data from fivc atria

E d r o p h o n i u m had but little effect on the AP prior to methylation; however postmethylation it consistently depressed the m a x i m u m rate of rise of the spike, the inward ionic current and k, the rate constant of the discharge of capacitative current. Thus it has not preserved the status quo of the excitatory system as unequivocally as did T T X . T h e possibility of cholinergic involvement in the protection against T M O led us to investigate the effect of acetylcholine (ACh) as a protecting agent. We were obliged to use the rather high concentration of 10 #M, since the animals we used proved to be resistant to the effects of A C h on the AP at lower concentrations. We noted previously [5] that shortening

of the AP required higher concentrations of A C h than did the negative inotropic response. Table 3 shows that 10 ~tM A C h was able to protect the AP parameters almost completely. Further application of A C h after methylation and washout was able to bring about a similar shortening of the AP to that found in the control period. The decrease in the time to 50% repolarization was quantitatively less, both compared to control I or control II, suggesting that the cholinoceptive site had undergone some change during methylation. Because of the structural similarity of T M O and tetramethyl a m m o n i u m ion (TMA) we carried out a similar series of experiments in which T M A was used to protect the tissue

TABLE 3. The effect of ACh on atrial potentials before and after metbylation of the atrium with T M O in the presence of ACh Spike height (mV)

Maximum diastolic potential (mV)

Overshoot (mV)

Maximum rate of rise (V/s)

Time to 50% repolarization (ms)

Rate constant k (m/s)

Voltage of maximum current (mV)

Maximum ionic current

88.3--+0.9

67.6-+0.6

20.7+0.8

156-+5,2

37-+0.4

4.2-+0.1

--4-+1.0

0.38 -+ 0.01

(50)

(50)

(50)

(50)

(50]

(5o)

(49)

(50)

ACh 10 gM

84,9-+1,1 (37)

72.6-+0.8 (37)

12.3-+0.5 (37)

155__.4.9 (37)

13-+0.5 (37)

4.2-+0.1 (37)

- - t 0 - + 1.0 (37)

0,36___0.01 (37)

Control II (after TMO)

89,3 -+ 1.0

69.2 -+ 0.8

20.6 -+ 0.7

140 -4- 4.7

31 -+ 0.4

3.6 _ 0.07

--8 + 1,2

0,34 • 0.01

(85)

(83)

(83)

(85)

(85)

(82)

(83)

ACh 10 ,uM (after T M O )

87.4_+ 1.6

70,7 _+ h0

3.8 -4-0.1

--13 • 1.3

(36)

(35)

(35)

(36)

(36)

(36)

(35)

(36)

0,5 0.2

0.2 0.2

0.9 0.001

0.04 0.04

0,001 0.001

I3.001 0.01

0.03 0.08

0.01 0.03

Control I

Probability Iv. 3 2v. 4 Data from four atria,

17.0•

135 -+8,0

20•

(mA/cmz)

(85) 0,31 •

Carboxyl Group M e t h y l a t i o n o f Na Channel in A t r i u m

I03

TABLE 4. The effect of TMA on atrial potentials before and after methylation with TMO in the presence of T M A diastolic potential

rate of rise

T i m e to 50% repolarization

Rate

constant k

Voltage of

Maximum

Overshoot

(mY)

(mY)

(mY)

(V/s)

(ms)

(m/s)

(mV)

(mA/em 2)

Control I

88.3++_1.2 (42)

72.84-1.0 (42)

15.5_+0.8 (42)

174 • 7.1 (42)

34• (42)

5.0_+03 (42)

-13+_1.8 (42)

0.41_+0.02 (42)

TMA 1 mM

90.0_+1.1 (41 )

76.4• (38)

13.8_+0.9 (38)

189_+6.8 (41)

13• (41)

4.4-t-0.1 (41)

-6• (38)

0.45_+0.02 (41)

Control II (after TMO)

86.6_+1.5 (46)

71.8_+1.2 (46)

14.8_+0.8 (46)

146_+6.9 (46)

284-0.6 (46)

4.04-1.4 (46)

-10• (46)

0.34_+0.02 (46)

TMA 1 mM (after TMO)

93.0_+1.6 (41 )

75.2• (40)

17.9_+0.9 (40)

