Lectins modulate calcium channels in chick sympathetic ganglia

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Neuroscience Vol. 69, No. 1, pp. 331 337, 1995 Elsevier Science Ltd

Pergamon

IBRO

Printed in Great Britain 0306-4522/95 $9.50+ 0.00

0306-4522(95)00266-9

LECTINS MODULATE CALCIUM CHANNELS SYMPATHETIC GANGLIA

IN C H I C K

A. G O L A R D * Howard Hughes Medical Institute, Center for Neurobiology and Behavior, Columbia University P&S, New York, NY 10032, U.S.A.

Abstract--Calcium currents were measured in dissociated chick sympathetic ganglia using the whole cell patch-clamp technique. Lectins (1 /~M) were applied by local superfusion. All lectins tested reversibly inhibited Ca currents. Two types of inhibition were observed: a speeding up of inactivation and a voltage-dependent inhibition with slowing of the activation kinetics. When guanosine 5'-0-(3thiotriphosphate) was substituted for guanosine triphosphate, the voltage-dependent inhibition was irreversible, while the acceleration of inactivation remained reversible. Guanosine 5'-O-(2-thiodiphosphate) suppressed the voltage-dependent inhibition, but had little effect on the speeding up of inactivation. Lectins with high affinities for a-L-fucose, N-acetylglucosamine or N-acetylgalactosamine preferentially induced a voltage-dependent inhibition, while lectins with a-D-galactose affinity produced an acceleration of inactivation. Lectins with D-mannose affinity produced both type of modulation. Differential effects of concanavalin-A and succinyl-Con-A indicate that the speeding up of inactivation may be due to cap formation. It is concluded that, depending on their carbohydrate specificity, some lectins activate G-proteincoupled receptors, and thereby inhibit calcium channels in a voltage-dependent manner. Other lectins speed up the inactivation of the same channels, possibly through a direct interaction. Key words: G-proteins, inactivation, glycoproteins, nervous system, concanavalin A, ion channels.

Lectin receptors are involved in n u m e r o u s processes, including T cell activation, 2° i n f l a m m a t i o n , 25 microbial a d h e s i o n 29 a n d o t h e r cell recognition processes.16 In the n e r v o u s system, lectins have been s h o w n to m o d u l a t e synaptic t r a n s m i s s i o n 4'24 a n d g r o w t h cone m o v e m e n t s . 7'~8'39 Some o f the actions of lectins are o n ion channels. They include inhibition of nicotinic receptors, 3~ m o d u l a t i o n o f p o t a s s i u m channels, 23 a n d block o f the desensitization o f kainate receptors. 32 C h a n n e l m o d u l a t i o n can occur t h r o u g h direct interaction between the lectin a n d channel, as in the block o f k a i n a t e receptor desensitization, 32 or be mediated by second messengers. 23 The fact t h a t sodium a n d calcium c h a n n e l s are glycosylated has m a d e lectins useful tools for their purification. 2'3°'37 Despite their glycosylation, a n d t h a t they are m a j o r targets for m o d u l a t i o n t h r o u g h second messengers, ~ calcium c h a n n e l m o d u l a t i o n by *Present address: Department Physiology and Biophysics, SJ-40, University of Washington, Seattle, WA 98195, U.S.A. Abbreviations: Con-A, concanavalin A; 3,4DAP, 3,4 diaminopyridine; EGTA, ethylene glycol-bis(fl-aminoethylether)N,N,N',N'-tetraacetic acid; GDP-fl-S, guanosine 5'-O-(2-thiodiphosphate); GTP-7-S, guanosine 5'-O-(3-thiotriphosphate); GTP, guanosine 5'-triphosphate; HEPES, N-[2-hydroxyethyl]piperazine-N'[ethanesulfonic acid]; Ic,, calcium current; TEA, tetraethylammoniurn; TTX, tetrodotoxin. 331

lectins has n o t been reported. The present study shows t h a t these c h a n n e l s are indeed m o d u l a t e d by lectins t h r o u g h two mechanisms: one involving G protein-coupled receptors, the o t h e r not.

