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Ligand interactions of crustacean axonal membranes
Neurochemistry International Vol.2, pp.53-60. Pergamon Press Ltd. ]980. Printed in Great Britain.
LIGAND INTERACTIONS OF CRUSTACEAN AXONAL MEMBRANES
Henry G. Mautner, James E. Jumblatt and Judith K. Marquis Department of Biochemistry and Pharmacology Tufts University School of Medicine 136 Harrison Avenue Boston, MA 02111
ABSTRACT The relative abilities of a series of local anesthetics s in which either or both the ester oxygens had been replaced with sulfur or selenium to block axonal or synaptlc preparations are compared with their abilities to displace SH-~icotine from lobster axon plasma membrane fragments. The relative affinities of a series of cholinergic agonists and antagonists for synaptic and axonal membranes are discussed as is the salt sensitivity and reversibility of their interactions. The utility of a conjugate of e-bungarotoxln and horse-radish peroxidase for the histoehemlcal visualization of binding sites of intact axons or axonal vesicles is discussed. Labelling of a peptide isolated from axon plasma fragments with MBTA is compared with labelling of peptides isolated from synaptie membranes. KEYWORDS Thlo and seleno analogs of local anesthetics; trimethylammonium analogs of local anesthetics; blocking action of local anesthetics on axonal and synaptic preparations; affinities of cholinergic agonists and antagonists for axonal membranes; axonal membrane vesicles; ~-bungarotoxin-horseradish peroxidase conjugate; ~ T A labelling. TEXT The proposal by Nachmansohn (1954) that attachment of acetylcholine to a protein receptor induces a change in the cation permeability of synaptic membranes has won general acceptance, however, the question of the involvement of this cycle in the functions of axonal membranes has remained controversial. It is known that most chollnergic agonlsts or antagonists don't affect axonal conduction, possibly because of membrane barriers limiting access to binding sites. However, it has been reported that at the nodes of Ranvier where permabillty barriers are minimal, chollnergic llgands can affect conduction (Dettbarn, 1960, Chang and Lee, 1966); similar effects have been noted in uumyelinated fibers (Armett and Ritehie, 1961; Ritchle, 1967). We approached this problem by means of a series of local anesthetics in which one or both of the ester group oxygens had been replaced by sulfur or selenium. It was found that such molecular modifications greatly altered the abilities of such local anesthetics to block electrical activity in squid giant axons (Rosenberg and Mautner, 1967) or to block the earbamylcholine induced depolarization of the electroplax preparation (Webb and Mautner, unpublished data). There was striking parallelism in the abilities of the ~arious isologs to block synaptlc or axonal preparations. The blocking actions of local anesthetics carrying a tertiary amino group exceeded those of analogous compounds carrying the trimethylarmnonium group in squid giant axons. However, analogous tertiary amino and trimethylammonium derivatives were equipotent blocking agents in the electroplax preparation in which structural barriers are minimal. The presence of "axonal eholinergic binding macromoleeules" in axonal membranes was reported several years ago (Denburg, Eldefrawi and O'Brien, 1972; Denburg and O'Brien, 1973). In extending this work, we studied the ester, thiolester and selenolester analogs of local 53
54
H.G.