165_+8.1 (41)

31 • (41)

4.2• (40)

0.5 0.01

0.01 0.03

0.001 0.001

0.5 0.4

Spike height

Maximum

Maximum

maximum current

ionic current

-8_+1.5 (40)

0.39-+0.02 (40)

0.2 0.4

0.01 0.04

Probability

0.4 0.1

lv. 3 2v. 4

0.5 0.3

Data from two atria.

during methylation. Like ACh, T M A proved to vary in its ability to shorten the AP during the control period. This effect was usually evident at 1 mM T M A (see Table 4) ; however we found that some preparations showed but little reduction in the time to 50% repolarization at that concentration, and we used 2 mM for these experiments. As may be seen from Table 4 T M A was able to protect the AP from the effects of methylation almost completely. However it is notable that when T M A was readmitted to the organ bath after methylat• and washout it was quite unable to bring about the usual shortening of the AP. It has been shown elsewhere[6] that aman-

tadine binds weakly to the muscarinic receptor of the guinea-pig atrium, but appears to reduce the outward K + current at approximately 10-fold greater dilution. Table 5 shows that relative to the first set of control data amantadine lengthened the AP, and reduced the maximum rate of rise of the spike and the inward ionic current. After methylat• in the presence of amantadine excitability returned on washing the preparation and the second group of control data showed a good level of recovery. The time to 50% repolarization did not recover totally; this effect was also seen in Tables 2 to 4. Readmission of amantadine produced an increase

TABLE 5. The effect of amantadine on atrial potentials before and after methylation of the atrium with TMO in the presence ofamantadine Voltage of maximum current

Maximum

repolarization

Rate constant k

(V/s)

(ms)

(m/s~

(mV)

(mA/cm 2)

16.1 + 0.5 (80)

123 + 3.4 (79)

42 -+ 0.7 (79)

5.7 + 0.2 (77)

- 2 0 + 0.9 (78)

0.31 • 0.01 (79)

69.2 • 1.4 (27)

19.7 + 0.7 (27)

107 • 4.2 (27)

58 • 1.6 (27)

5.5 + 0.2 (27)

- 2 6 + 1.5 (27)

0.26 • 0.04 (27)

86.8_+0.9 (95)

69.2 • 0.7 (95)

17.620.5 (95)

112_+3.2 (95)

37_.+0.8 (95)

4.8_+0.2 (93)

--17-t-0.8 (90)

0.30_+0.01 (92)

86.4_+2.3 (23)

68.2_+1.9 (23)

18.2_+1.1 (23)

102+6.2 (23)

53_+1.6 (23)

5.1_+0.3 (23)

--164-1.5 (23)

0.26_+0.02 (23)

0.4 0.4

0.02 0.6

0.03 0.2

0.02 0.5

0.001 0.03

0.001 0.2

0.01 0.001

0.5 0.9

Spike height

Maximum diastolic potential

(mV) Control I Amantadine

Maximum Overshoot

rate of rise

(mV)

(mY)

88.2 + 1.0 (80)

71.9 • 0.8 (80)

0.2 mM

88.9 + 1.8 (27)

Control II (after TMO) Amantadine

0.2 mM

Time to 50%

ionic current

(after TMO) Probability

! v. 3 2 v. 4 Data from three atria.

I04

S . E . F r e e m a n et al.

in the time to 50% repolarization almost identical to the effect in the control period, and a similar reduction in rate of rise of the spike and in ionic current. Experiments in which we attempted to protect the excitability of the preparation with atropine (0.2 #M), QNB (0.2 #M) or neostigmine (1 #M) were reported previously [8]. These drugs, which bind specifically to the muscarinic receptor (atropine and QNB) and acetylcholinesterase, (neostigmine) afforded very little protection. After approximately 2 h wash post-TMO treatment, AP could be recorded in scattered areas of preparations, but they were smaller than usual, and totally lacked plateaux. Taking into account the slow wash-out of these drugs from preparations, they must still be considered to offer very little protection to the carboxyl group(s) which are critical for excitability. The drugs studied in Tables 1 to 5 have a wide range of pharmacological actions in the atrium. If their protective action results from binding to the same carboxyl group(s) then such binding must produce totally different configurational effects on the membrane. Thus T T X reduces the Na + current, and ACh increases the outward K + current, presumably via an action on the muscarinic receptor. If in spite of diverse pharmacological effects they bind to the same carboxyl group(s) then the Na + channel and a cholinoceptive site must be part of the same macromolecular complex. In order to test this hypothesis we carried out a series of experiments in which we tested the reactivity of the treated atrium to a drug after methylation in the presence of a different drug.