EXPERIMENTAL PROCEDURES Chick sympathetic ganglia were prepared according to Role (1988)/~4 Briefly, lumbar sympathetic ganglia were isolated from embryonic day 11 chicks. Tissue collection from these embryos only produces momentary pain or discomfort. The ganglia were dissociated by trituration after a 30 rain treatment with 0.01% trypsin. The cells were plated in polylysine-coated dishes at a density of one sympathetic chain (5~i ganglia) per dish. The cells were maintained at 37°C in a 5% CO2 atmosphere, in Dubelcco's modified Eagle's medium supplemented with 10% horse serum, 2% chick embryo extract, 2 rnM glutamine, penicillin (50 U/ml), streptomycin (50 mg/ml), and nerve growth factor (0.1 g/ml). Cells were used three to 10 days after plating, thereby minimizing the number of preparations needed. Prior to recording the cells were rinsed with a solution containing (in mM) NaC1, 138; KCI, 5; MgCI2, 0.8; CaC12, 0.5; HEPES, 15; glucose, 5; TTX, 0.002. The pH was adjusted to 7.35 with NaOH. Glucose and TTX were added just before use. The lectins were dissolved in a buffer containing (in mM) NaCl, 132; CaCI2, 2.5; MgCI2, 0.8; HEPES, 10; TEA, 10; 3,4DAP, 3; glucose, 5; TTX, 0.002. The pH was adjusted to 7.35 with NaOH. All lectins were applied at a concentration of 1 stM, by local superfusion onto the cell body and nearby processes.

332

A. Golard

Whole cell patch-clamp recordings were performed at room temperature ,,vith a List EPC7 amplifier (List Electronics). Patch electrodes contained (in mM) CsMethanesulfonate, 100; CsC1, 20; MgCI2, 2, EGTA, 10; CaCI2, 1; HEPES, 15. The pH was adjusted to 7.35 with CsOH. ATP (2 mM) and GTP (200 M) were added just before use. The current signal was filtered at 1 kHz with an 8-pole Bessel filter (Frequency devices model 902), and digitized with the pClamp software and TL-1 interface (Axon Instruments). Leak subtraction was performed with a P/8 protocol. Typical currents ranged from 0.6 to 1.4 nA, electrode resistances were 2.5-4 MfL series resistance compensation (50-80%) was used. Chemicals were obtained from Sigma, cell culture media were obtained from GIBCO.

n o u n c e d at 0 to + 2 0 mV, a n d smaller at more depolarized potentials (Fig. 1B). This type of inhibition is well characterized in sympathetic ganglia for a n u m b e r o f neurotransmitters. 14 O t h e r lectins, such as Lens culinaris lectin, produce a different type of inhibition. Here, the activation kinetics remain u n c h a n g e d , peak current is reduced, a n d inactivation appears to be faster (Fig. 1 C,D). This inhibition is completely reversible. The ratio of the peak current to the current at the end of a 200 ms pulse to 0 m V provides a stable estimate of this type of inhibition (Fig. 1 D), notice the ratio is stable during current r u n - u p at the onset of recording. In order to screen lectins for their ability to produce one effect or the other, a single protocol which c a n test for b o t h effects was used. The v o l t a g e - d e p e n d e n t inhibition can be relieved by strong depolarization, 3 a n d the removal of inhibition outlasts the depolarization. 6'~2 A double pulse protocol was optimized in these cells in order to study the voltage-dependent inh i b i t i o n a n d minimize the effects o f current rundown. 11 F r o m a holding potential o f - 8 0 mV, a n initial 20 ms pulse to 0 m V is followed by a 15 ms pulse to + 100 mV, a 5 ms repolarization to -80 mV, a n d a second 20 ms pulse to 0 m V (Fig. 2, lower left).

RESULTS

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Calcium c h a n n e l s in chick sympathetic ganglia are mostly N-type. ~ A n u m b e r o f lectins (see below) reversibly inhibit these c h a n n e l s in a voltage-dependent m a n n e r . This inhibition, s h o w n in figure 1 for C o n c a n a v a l i n A (Con-A), is a c c o m p a n i e d by a slowing of the current activation (Fig. 1A), is most pro-

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Fig. 1. Lectins have two effects o n the Ca current. A. The cell was held at -80 rnV and depolarized to 0 mV for 70 ms. Con A (1 #M) slows the activation kinetics of ICa. B. The inhibition by Con-A is voltage-dependent. The same cell was depolarized to various potentials for 70 ms. Peak currents are shown in the absence (open circles) and presence (filled circles) of 1 pM Con-A.. C. A different cell was held at -80 rnV and depolarized to + 10 mV for 200 ms every 10 s. A 35 s application of 1 pM Lens culinaris lectin induces a reversible reduction of peak current and speeding of inactivation. Individual records are shown before, during, and after lectin application. D. Lens culinaris lectin (1 pM, hatched bar) inhibits peak current (down triangles) by 17%, and the current at the end of a 200 ms pulse (up triangles) by 4 0 0 . The ratio of the two currents is plotted (squares).