Mautner, J. E. Jumblatt and J. K. Marquis
~ nesthetics
as well as their trimethyla~onium analogs for their abilities to displace H-nicotine from vesicular lobster plasma membrane fragments. The relative abilities of oxo-, thio- and seleno-compounds to displace 3H-nicotine from such fragments parallelled their abilities to block synaptic or axonal preparations. Quaternization of the tertiary amino groups did not alter ability to displace nicotine (Marquis,_Hilt, Papadeas and Mautner, 1977). The ability of the membrane fragments to bind iZDl-a-bungarotoxin, a ligand widely believed to be a specific marker of binding sites for cholinergic ligands, could be demonstrated (Marquis, Hilt and Mautner, 1977). Use of a conjugate of a-bungarotoxin and horseradish peroxidase (Lentz, Hazurkiewicz and Rosenthal, 1977) permitted the electron microscopic visualization of a-bungarotoxin binding sites of axonal membrane vesicles or intact nerve fibers from lobster or spider crab. The binding of the conjugate could be prevented by pretreatment with d-tubocurarine or with native toxin (Chester, Lentz, ~quls and Mautner, 1979). Studies of Rawlins and Villegas (1978) showed the binding of ~ l-a-bungarotoxin at the axon-Schwann cell boundary of squid nerve fibers. Use of the bungarotoxin-peroxidase conjugate demonstrated that binding sites were localized primarily on the axolemma rather than on the Schwann cell membrane (Chester, Lentz, Marquis and Mautner, 1979). While axonal membrane fragments can be labelled by several ligands believed to be bound specifically to acetylcholine receptors (Denburg, Eldefrawi and O'Brien, 1972; Denburg and O'Brien, 1973; Marquis, Hilt, Papadeas and Mautner, 1977), it was recognized early that the relative affinities of these ligands for axonal membranes were very different from those seen with synaptic membranes. For instance, Denburg and O'Brien (1973) noted that procaine was a competitive antagonist of n i c o t ~ binding. As can be seen in the following Table, the abilit~ of ligands to displace l-a-bungarotoxin, while it parallels the ability to displace H-nicotine, is entirely different from the binding behavior of cholinergic ligands interacting with "nicotinic" aeetylcholine receptor (Cohen and Changeux, 1975) or with "muscarinic" acetylcholine receptor (Heilbronn and Bartfai, 1978). Furthermore, in contrast to synaptie membranes, the binding of a-bungarotoxin to axonal membranes is relatively loose with a K D of 3 10-7M (Marquis, Hilt and Mautner, 1977), is completely reversible and is very sensitive to the presence of salts (Jumblatt and Mautner, unpublished data).
INHIBITION OF BINDING OF [1251] a-BUNGAROTOXIN (2-4xlO-8M) TO AXON PLASMA HEMBRANE FRAGHENT BY VARIOUS DRUGS DRUG (10-4H) d-Tubocurarine Atropine Nicotine Carbamylcholine Choline Acetylcholine (±) Quinuclidinyl Benzilate Physostigmine Neostigmine Procaine Tetracaine (3 °) Tetracaine (4 °) Lidocaine GABA Bicuculline Octopamine Tetrodotoxin 4-Aminopyridine Veratridine
% INHIBITION 69 84 43 15 17 i0 0 74 16 82 37 50 29 0 0 0 0 0 0
Recently, we found that 3H-4-(N-maleimido) benzyltrimethylammonium (MBTA), a ligand believed to be capable of selective attachment to the a-subunit of the acetylcholine receptor (Karlin and Winnik, 1968; Reiter, Cowburn, Prives and Karlin, 1972), can label a peptide with a molecular weight of 38,000 Daltons derived from axonal membranes. However, in contrast to synaptic membranes, labelling with ~II-MBTA is not prevented by nicotine or a-bungarotoxin in the axonal membrane preparation.
Ligand Interactions of Crustacean Axonal Membranes
Lobster walking leg nerve membrane fraction incubated in HRP-~-BuTx donjugate and assayed for peroxidase activity.
55
56
H.G.
Mautner,
J. E. Jumblatt and J. K. Marquis
'++
WLCT /
SC
From Chester, Lentz, Marquis, Mautner,
(1979).
With permission.
A.
Lobster axon (Ax) enveloped by Schwann cell (SC). was incubated in HRP-~-BuTx conjugate.
B.
Portion of large crab axon (Ax) and adjacent conjugate and HRP assay.
N, Schwann cell nucleus.
The nerve
Schwann cell (SC) after incubation with
Ligand Interactions of Crustacean Axonal Membranes
45.
2~
Ax
Tp CB
Ax
Tp
$ H'MBTA
SDS slab gel electrophoresis of membrane fragments derived from lobster axons Ax) or Torpedo nobiliana electroplax (Tp) CB Polypeptides stained with Coomassie Blue 3H-MBTA Polypeptide labelling visualized by autofluorography
57
Ligand Interactions of Crustacena Axonal Membranes
59
In summary, it appears that axonal membranes contain a protein capable of binding several cholinergic ligands with the abundance of this protein greatly exceeding that of the "sodium channel" biopolymer isolated from Electrophoru 9 electricus membranes (Agnew and Raftery, 1979) or from garfish olfactory nerve (Henderson and Wang, 1972). While the functional significance of the axonal biopolymer capable of binding cholinergic ligands has not been established, the possibility that it may contain subunits related to at least some of the subunits of the acetylcholine receptor should be explored. The presence of this protein as well as that of acetylcholinesterase (Villegas and Villegas, 1974) on the axolemma, coupled with reports that higher levels of acetylcholine are found in surrounding Schwann cells than in the squid giant axon (Villegas and Jenden, 1979), raises further questions regarding the roles of the axolemmal cholinergic ligand binding material.
