Acta physiol. scand. 1978. 103. 154-159 FroniIthe Department of Pharmacology, Karolinska Institutet, Stockholm, Research Institute of National Defence, Unit of Biochemistry, Sundbyberg and the Department of Physiology 11, Karolinska Institutet, Stockholm, Sweden

Binding of thiamine to nicotinic acetylcholine receptor in Torpedo marmorata and the frog end plate BY

LENNART WALDENLIND, LENAELFMAN and Bo RYDQVIST Received 29 November 1977

Abstract WALDENLIND, L., L. ELFMA?:and B. RYDQVIST. Bitiditig of thiamine to tricotitiic ucetvlckolitie receptor itr Torpedo murmorata utid the .frog erid plate. Acta physiol. scand. 1978. f03. 154--159. Thiamine was found to bind t o the isolated nicotinic receptor (nAChR) from Toqwfo niarnioi.nfa (K, 3 5 M). The binding was reversible and inhibited by @-neurotoxinfrom Naja naja siatnensiu. Acetjlcholine hinding was not inhibited by thiamine. When thiamine ( 5 . lo-&M) was applied to frog sartorius muscles a decrease of miniature end plate potential amplitudes was seen. This effect was readily re\ersihle after the perfusion fluid of the muscle was changed to normal Ringer solution. Thus both biological and electrophysiological studies indicated a binding of thiamine to the nAChR. Ahbrc\ iations: ACh -acetylcholine, MEPP =miniature end plate potential, nAChR-nicotinic choline receptor and PTP post-tetanic potentiation.

acetyl-

~

It has been shown that post-tetanic potentiation (PTP) i s abolished in thiamine deficiency induced by pyrithiainine (Waldenlind 1977). The PTP was then recovered by thiamine injections. The importance of thiamine for PTP was not correlated to the cocarboxylase functions of thiamine-diphosphate. Besides it was shown that d-tubocurarine in doses (4 /tg/kg) which left the ordinary supramaximal muscle twitches unaffected, blocked the PTP. Therefore it was assumed that thiamine was necessary for curare-sensitive receptors mediating PTP. This assumption led us to study the binding of thiamine t o the purified nAChR of Torpedo marmoratci and the electrophysiological effects of thiamine on miniature end plate potentials ( M E P P s ) registered from the muscle of the frog R a m pipiens.

Methods Binding u.f rhianiine t o 11.4Ch R The nAChR from Torpedo murniorafa electric organ was purified by biospecific chromatography on acetylated Naju najci sianiensis toxin bound to Sepharose 4 B (Heilbronn and Mattsson 1974). Carbachol elution gave a protein peak, which was further purified on a DEAE-ion exchanger in order to get rid of the carbachol and traces of acetylcholine esterase.

154

THIAMINE BINDING TO NICOTINIC RECEPTOR

155

Binding of (35S)thiamine hydrochloride (10-8-10-3 M) to the nAChR was tested as earlier described, using both the Sartorius membrane filter method (Heilbronn and Mattsson 1974) and equilibrium dialysis (Heilbronn et al. 1974). The nAChR (1 mg/ml) was incubated with nonradioactive cc-neurotoxin, a specific antagonist for nAChR, for I5 min at room temperature before the binding of thiamine was studied. In one experiment the receptor was incubated with oxythiamine and pyrithiamine (up to M) prior to thiamine-binding studies. Oxythiamine inhibits cocarboxylase function of thiamine-diphosphate but never induces neurological symptoms while pyrithiamine induces neurological symptoms reminding of thiamine deficiency without any major direct effect on cocarboxylase functions of thiamine-diphosphate (SteynParve 1967). T o study whether thiamine could inhibit the binding of ACh, the receptor protein was first incubated with 10V M Sarin for 60 min at room temperature in order to block traces of acetylcholine esterase. Thiamine (10-8-10-3 M) was then added and the incubation continued for another 30 min. (3H)ACh (10V M) in thiamine containing buffer was then added and equilibrium dialysis was performed. Protein was determined according to Lowry et al. (1951).