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10 15 20 time, min Fig. 2. Inhibition of Ica by various lectins, using a double pulse protocol (lower left). Seven lectins were applied twice to a cell. The record numbers indicate the time at which individual records were acquired (lower right), letters indicate the lectin used, bars show the time of application. Lectins (all 1 /~M): a: Con-A, b: succinyl-Con-A, c: wheat germ agglutinin, d: Anguilla anguilla, e: Ptilota plumosa (red marine algae), f: Pisum sativum (pea), g: Arachis hypogaea (peanut). The voltage-dependent inhibition by ConA (a, records 1-3) is largely removed by a 15 ms pulse to + 100 mV, resulting in a decrease I1/12 ratio. The same pulse protocol does not antagonize the inhibition by Ptilota plumosa (e, records 4~6). Here, due to faster inactivation, the 11/12 ratio increases. Similar to what was observed with neurotransmitters, 11 the current during the first pulse to 0 mV (I 1) is reversibly inhibited by 1 #M Con-A (Fig. 2, 1-3). The strong depolarization largely removes this inhibition, resulting in a larger current during the second pulse to 0 mV (I2). The ratio of these two currents ( I 1 / I 2 ) decreases in the presence of a voltage-dependent inhibition (Fig. 2, lower panel). This decrease is a signature of voltage-dependent inhibition. In the absence of inhibition, the I1/I2 ratio is higher than one, due to channel inactivation. Lectins that increase the inactivation further increase this ratio, here from 1.1 to 1.24 for Ptilota plumosa lectin (Fig. 2, 4-6). Various lectins were tested using this protocol. An example is shown in Fig. 2, lower right. The individual records (1-3, 4-6) are taken at the times indicated. All lectins were

applied twice. Similar to neurotransmitter-mediated inhibition, lectin-induced voltage-dependent inhibition shows desensitization. For all the lectins which initially produce a voltage-dependent inhibition, a second application produces a much smaller inhibition than the first. In the case of Con-A (a), the effect reverses, going from a dominant voltage-dependent inhibition to a dominant speeding up of inactivation. Interestingly, a subsequent application of succinyl Con-A (b, a chemically modified Con-A with the same carbohydrate specificity but which is a dimer instead of a tetramer 13) still produces a voltage-dependent inhibition. Responses to wheat germ agglutinin (c) and Anguilla anguilla (eel, d) lectin desensitize but still show voltage-dependent inhibition during the second application. Repeated applications of Ptilota plu-

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time, m i n Fig. 3. Inhibition of tCa with 400 /~M GTP-~,-S in the pipette. A: the inhibition by l #M Datura stramonium lectin was monitored using the protocol in Figure 2. Tonic inhibition is present before tectin application (1). A 20 s application of Datura stramonium lectin further inhibits the current.(2), which does not recover upon washout (3). A long pulse protocol (Fig. l C,D) was then used in the same cell to monitor the speeding of inactivation of Ica by Lens culinaris lectin (l /xM). A 20 s application of the lectin produces a reversible speeding of inactivation (4-6, see current at the end of the pulse). A second application has a smaller effect.

mosa (red m a r i n e algae, e) a n d Pisum sativum (pea, f ) lectins consistently p r o d u c e a speeding u p o f inactivation. The v o l t a g e - d e p e n d e n t inhibition by Arachis hypogaea (peanut, g) is small a n d completely desensitizes. Carbohydrate vation

specificity o f inhibition and inacti-

Experiments such as the one in Fig. 2 were repeated with various lectins. Responses were cate-

gorized as v o l t a g e - d e p e n d e n t inhibition, acceleration o f inactivation, or no effect o n kinetics. Only the first response to a given lectin was t a k e n into account. F o r example in Fig. 2, C o n - A would be categorized as inducing a v o l t a g e - d e p e n d e n t inhibition. The results are s h o w n in T a b l e 1, along with the p r i m a r y c a r b o h y d r a t e specificities of the lectins tested. Lectins with affinities for c~-L-fucose, NAcetyl-glucosamine and N-Acetyl-galactosamine preferentially induce a v o l t a g e - d e p e n d e n t inhibition.