Crossover experiments The protocol followed in these cross-over experiments was to collect control data, then data in the presence of drug A. This was washed out, drug B was admitted and data collected to ensure it was acting as expected. Methylation and washout were then carried out, and more controls obtained. Drug A was then readmitted to ascertain its action postrnethylation. In the first of these experiments the action of ACh was studied before and after methylation in the presence of TTX. Acetylcholine (ACh) had a qualitatively similar

effect during the control period and after methylation. Thus the time to 50% repolarization was shortened from 36 + 1.1 ms ( n = 4 8 ) to 1 2 _ 0 . 7 ms ( n = 5 0 ) prior to methylation in the presence of T T X . After methylation the time to 50% repolarization was shortened from 28 _ 0.5 ms (n = 62) to 17-t-0.5 ms (n = 59). A similar result was noted in Table 3, when methylation was carried out with ACh as protecting drug. Thus T T X had protected the cholinoceptive site as well as did ACh. Similar experiments were carried out with T T X as the protecting drug in which we compared the action of T M A before and after methylation. It was noted above that when methylation was carried out in the presence of T M A this drug lost its ability to shorten the AP. However when T T X was used as protecting drug T M A partially retained its ability to shorten the AP. Prior to methylation T M A shortened the time to 50% repolarization from 35_+ 0.6 ms ( n = 5 6 ) to 1 9 _ 0 . 6 ms ( n = 4 8 ) , after methylation the times were shortened from 33 + 0.7 ms (n = 55) to 27 _ 0.3 ms (n = 25). The procedure was then reversed, and the effects of T T X were determined before and after methylation in the presence ofACh. Tetrodotoxin produced essentially the same effects after methylation as before. The only significant difference was a reduction in the time to 50% repolarization after methylation. This reflects, however, the shorter control potentials that were recorded after methylation in the presence of ACh, and not a different action of T T X . It was noted above that we frequently, but not invariably, found postmethylation potentials to be of shorter duration than controls prior to methylation. Similarly, we tested the ability of ACh to shorten AP before and after methylation in the presence of TMA. Prior to methylation the mean time to 50% repolarization of 37 • 1.3 ms ( n = 36) was shortened to 21 • 1.3 ms (n = 32) by ACh. Post methylation controls were 37 • 0.9 ms (n = 23), and shortened to 22 -t- 1.5 ms (n = 21). Thus ACh has shortened the time to 50% repolarization by the same amount during the control period and after methylation in the presence of TMA. It will be recalled (Table 4) that T M A was found consistently to be unable to shorten AP after methylation with T M A as protecting

Carboxyl Group Methylation of Na Channel in Atrium drug. It appears however that T M A can protect the A C h receptor. We determined in further experiments that methylation in the presence of T M A does not alter the ability of T T X to reduce the inward ionic current. Tetrodotoxin reduced spike height, overshoot, m a x i m u m rate of rise, k, and inward current to an extent comparable to that shown in Table 1. We have noted elsewhere that amantadine is a weak antagonist of the muscarinic receptor, but appears to be effective in reducing the outward K + current at lower concentrations [6]. It was of interest therefore to determine if methylation in the presence of amantadine protected the site whereby A C h shortens the AP. Amantadine (0.2 mM) afforded a reasonable measure of protection, and the effect of A C h (10 /~M) on the time to 50% repolarization was somewhat reduced. Prior to methylation the time to 50% repolarization was 42 _+ 0.7 ms (n = 77), A C h reduced this to 17 _+ 0.4 ms (n = 51). After methylation this time was 37 + 0.8 ms (n = 93), and this was reduced to 20 + 0.8 (n = 53) by ACh. Relative to controls after methylation A C h reduced spike height as well as time to 50% repolarization but had little effect on other parameters of the AP. Further experiments were carried out in which the effects of T T X were determined prior to and after methylation in the presence of amantadine. Tetrodotoxin had a closely similar action after methylation to that seen beforehand. D a t a collected in conjunction