Electroplt~ssiologicu~ recordings The recording of MEPP was performed on sartorius muscle fibres from small Rana pipiens. The muscle was dissected free and placed in a perspex chamber (volume 5 ml) with its deep surface upward. The Ringer solution contained 115 m M NaCI, 2.5 m M KCI, 1.8 m M CaCI,, 2.15 mM Na,HPO, and 0.85 mM NaH,PO,, and the p H was 7.0-7.2 (Adrian 1956). The solution in the chamber was changed carefully with continuous recording from muscle fibres. The location of the end-plate region was determined by visual inspection using a binocular zoom microscope % 100 with dark field illumination. Intracellular potential changes were rzcorded using glass micropipettes filled with 3 M KCI and with a tip resistance of 8-10 M a . The micropipette was connected to a high input impedance ( % 10) amplifier. The output from this amplifier was fed into an oscilloscope (Tektronix 5103 N), a digital voltmeter (to record resting membrane potential) and a high gain :* 250 amplifier (AC-coupled, with lower cut-off frequency (- 3 dB point) of 0.3 Hz) the output of which was fed to a tape-recorder (Hewlett-Packard 3960). The speed of the tape-recorder was set to 3 3/4 inch/s which is equivalent to a bandwidth of 0-1250 Hz. The MEPP’s thus stored on tape could later be displayed on a n oscilloscope and recorded on photographic film.

Materials (3H)ACh (250 mCi/mmol) and (35S)thiamine (150 mCi/mmol) were purchased from the Radiochemical Centre, Amersham, U.K. Oxythiamine and pyrithiamine were obtained from Sigma Chemical Co. Other chemicals were from usual commercial sources.

Results Binding of thiamine to nAChR The purified nAChR protein from Torpedo marmorata which has a specific affinity for cholinergic ligands (Chang and Lee 1966, Heilbronn and Mattsson 1974, Meunier et al. 1974) is also able to bind thiamine in uitro (Fig. 1 and 2). The K,-value for the binding of M when measured by the Sartorius membrane thiamine was shown to be 3.05 5-0.87 x M when measured by equilibrium dialysis filter technique (Fig. 1) and 5.10k2.63 x (Fig. 2). The maximum binding capacity was 0.84 kO.10 nmol/mg receptor protein when the Sartorius filter technique was used (Fig. 1) and 2.57k0.79 nmol!mg protein when measured by equilibrium dialysis (Fig. 2). All values are mean S.D. The binding of thiamine to the nAChR was inhibited by the nAChR antagonist cc-neurotoxin from Naja naja siamensis at a concentration of M. This was proven by both techniques. The binding of ACh M) was not inhibited by thiamine (10-*-10-3 M), nor did the presence of thiamine (lo-* M) change the K,-value (2.3 M) for the binding of ACh (checked by equilibrium dialysis).

I56

LENNART WALDENLIND, LENA ELFMAN AND BO RYDQVIST

Fig. 1

Fig. 2

Fig. 1. Lineweaver-Burk plot showing binding of thiamine to nAChR using the Sartorius membrane filter method. The K,-value calculated from the curve is 3.05k0.87Y M and the maximal binding capacity 0.842 0.10 nmol of thiamine per mg of protein. Fig. 2. Lineweaver-Burk plot showing binding of thiamine to nAChR using equilibrium dialysis. The KDvalue calculated from the curve is 5.1012.63 :. M and the maximal binding capacity 2.57k0.79 nmol of thiamine per mg protein.