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time, s time, s Fig. 4. Inhibition of Ic, with 1 mM GDP-fl-S in the pipette. A: In control cells (200/~M GTP inside, open circles, n = 3), 1 /~M Solanum tuberosum (potato) lectin produces a reversible, voltage-dependent inhibition of Ica. When GTP is replaced with GDP-fl-S, no voltage-dependent inhibition is seen (filled circles, n=4). B: With GTP inside, 1 #M Phaseolus limensis (lima bean) lectin produces a reversible voltage-dependent inhibition of Ic, (open circles, n = 4). GDP-fl-S abolishes the voltage-dependent inhibition and reveals a reversible speeding of inactivation (filled circles, n = 3).

Lectins with affinity for ~-D-galactose preferentially induce a speeding o f inactivation. Lectins with a-Dm a n n o s e affinity p r o d u c e b o t h effects.

Involvement of G-proteins

In order to assess the possible role o f G - p r o t e i n s in the two forms o f inhibition, G T P analogs were substituted for G T P . First, G T P was replaced with the n o n - h y d r o l y s a b l e a n a l o g GTP-7-S (Fig. 3). In these conditions, some tonic v o l t a g e - d e p e n d e n t inhib i t i o n o f the c u r r e n t is typically seen (Fig. 3, A1). A p p l i c a t i o n o f 1 /tM D a t u r a S t r a m o n i u m lectin resulted in further i n h i b i t i o n (A2), which did n o t recover (A3). A t this p o i n t (2 m i n from the start o f recording), a pulse p r o t o c o l which is m o r e sensitive to changes in i n a c t i v a t i o n kinetics was used (Fig. 3B). This consisted o f 200 ms pulses to 0 mV,

every 10 s. A p p l i c a t i o n o f 1 /~M L e n s culinaris lectin resulted in a reversible speeding u p o f inactivation (B, 4-6). Peak c u r r e n t is reduced by 3 1 % while the current at the end o f the pulse is reduced by 65%. A second application o f Lens culinaris lectin h a d a m u c h smaller effect. The G T P a n a l o g GDP-fl-S, which prevents G p r o t e i n activation, was used in a similar m a n n e r . T h e d o u b l e pulse p r o t o c o l o f Fig. 2 was used for these experiments. W i t h G T P in the pipette, application o f 1 /~M S o l a n u m t u b e r o s u m (potato) lectin reliably induces a v o l t a g e - d e p e n d e n t inhibition (Fig. 4A, n = 3). W h e n GDP-fl-S is substituted for G T P , n o inhibition is seen ( n = 4 ) . Similarly, application o f 1 # M P h a s e o l u s limensis (lima bean) lectin p r o d u c e s a v o l t a g e - d e p e n d e n t inhibition in the presence of GTP (Fig. 4B, n = 4). R e m a r k a b l y , GDP-fl-S reveals a speeding u p of inactivation (n = 3).

Table 1. Carbohydrate specificity for inhibition and inactivation Lectin

Specificity

Inhibition

Inactivation

Anguilla anguilla Tetragonolobus Ulex europeaus Datura stramonium Triticum vulgaris Phytolacca americana Solanum tuberosum

c~-L-Fuc> > g-D-man ~-L-Fuc L-Fuc (D-glcNAc)z, D-IacNAc, fetuin (D-glcNAc)2, D-glcNAc (D-glcNAc)3 (D-glcNAc)3 g-D-man, g-D-glc > g-D-gal a-D-man, ~-D-glc a-D-man, g-D-glc D-galNAc > D-gal D-galNAc a-D-man, D-man a-D-man, D-man > D-glcNAc g-D-gal g-D-gal, g-D-galNAc

5/6 3/5 1/3 4/4 3/8 5/5 4/4 18/30 3/3 8/9 7/7 4/4 0/5 2/9 0/6 0/3

1/6 1/5 2/3 0/4 2/8 0/5 0/4 2/30 0/3 1/9 0/7 0/4 5/5 6/9 6/6 3/3

Concanavalin A succinyl Con/A Pisum sativum Glycine max Phaseolus limensis Lathyrus odoratus Lens culinaris Ptilota plumosa Maclura pomifera

fucose: N-acetylglucosamine: glcNAc, N/acetyllacttosamine: lacNAc, mannose: man, glucose: glc, galactose: gal, N-acetylgalactosamine: galNAc separates lectins which predominantly induce voltage-dependent inhibition (decrease 11/I2 ratio, see Fig. 2) from those which predominantly increase inactivation (increase 11/12).