105

with both exposures to T T X were within the range shown in Table 1. These experiments suggest strongly that there is a cholinoceptive site situated close to the N a + channel, and that T T X and the cholinergic drugs bind to the same carboxyl group(s). This hypothesis suggests that there should be some interaction between T T X and the cholinergic drugs in untreated atria. Experiments were therefore carried out in which atria were exposed to A C h (10 /~M), then T T X (5 /~M), then both drugs together. The order of drug addition was randomized; the results showed that the order of drug addition had no effect. T h e pooled results are shown in Table 6. Acetylcholine and T T X produced their usual effects on the AP. However when present together A C h modified the effects of T T X . T h e rate of rise of the AP and the inward ionic current were greater in the presence of both drugs than when T T X was the sole drug. Spike height and overshoot were however significantly reduced in the presence of both drugs compared to the control data on either drug alone. O n the other hand the shortening in the time to 50% repolarization by A C h was not affected by the presence of T T X . Thus A C h is able to antagonize significantly the reduction in Na + current due to TTX.

[ 3H]-QNB binding studies Binding studies with [ S H ] - Q N B were carried out to determine whether methylation of the

TABLE 6. Interactions between ACh and T T X in untreated atria Spike height (mY)

Maximum diastolic potential (mY)

Overshoot (mV)

Maximum rate of rise (V/s)

Time to 50% repolarization (ms)

Control

96.4_+1.0 (78)

76.2+1.0 (78)

20.2+0.5 (78)

162+5.1 (78)

47+0.8 (76)

ACh 1 0 / 2 M

89.8--+0.9 (41)

74.6-+ 1.0 (4t)

15.1 -+ 1.0 (41)

156-+4.4 (41)

T T X 5 #M

86.0_+1.2 (59)

71.1--+0.9 (59)

14.9-+0.5 (59)

ACh + T T X

79.4 -+ 1.1

75.2-+0.9

(50)

(50)

0.001 0.001 0.001

0.3 0.001 0.01

Probability 1 v. 2 1 v. 3 3 v. 4

Rate constant k

Voltage of maximum current (mV)

Maximum ionic current (mA/cm 2)

4.1+0.1 (78)

--8_+0.9 (78)

0.39+_0.01 (78)

I3 + 0.5 (41)

4.2 + 0. l (41)

--12-+ 1.3 (41)

0.36-+0.01 (41)

70-+3.1 (59)

44-+1.2 (48)

2.0-+0.1 (59)

--16-+1.1 (59)

0.17-+0.01 (59)

4.2-+0.9

97-+4.7

14 -+ 0.6

2.3-+0.1

- 1 4 - + 1.3

0.24-+0.01

(50)

(50)

(50)

(50)

(50)

(50)

0.001 0.001 0.001

0.5 0.001 0.001

0.001 0.04 0.001

0.3 0.001 0.04

0.03 0.001 0.2

0.2 0.001 0.001

(m/s)

106

S . E . F r e e m a n et aL

TABLE 7. Effect of methylation on binding of [3H]-QNB to atrial homogenates Protective Agent None ACh 10 /~M TTX 5 /~M Pooled results

Number of experiments 2 4 3 9

K D (pM)

gmax

(fmol/mg protein)

Left (treated)

Right (control)