We are indebted to the National Science Foundation (BNS-79-06188) for support of this work.
Agnew, W.S. and M.A. Raftery (1979) Solubilized tetrodotoxin binding component from the electroplax of Electrophorus electricus. Stability as a function of mixed lipiddetergent micelle composition. Biochem. 18, 1912-9. Armett, C.J. and J.M. Ritchie (1961) The action of acetylcholine and some related substances on conduction in mammalian non-myelinated nerve fibres. J. Physiol., 155, 372-384. Chang, C.C. and C.J. Lee (1966) Electrophysiological study of neuromuscular blocking action of cobra neurotoxin. Brit. J. Pharmacol. 28, 172-181. Chester, J., T.L. Lentz, J.K. Marquis and H.G. Mautner (1979) Localization of horseradish peroxidase-~-bungarotoxln binding in crustacean axonal membrane vesicles and intact axons. Proc. Nat. Acad. Sci. USA, 76, 3542-3546. Cohen, J.B. and J.P. Changeux (1975) The cholinergic receptor protein in its membrane environment. Ann. Rev. Pharmacol. 15, 83-103. Denburg, J.L. (1972) An axon plasma membrane preparation from the walking legs of the lobster Homarus americanus. Biochim. Biophys. Acta, 282, 453-458. Denburg, J.L., M.E. Eldefrawi and R.D. O'Brien (1972) Macromolecules from lobster axon membranes that bind cholinergic ligands and local anesthetics. Proc. Nat. Acad. Sci. USA 6__9, 177-181. Denburg, J.L. and R.D. O'Brien (1973) Axonalcholinergic binding macromolecule. Response to neuroactive drugs. J. Med. Chem. 16, 57-60. Dettbarn, W.D. (1960) Effect of curare on conduction in myelinated isolated nerve fibres of the frog. Nature, 186, 891-892. Henderson, R. and J.H. Wang (1972) Solubilization of a specific tetrodotoxin-blnding component from garfish olfactory nerve membrane. Biochem. ii, 4565-4569. Heilbronn, E. and T. Bartfai (1978) Muscarinic acetylcholine receptor. Progress Neurobiol. ii, 171-188. Karlin, A. and M. Winnik (1968) Reduction and specific alkylation of the receptor for acetylcholine. Proc. Nat. Acad. Sci. USA 60, 668-674. Lentz, T.L., J.E. Mazurkiewlcz and J. Rosenthal (1977) Cytochemical localization of acetylcholine receptors at the neuromuscular junction by means of horseradish peroxidaselabeled ~-bungarotoxin. Brain Res. 132, 423-442. Marquis, J.K., D~C. Hilt and H.G. Mautner (1977) Direct binding studies of 1251-~-bungarotoxin and -H-quinuclidinyl benzilate interaction with axon plasma membrane fragments. Biochem. Biophys. Res. Comm. 78, 476-482. Marquis, J.K., D.C. Hilt, V.A. Papadeas and H.G. Mautner (1977) Interaction of cholinergic ligands and local anesthetics with plasma membrane fragments from lobster axon. Proc. Nat. Acad. Sci. USA 74, 2278-2282. Nachmansohn, D. (1954) Metabolism and function of the nerve cell. Harvey Lectures, 19531954, Academic Press, New York. pp. 57-99. Rawlins, F.A. and J. Villegas (1978) Autoradiographic localization of acetylcholine receptors in the Schwann cell membrane of the squid nerve fiber. J. Cell. Biol. 77, 371-376. Relter, M.J., D.A. Cowhurn, J.M. Prives and A. Karlin (1972) Affinity labeling of the acetylcholine receptor in the electroplax: Electrophoretic separation in sodium dodecyl sulfate. Proc. Nat. Acad. Sci. USA 69, 1168-1172.