Oxythiamine and pyrithiamine (two thiamine antagonists) were tested in order to determine whether thiamine-binding to nAChR could be prevented. No inhibition could be seen at concentrations up to lo-'' M. Elwtroplij s iologicrrl rc~orrlitig~

Thiamine (10+-5 10 ' M) was applied to frog sartorius muscles for different periods of time (2-10 min mainly depending on the concentration used). U p to a concentration of 10 M no changes in mean amplitude of the M E P P s could be seen. At higher concentration ( 1-5 M ) , a decrease in MEPP amplitude was observed (Fig. 3). Amplitude histograms for spontaneous MEPP's in the same fibre as in Fig. 3 are shown in Fig. 4 at two conand 5 M). At 5 M, the mean amplitude is centrations of thiamine (5 unchanged (at 95 O O significance level) compared with the mean amplitude in control measurements both before and after application of thiamine (Fig. 4 A, B and C). After application of 5 lo-' M thiamine, the mean MEPP amplitude was drastically reduced from 0.9 mV (Fig. 4 C ) to 0.3 mV (Fig. 4 D). At this concentration, reduction occurred within about 1 min, the total time of application being 3 min. After 5 min in normal Ringer solution the MEPP amplitude had returned to the same value (0.9 mV) (Fig. 4 E) as before application of thiamine (no difference at a 95 O; significant level). The MEPP frequency seems to be somewhat increased at both concentrations of thiamine compared with the frequency in Ringer solution (Fig. 4).

157

THIAMINE BINDING TO NICOTINIC RECEPTOR

A

Normal Ringer

.

-.r

,..

.L\ L . - .

B

Thiamine 5~10-~M

Normal Ringer

C

I

-

I

1

.

1

I

-

?-

c ”

r L

-

_

I

-

1

5

-71 -

Fig. 3. Effect of thiamine on MEPP‘s. A. Before application of thiamine. B. Two min after application of M). C . After return to Normal Ringer. Resting membrane potential: (A) - 93 mV, (B) thiamine ( 5 x - 95 mV, (C) - 94 mV. Time bar: 0.5 s, Vertical bar: 2 mV.

The resting membrane potential was not affected in thiamine solutions, the values ranging from about - 90 mV to - 95 mV. After the final measurements in normal saline, the micropipette was withdrawn from the fibre and the resting membrane potential was found to be -94 mV.

Discussion The isolated nAChR from Torpedo marmorata binds thiamine at a concentration of M. The binding of thiamine is prevented by a-neurotoxin from Naja naja siamensis, which is a

C

B

A

Thiamine 5

Normal Ringer

M

Normal Ringer

f = 0.33

m-1.0

0.5 Thiamine 5

1.0

1.5

0.5

1.0

1.5

mV

E

D

m.0.9 a . 2

0.3

Normal Ringer

M

n.22

I

f =0.55 rns 0.3f 0.1

w 5

0.5

lo

65

li

10

115

mV Fig. 4. Histograms from experiments as illustrated in Fig. 3 showing distribution of amplitudes of MEPP’s in a muscle fibre treated with two concentrations of Thiamine ( 5 x M and 5 x lo-* M). n is number of fibres;fis mean MEPP frequency (s-l); rn is mean i standard deviation of MEPP amplitude (mV). Resting membrane potential: (A) -92 mV, (B) -90 mV, (C) - 93 mV, (D) -95 mV, (E) -94 mV.