Left

Right

Left/Right

50.6 _ 0.5 85.8 _+6.4 64.5 _ 6.0

65.4 ___3.9 59.0 ___7.4 55.8 + 2.2 59.2 -t- 3.2

64 -I- 8.0 60 _+9.1 66 ___15.0

340 _+ 15.7 290 -t- 17.4 279 __ 10.5 298 +_ 11.6

0.19 -I- 0.02 0.21 _+0.03 0.24 ___0.06

atrium, with or without the addition of a protective drug, affected the muscarinic receptor. The results of these studies are shown in Table 7. Methylation with T M O reduced the concentration of [ a H ] Q N B binding sites, but did not abolish binding. Assays ofhomogenates of left atrium (treated) were always carried out simultaneously with assays of right atrium (control). Further, apart from the process of methylation, both atria were subjected to the same drug treatment in the same organ bath. Three groups of experiments were carried out (a) without protective drug; (b) with A C h protecting and (c) with T T X protecting. Action potentials were monitored prior to binding assays to ascertain that the preparations were comparable in their response to that reported above. T h e results were essentially the same whether the left atrium was unprotected, or protected with either T T X or ACh. The dissociation constant of the ligand-binding site complex, KD, was largely unchanged while Bin, X (total concentration of binding sites) for the left atrium was reduced to 19% to 24% of the value for the right atrium. U n d e r control conditions values of Bmax for left and right atria are statistically indistinguishable, as are the corresponding values of KD [6]. I n the case of protection with ACh the value for KD for the left atrium (85.8 pM) is statistically different from the value of the right atrium (59.0 pM; P < 0.05). The effect is a small one, and m a y reflect the difficulty in determining Kt) accurately when the total binding of [ 3 H ] Q N B is not much higher than the non-specific binding in T M O - t r e a t e d preparations. Discussion

Unlike skeletal muscle and crab nerve [1, 14] guinea-pig atrial muscle cannot be methylated without loss of excitability. As we report-

ed previously [7, 8] the critical carboxyl group(s) can be protected with tetrodotoxin ( T T X ) and a n u m b e r of cholinergic drugs. Excitability and contractility return on washout of the protecting drug and the products of the methylation reaction. Some process of restitution of ionic gradients m a y occur, since the return of full excitability was sometimes delayed by up to 60 rnin. Spontaneous demethylation appears not to occur, since in no instance did prolonged washing of the unprotected preparation lead to a return of excitability. Since the carboxyl group which binds T T X and saxitoxin (STX) in muscle and nerve is not critical for the preservation of excitability, but cannot be methylated in atrium without loss of excitability, it appears that the N a + channel architecture must differ in the two situations. Tetrodotoxin is less effective in reducing the Na + current in heart than in skeletal muscle or nerve; this m a y reflect either less tight binding, or binding that leads to a less effective blockade of the N a + channel. Tetrodotoxin m a y produce a configurational change in the N a + channel which reduces N a § conductance rather than causing channel occlusion [9]. T h e present results suggest the proximity of the N a + channel and a cholinoceptive site, since the critical carboxyl group(s) can be protected by T T X or cholinergic drugs. It is unlikely that the protective effect of the cholinergic drugs is non-specific and due only to the binding of the cationic drug to all carboxyl groups. T h e drugs were used at concentrations which produce specific pharmacological effects, suggesting specific binding. Also the concentration of trimethoxonium ion ( T M O ; 25 raM) would be expected to dislodge the drugs from non-specific binding sites. T h e highest concentrafion of p r o ~ c t i n g drug used was 2 mM t r i m e t h y l a m m o n i u m ion (TMA).

Carboxyl Group Methylation of Na Channel in Atrium

A non-specific effect would also be inconsistent with the failure of neostigmine to protect, when the structurally related edrophonium did so. Further, methylation was carried out in the presence of 150 m~ Na +. The crossover experiments, in which the preparation was methylated in the presence of one drug, and then exposed to another, further strengthen the evidence that at least one carboxyl group binds in common both T T X and the cholinergic drugs. The wide range of pharmacological effects brought about by the different drugs would reflect membrane perturbations due to binding of the non-cationic part of the molecule to appropriate active sites. The experiments with T M A are of interest in this regard. The simple structure of this compound and its resemblance to T M O suggest that both its pharmacological effect and its protection against methylation reflect binding only to appropriate, accessible anionic sites. It will be recalled that when used as the protecting drug, with admission of 1 or 2 mM T M A after washout it was then unable to shorten the action potential (AP). Thus it can preserve excitability, but not protect the site which produces AP shortening. However, crossover experiments in which T M A protected the tissue during methylation followed by exposure to T T X or acetylcholine (ACh) after washout showed that the site of action of both these drugs had been protected. Acetylcholine shortened the AP to the same extent before and after methylation. T h e difference between the actions of T M A and ACh suggest either that T M A shortens the AP at a site different to ACh, or that T M A binds co-operatively to two anionic sites in order to shorten the AP. One of these (the site which binds ACh or T T X ) is protected by TMA, the other is not. Experiments in which we studied interactions between ACh and T T X in untreated preparations are consistent with the concept that these drugs bind to the same carboxyl group (Table 6), although these data could also be interpreted in other ways. Bartfai et al. [2] have reported experiments in which they found that T T X modified the binding of carbamyl choline to muscarinic receptors in brain. They suggest that binding of T T X to the Na + channel causes a conformational change in this protein which is sensed by the