60
H.G.
Mautner, J. E. Jumblatt and J. K. Marquis
Ritchie, J.M. (1967) On the role of acetylcholine in conduction in mammalian non-myelinated nerve fibers. Ann. N. Y. Acad. Sci. 144, 504-516. Rosenberg, P. and II.G. Mautner (1967) Acetylcholine rece tot: Similarity in axons and junctions. Science 155, 1569-1571. Villegas, G. M. and J. Villegas (1974) Acetylcholinesterase localization in the giant nerve fiber of the squid. J. Ultrastruct. Res. 46, 149-163. Villegas, J. and D.J. Jenden (1979) Acetylcholine content of the Schwann cell and axon in the giant nerve fibre of the squid. J. Neurochem. 32, 761-766.
LIGAND INTERACTIONS OF CRUSTACEAN AXONAL MEMBRANES
Henry G. Mautner, James E. Jumblatt and Judith K. Marquis Department of Biochemistry and Pharmacology Tufts University School of Medicine 136 Harrison Avenue Boston, MA 02111
ABSTRACT The relative abilities of a series of local anesthetics s in which either or both the ester oxygens had been replaced with sulfur or selenium to block axonal or synaptlc preparations are compared with their abilities to displace SH-~icotine from lobster axon plasma membrane fragments. The relative affinities of a series of cholinergic agonists and antagonists for synaptic and axonal membranes are discussed as is the salt sensitivity and reversibility of their interactions. The utility of a conjugate of e-bungarotoxln and horse-radish peroxidase for the histoehemlcal visualization of binding sites of intact axons or axonal vesicles is discussed. Labelling of a peptide isolated from axon plasma fragments with MBTA is compared with labelling of peptides isolated from synaptie membranes. KEYWORDS Thlo and seleno analogs of local anesthetics; trimethylammonium analogs of local anesthetics; blocking action of local anesthetics on axonal and synaptic preparations; affinities of cholinergic agonists and antagonists for axonal membranes; axonal membrane vesicles; ~-bungarotoxin-horseradish peroxidase conjugate; ~ T A labelling. TEXT The proposal by Nachmansohn (1954) that attachment of acetylcholine to a protein receptor induces a change in the cation permeability of synaptic membranes has won general acceptance, however, the question of the involvement of this cycle in the functions of axonal membranes has remained controversial. It is known that most chollnergic agonlsts or antagonists don't affect axonal conduction, possibly because of membrane barriers limiting access to binding sites. However, it has been reported that at the nodes of Ranvier where permabillty barriers are minimal, chollnergic llgands can affect conduction (Dettbarn, 1960, Chang and Lee, 1966); similar effects have been noted in uumyelinated fibers (Armett and Ritehie, 1961; Ritchle, 1967). We approached this problem by means of a series of local anesthetics in which one or both of the ester group oxygens had been replaced by sulfur or selenium. It was found that such molecular modifications greatly altered the abilities of such local anesthetics to block electrical activity in squid giant axons (Rosenberg and Mautner, 1967) or to block the earbamylcholine induced depolarization of the electroplax preparation (Webb and Mautner, unpublished data). There was striking parallelism in the abilities of the ~arious isologs to block synaptlc or axonal preparations. The blocking actions of local anesthetics carrying a tertiary amino group exceeded those of analogous compounds carrying the trimethylarmnonium group in squid giant axons. However, analogous tertiary amino and trimethylammonium derivatives were equipotent blocking agents in the electroplax preparation in which structural barriers are minimal. The presence of "axonal eholinergic binding macromoleeules" in axonal membranes was reported several years ago (Denburg, Eldefrawi and O'Brien, 1972; Denburg and O'Brien, 1973). In extending this work, we studied the ester, thiolester and selenolester analogs of local 53
54
H.G.