336

A. Golard DISCUSSION

Two types o f inhibition

Con-A has two effects: a voltage-dependent inhibition and a scaling/speeding of inactivation. Con-A is a tetramer, which allows binding to sugar residues on different glycoproteins, thereby linking these proteins. This crosslinking is greatly reduced or absent when Con-A is transformed into a dimer by succinylation. A first application of Con-A to a cell produces a voltage-dependent inhibition. As this response desensitizes, speeding of inactivation becomes the dominant effect (Fig. 2). A subsequent application of succinylCon-A still produces a voltage-dependent inhibition. This indicates that the acceleration of inactivation may be due to cap formation, while the voltagedependent inhibition is not. Con-A which produces both types of inhibition, binds a ubiquitous sugar residue, D-mannose. Other lectins, such as Phaseolus limensis (Fig. 4) can also produce both effects. Lectins from different sources (plant and fish, plant and marine algae) with the same primary affinities preferentially induce the same type of inhibition (Table 1). While sugar specificity patterns emerge from this table, factors other than the primary affinities, such as differences in the ability to form caps and non-carbohydrate binding,9"36 may come into play. Importantly, sugar specificity does not prove interaction with sugar residues, as protein epitopes can show similar lectin binding patterns. 36 Another difference between the two types of inhibition lies in the involvement of G-proteins. The voltage-dependent inhibition is irreversible with GTP-y-S, and abolished by GDP-fl-S, indicating that G-proteins are involved. In contrast, the speeding up of inactivation is still reversible with GTP-7S, and present with GDP-fl-S, indicating that this effect is not mediated by G-proteins.

been demonstrated in Aplysia neurons, 23 mouse leukocytes 35 and human T lymphocytes. 2~ Thus, lectin binding may activate G-protein-coupled receptors as efficiently as neurotransmitters do. The identity of the receptor(s) involved here remains to be determined. An alternate interpretation of the results would be the direct activation of G-proteins by lectins. However the onset and recovery of inhibition by lectins are fast (within a few seconds), comparable to the values obtained with neurotransmitters. Rapid translocation across the cell membrane of large glycoproteins such as lectins is unlikely. Speeding up o f inactivation

In these cells, substance P increases the inactivation of Ica by activation of an NK-1 receptor.l° However lectin-mediated inactivation does not require G-proteins. Lectins may act directly on the channel, similar to their actions on kainate 15"32 and quisqualate38 receptors. Con-A has been shown to alter the distribution of acetylcholine receptors, while succinyl-ConA does not.19 Calcium channels may appear as clusters. 27 Cytoskeletal disruption alters channel inactivation.~7 Taken together these findings suggest that lectins may alter Ca channel inactivation by acting on channel distribution. Alternatively, lectins may affect inactivation kinetics by activation of receptors which do not couple to G-proteins, such as receptor protein-tyrosine kinases. Glycosylation of these receptors also affects their function.22 Most lectins used here were from plant, however the list of animal lectins has been expanding rapidly. 5 Plant and animal lectins share many similarities, including homologies in their primary amino acid sequences.8

CONCLUSIONS

Voltage-dependent inhibition

The lectin-induced, voltage-dependent inhibition has all the hallmarks of a neurotransmitter and Gprotein-mediated modulation. This inhibition is comparable in magnitude to that obtained with dopamine, norepinephrine2s and somatostatin II in these cells. Similar to neurotransmitter-mediated inhibition, the lectin-induced inhibition desensitizes. Many, if not all, G-protein-coupled receptors are glycosylated. One glycosylation site on somatostatin receptors is close to the ligand binding domain and glycosylation is required for high affinity ligand binding.33 Activation of second messenger systems by lectins has

Calcium channels are central to many cellular functions.26 Their modulation by lectins is likely to be important in vivo, either through cell contacts or through the action of solubles lectins. Perhaps more importantly, the present study shows that lectins activate G-protein-coupled receptors efficiently, and also interact with, amt alter the function of, other membrane components. This may enable lectins to perform broad physiological functions beyond cellular recognition events.

Acknowledgement--The

author

is

grateful

to

Dr

S.

Siegelbaum, in whose laboratory this work was performed.

REFERENCES

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Lectins modulate calcium channels

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