107

muscarinic receptor. Their results differ from ours in that they found that T T X abolished high affinity carbamyl choline binding, whereas we found that ACh affected the actions of T T X , but not vice versa. However the thesis that the Na + channel is co-located with a cholinoceptive site seems well founded. It remains to determine whether this cholinoceptive site can be equated with the muscarinic receptor. The evidence to hand suggests that the cholinoceptive site in the proximity of the Na + channel is not the muscarinic receptor. Earlier work from this laboratory [5] showed that the negative inotropic effect of ACh was manifest at approximately one tenth the concentration that was required to shorten the AP. Further, reduction in AP duration was variable, and not always evident even at very high concentrations of ACh. This suggested that the two phenomena were not causally related, and could possibly be mediated by separate receptor systems. Our present finding that atropine and quinuclidinyl benzilate (QNB) offer little protection against the loss of excitability following methylation also suggests separate receptor systems. More direct evidence for this thesis is given by the [3H]-ONB binding studies. Here it was found that approximately 80% of [3H]-ONB binding sites were lost during methylation, regardless of whether or not the excitability of the atrium was preserved by T T X or ACh. Thus the excitability of the atrium is not dependent upon the maintenance of a normal population of [3H]-QNB binding sites. The reason for the survival of approximately 20% of the binding sites may be that [3H]-QNB binding was assayed in an homogenate, while methylation was carried out on the intact left atrium. Some binding sites deep in the preparation may well have escaped methylation during the usual 5 min treatment. It is of interest that 10 p~ ACh was not able to protect the muscarinic receptor from methylation. Further evidence for the existence of two sets of cholinoceptive sites comes from the amantadine studies. This drug has been shown to be a weak inhibitor of the muscarinic receptor, but to prolong the AP and increase contractility at approximately one tenth the concentration [6]. These latter effects were considered to be due to a delay in

108

S . E . F r e e m a n et aL

the o u t w a r d flow o f K +' c u r r e n t r e a d i n g to r e p o l a r i z a t i o n . T h e r e a p p e a r e d to be c o m petitive and noncompetitive interaction b e t w e e n a m a n t a d i n e a n d A C h . I n the p r e s e n t study a m a n t a d i n e was f o u n d to be an effective d r u g in p r o t e c t i n g e x c i t a b i l i t y d u r i n g m e t h y l ation, a n d was also effective in p r o t e c t i n g the T T X a n d A C h sites. T h u s T T X a n d A C h p r o d u c e d their usual effects on the A P after m e t h y l a t i o n in the p r e s e n c e o f a m a n t a d i n e . I f o u r p r e v i o u s hypothesis t h a t a m a n t a d i n e delays the o u t w a r d flow o f K + c u r r e n t is c o r r e c t [6] t h e n p r e s u m a b l y it binds to a v o l t a g e sensitive K + c h a n n e l , e i t h e r on the o u t e r or i n n e r m e m b r a n e surface, or to a site close to this K + c h a n n e l . S h o u l d this site be c h o l i n o c e p t i v e t h e n the ability o f A C h to s h o r t e n the A P a n d h y p e r p o l a r i z e the m e m b r a n e c o u l d be m e d i a t e d here. T h e n e g a t i v e i n o t r o p i c effect of A C h c o u l d well be m e d i a t e d via the m u s c a r i n i c r e c e p t o r , a n d m i g h t or m i g h t n o t b e i n d e p e n d e n t of the a c t i o n o f A C h on the K + c h a n n e l . T h e p r o p o s a l is t h e r e f o r e p u t f o r w a r d t h a t a c h o l i n o c e p t i v e site is s i t u a t e d close to the N a +