Mautner, J. E. Jumblatt and J. K. Marquis
~ nesthetics
as well as their trimethyla~onium analogs for their abilities to displace H-nicotine from vesicular lobster plasma membrane fragments. The relative abilities of oxo-, thio- and seleno-compounds to displace 3H-nicotine from such fragments parallelled their abilities to block synaptic or axonal preparations. Quaternization of the tertiary amino groups did not alter ability to displace nicotine (Marquis,_Hilt, Papadeas and Mautner, 1977). The ability of the membrane fragments to bind iZDl-a-bungarotoxin, a ligand widely believed to be a specific marker of binding sites for cholinergic ligands, could be demonstrated (Marquis, Hilt and Mautner, 1977). Use of a conjugate of a-bungarotoxin and horseradish peroxidase (Lentz, Hazurkiewicz and Rosenthal, 1977) permitted the electron microscopic visualization of a-bungarotoxin binding sites of axonal membrane vesicles or intact nerve fibers from lobster or spider crab. The binding of the conjugate could be prevented by pretreatment with d-tubocurarine or with native toxin (Chester, Lentz, ~quls and Mautner, 1979). Studies of Rawlins and Villegas (1978) showed the binding of ~ l-a-bungarotoxin at the axon-Schwann cell boundary of squid nerve fibers. Use of the bungarotoxin-peroxidase conjugate demonstrated that binding sites were localized primarily on the axolemma rather than on the Schwann cell membrane (Chester, Lentz, Marquis and Mautner, 1979). While axonal membrane fragments can be labelled by several ligands believed to be bound specifically to acetylcholine receptors (Denburg, Eldefrawi and O'Brien, 1972; Denburg and O'Brien, 1973; Marquis, Hilt, Papadeas and Mautner, 1977), it was recognized early that the relative affinities of these ligands for axonal membranes were very different from those seen with synaptic membranes. For instance, Denburg and O'Brien (1973) noted that procaine was a competitive antagonist of n i c o t ~ binding. As can be seen in the following Table, the abilit~ of ligands to displace l-a-bungarotoxin, while it parallels the ability to displace H-nicotine, is entirely different from the binding behavior of cholinergic ligands interacting with "nicotinic" aeetylcholine receptor (Cohen and Changeux, 1975) or with "muscarinic" acetylcholine receptor (Heilbronn and Bartfai, 1978). Furthermore, in contrast to synaptie membranes, the binding of a-bungarotoxin to axonal membranes is relatively loose with a K D of 3 10-7M (Marquis, Hilt and Mautner, 1977), is completely reversible and is very sensitive to the presence of salts (Jumblatt and Mautner, unpublished data).
INHIBITION OF BINDING OF [1251] a-BUNGAROTOXIN (2-4xlO-8M) TO AXON PLASMA HEMBRANE FRAGHENT BY VARIOUS DRUGS DRUG (10-4H) d-Tubocurarine Atropine Nicotine Carbamylcholine Choline Acetylcholine (±) Quinuclidinyl Benzilate Physostigmine Neostigmine Procaine Tetracaine (3 °) Tetracaine (4 °) Lidocaine GABA Bicuculline Octopamine Tetrodotoxin 4-Aminopyridine Veratridine
% INHIBITION 69 84 43 15 17 i0 0 74 16 82 37 50 29 0 0 0 0 0 0
Recently, we found that 3H-4-(N-maleimido) benzyltrimethylammonium (MBTA), a ligand believed to be capable of selective attachment to the a-subunit of the acetylcholine receptor (Karlin and Winnik, 1968; Reiter, Cowburn, Prives and Karlin, 1972), can label a peptide with a molecular weight of 38,000 Daltons derived from axonal membranes. However, in contrast to synaptic membranes, labelling with ~II-MBTA is not prevented by nicotine or a-bungarotoxin in the axonal membrane preparation.
Ligand Interactions of Crustacean Axonal Membranes
Lobster walking leg nerve membrane fraction incubated in HRP-~-BuTx donjugate and assayed for peroxidase activity.
55
56
H.G.
Mautner,
J. E. Jumblatt and J. K. Marquis
'++
WLCT /
SC
From Chester, Lentz, Marquis, Mautner,
(1979).
With permission.
A.
Lobster axon (Ax) enveloped by Schwann cell (SC). was incubated in HRP-~-BuTx conjugate.
B.
Portion of large crab axon (Ax) and adjacent conjugate and HRP assay.
N, Schwann cell nucleus.
The nerve
Schwann cell (SC) after incubation with
Ligand Interactions of Crustacean Axonal Membranes
45.