158

LENNART WALDENLIND, LENA ELFMAN A N D BO RYDQVIST

specific antagonist of the nAChR (Chang and Lee 1966). The binding of thiamine to the isolated nAChR occurs in a similar dose range to that which inhibits nicotine-induced contractions of the guinea-pig ileum (Waldenlind 1977). The &,-value for thiamine in the present study is based on in ritro experiments. The binding constants need not be the same in r-iro. It is known that the binding of e g . ACh to the isolated nAChR occurs at a dose which is higher than that which is active on isolated organ preparations. Extrinsic factors, such as temperature, pH, intact membrane structure and possible co-factors present, which are defined in the non-injured tissue, may change the binding constant. Therefore the K,value gives no information concerning the concentration of thiamine which could be active hi t7ir)o. Thiamine decreased the mean MEPP amplitude in the frog end plates without affecting the membrane potential when added in a concentration of 5 lo-$ M (Fig. 3). At this concentration about 8 5 9 , of all thiamine-binding sites of the nAChR from Torpedo marmorata are occupied. This electrophysiological effect may therefore be explained as being due to binding of thiamine to the nAChR of the end plates. The bulk concentration of thiamine ( 5 LO-& M ) necessary to block the MEPP’s is probably higher than the concentration at the end plate region due to the high polarity of the thiamine molecule which makes it difficult to penetrate tissue barriers. In the purified nAChR the high affinity site (K, - 3 i M) is changed to a low-affinity site (K, --2 M) for ACh. Thiamine could not inhibit the low-affinity binding of ACh to purified nAChR but in electrophysiological studies where only the high-affinity site is present, the results indicate an effect on either the acetylcholine bind- or the ionophore activity of the nAChR. The electrophysiolo$cal studies do not reveal any certain physiological role for thiamine at the post-synaptic nAChR. However, it seems clear that thiamine binds to nAChR since this has been shown by binding studies on isolated receptors and by electrophysiological recordings. PTP is mediated by the activation of presynaptic curare-sensitive receptors and thiamine is necessary for the function of these receptors (Waldenlind 1977, Waldenlind. To be published). It is thus possible that the physiological role of thiamine may be in the mediation of PTP through activation of curare-sensitive receptors. We aish to thank Profescor E. Heilhronn for helpful comments on the manuscript.

References A D K I ~ SR. . H., The effects of internal and external potassium concentration on the membrane potential of the frog muscle. J. Pltj~siol.(Lond.) 1956. 133. 631-658. CHAW;,C. C. and C. Y. LEE,Electrophysiological study of neuromuscular blocking action of cobra neurotoxin. Brit. J . Pharmacoi. 1966. 28. 172-181. H~ILBIIONN, E. and CH. MATTSSON, The nicotinic cholinergic acetylcholine receptor protein. Improved purification method, preliminary amino acid composition and observed autoimmune response. J. Neurochrm. 1974. 22. 3 15-3 17. HFII.HKONN, E.. CH. MATTSSONand L. ELFMAN,Biochemical and physical properties of the nicotinic acetylcholine receptor from Torpedo mornlorota. Proc. 9th FEB’s Meet. Ed. Gardos. Budapest 1974. pp. 29-37. LOWRY,0. H., H. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL, Protein measurement with the Folin phenol reagent. J. bid. Clrrm. 1951. 193. 265-275.

THIAMINE BINDING TO NICOTINIC RECEPTOR

159

MEUNIER, J.-C., R. SEALOCK, R. V. OLSENand J.-P. CHANGEUX, Purification and properties of the cholinergic receptor protein from Electrophorus electric tissue. Europ. J. Biochem. 1974. 45. 371-393. STEYN-PARVE, E. P., The mode of action of some thiamine analogues with antivitamin activity. Thiamine Deficiencv. Ciba Foundation Study Group No. 28. Ed. G. E. W. Wolstenholme. London: Churchill Ltd. 1967. pp. 26-42. WALDENLIND, L., Release of thiamine and the formation of a methylthiamine-like substance in the phrenic nerve-diaphragm preparation of the rat. Acta physiol. scand. 1977. 10I. 22-27. WALDENLIND, L., Importance of thiamine for presynaptic nicotinic receptors mediating post-tetanic potentiation (PTP). Acta pharmacol. (Kbh.) 1977. 41. Supplement 1V. p. 79. WALDENLIND, L., Possible role of thiamine for neuromuscular transmission. To be published.

Binding of thiamine to nicotinic acetylcholine receptor in torpedo marmorata and the frog end plate.

Acta physiol. scand. 1978. 103. 154-159 FroniIthe Department of Pharmacology, Karolinska Institutet, Stockholm, Research Institute of National Defence...
307KB Sizes 0 Downloads 0 Views