c h a n n e l in g u i n e a - p i g a t r i u m . T e t r o d o t o x i n a n d A C h b i n d to a c o m m o n c a r b o x y l g r o u p w h i c h is an i n t e g r a l a n d critical p a r t of the m a c r o m o l e c u l a r c o m p l e x . T h e effects o f T M A c a n best be e x p l a i n e d b y a s s u m i n g t h a t there is a second, n o n - c r i t i c a l c a r b o x y l g r o u p associated w i t h the c o m p l e x . T h e c h o l i n o c e p tive site is c o n s i d e r e d to be p a r t of or associa t e d w i t h the K + c h a n n e l , a n d a p p e a r s to be d i s t i n c t f r o m the m u s c a r i n i c receptor. E v i d e n c e exists [11] t h a t the N a + c h a n n e l a n d K + c h a n n e l are l o c a t e d t o g e t h e r in skeletal muscle. S u c h a n association m a y also o c c u r in c a r d i a c muscle. T h e o n l y a d d i t i o n a l a s s u m p t i o n n e e d e d to e x p l a i n o u r results is t h a t the K + c h a n n e l is p a r t of or associated w i t h a c h o l i n o c e p t i v e site.

Acknowledgements W e are g r a t e f u l to C i b a - G e i g y A u s t r a l i a for their gift o f a m a n t a d i n e a n d to R o c h e P r o ducts A u s t r a l i a for their gifts of e d r o p h o n i u m a n d Q N B . Skillful t e c h n i c a l assistance was p r o v i d e d by Miss M . H . D o w l i n g .

References i

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2 BARTFAI,T., NORDSTR6M,HEDI.UND,B., UNDEN,A., GRYNrARB,M. Pre- and postsynaptic muscarinic receptors. 3 4 5 6 7 8 9 10 11 12

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In ChemicalNeurotransmission,75years. L. Stjiirne, P. Hedqvist, H. Lagercrantz, A. Wennmalm (Eds), London: Academic Press ( 1981 ). CHAO, Y., VANOELN,R. L., RAFTERY, M. A. Preferential chemical modification of a binding sub-site on the acetylcholine receptor. Biochem Biophys Res Commun 63, 301~307 (1975). DAWSON,R. M., PORETSKI,M. A Comparison of the muscarinic cholinoceptors and benzodiazepine receptors of guinea-pig brain and rat brain. Neurochem Int 5, 369 374 (1983). FREEMAN,S. E. Cholinergic mechanisms in heart : interactions with 4-aminopyridine. J Pharmacol Exp Ther 210, 7 14 (1979). FREEMAN,S. E., DAWSON,R. M., CULVENOR,A.J., KEEGHAN,A. M. Interactions of amantadine with the cardiac muscarinic receptor. J Mol Cell Cardio117, 9-21 (1985). FREEMAN,S. E., GRAY, P. J., BLADEN,M. P. Chemical modification of the sodium channel in the guinea-pig atrium. Proc. International Union ofPhysiol Sci 15, 309 (1983). FREEMAN,S. E., GRAY,P.J., KEEGHAN,A. M., BLADEN,M. P. The use of toxins in the characterization of the Na § channel in cardiac muscle. Toxicon [-Suppl 3], 153 156 (1982). FREEMAN,S. E., LEAKE,B., SADEDIN,D. R., GRAY,P.J. The effect of tetrodotoxin on the inward ionic current of the guinea pig atrium. Cardiovasc Res 18, 233-243 (1984). FREEMAN,S. E., TURNER, R. J. Phase-plane trajectories of atrial cell action potentials: effects of temperature reduction. Cardiovasc Res 8, 451 459 (1974). KAO, C. Y., WALKER, S. E. Active groups of saxitoxin and tetrodotoxin as deduced from actions of saxitoxin analogues on frog muscle and squid axon. J Physiol [Lond] 323, 619-637 (1982). NAKAYAMA,H., TANIZAWA,K., KANOAKA,Y. Modification of carboxyl groups in the binding site of trypsin with the Meerwein Reagent. Biochem Biophys Res Commun 40, 537-541 (1970). RAWN,J. n., LIENHARD,G. E. The inactivation of acetylcholinesterase by trimethyloxonium ion, an active-sitedirected methylating agent. Biochem Biophys Res Commun fi6, 654-660 (1974). SPALDING,B. Properties of toxin-resistant sodium channels produced by chemical modification in frog skeletal muscle. J Physiol [Lond] 305,485-500 (1980).