2~
Ax
Tp CB
Ax
Tp
$ H'MBTA
SDS slab gel electrophoresis of membrane fragments derived from lobster axons Ax) or Torpedo nobiliana electroplax (Tp) CB Polypeptides stained with Coomassie Blue 3H-MBTA Polypeptide labelling visualized by autofluorography
57
Ligand Interactions of Crustacena Axonal Membranes
59
In summary, it appears that axonal membranes contain a protein capable of binding several cholinergic ligands with the abundance of this protein greatly exceeding that of the "sodium channel" biopolymer isolated from Electrophoru 9 electricus membranes (Agnew and Raftery, 1979) or from garfish olfactory nerve (Henderson and Wang, 1972). While the functional significance of the axonal biopolymer capable of binding cholinergic ligands has not been established, the possibility that it may contain subunits related to at least some of the subunits of the acetylcholine receptor should be explored. The presence of this protein as well as that of acetylcholinesterase (Villegas and Villegas, 1974) on the axolemma, coupled with reports that higher levels of acetylcholine are found in surrounding Schwann cells than in the squid giant axon (Villegas and Jenden, 1979), raises further questions regarding the roles of the axolemmal cholinergic ligand binding material.
We are indebted to the National Science Foundation (BNS-79-06188) for support of this work.
Agnew, W.S. and M.A. Raftery (1979) Solubilized tetrodotoxin binding component from the electroplax of Electrophorus electricus. Stability as a function of mixed lipiddetergent micelle composition. Biochem. 18, 1912-9. Armett, C.J. and J.M. Ritchie (1961) The action of acetylcholine and some related substances on conduction in mammalian non-myelinated nerve fibres. J. Physiol., 155, 372-384. Chang, C.C. and C.J. Lee (1966) Electrophysiological study of neuromuscular blocking action of cobra neurotoxin. Brit. J. Pharmacol. 28, 172-181. Chester, J., T.L. Lentz, J.K. Marquis and H.G. Mautner (1979) Localization of horseradish peroxidase-~-bungarotoxln binding in crustacean axonal membrane vesicles and intact axons. Proc. Nat. Acad. Sci. USA, 76, 3542-3546. Cohen, J.B. and J.P. Changeux (1975) The cholinergic receptor protein in its membrane environment. Ann. Rev. Pharmacol. 15, 83-103. Denburg, J.L. (1972) An axon plasma membrane preparation from the walking legs of the lobster Homarus americanus. Biochim. Biophys. Acta, 282, 453-458. Denburg, J.L., M.E. Eldefrawi and R.D. O'Brien (1972) Macromolecules from lobster axon membranes that bind cholinergic ligands and local anesthetics. Proc. Nat. Acad. Sci. USA 6__9, 177-181. Denburg, J.L. and R.D. O'Brien (1973) Axonalcholinergic binding macromolecule. Response to neuroactive drugs. J. Med. Chem. 16, 57-60. Dettbarn, W.D. (1960) Effect of curare on conduction in myelinated isolated nerve fibres of the frog. Nature, 186, 891-892. Henderson, R. and J.H. Wang (1972) Solubilization of a specific tetrodotoxin-blnding component from garfish olfactory nerve membrane. Biochem. ii, 4565-4569. Heilbronn, E. and T. Bartfai (1978) Muscarinic acetylcholine receptor. Progress Neurobiol. ii, 171-188. Karlin, A. and M. Winnik (1968) Reduction and specific alkylation of the receptor for acetylcholine. Proc. Nat. Acad. Sci. USA 60, 668-674. Lentz, T.L., J.E. Mazurkiewlcz and J. Rosenthal (1977) Cytochemical localization of acetylcholine receptors at the neuromuscular junction by means of horseradish peroxidaselabeled ~-bungarotoxin. Brain Res. 132, 423-442. Marquis, J.K., D~C. Hilt and H.G. Mautner (1977) Direct binding studies of 1251-~-bungarotoxin and -H-quinuclidinyl benzilate interaction with axon plasma membrane fragments. Biochem. Biophys. Res. Comm. 78, 476-482. Marquis, J.K., D.C. Hilt, V.A. Papadeas and H.G. Mautner (1977) Interaction of cholinergic ligands and local anesthetics with plasma membrane fragments from lobster axon. Proc. Nat. Acad. Sci. USA 74, 2278-2282. Nachmansohn, D. (1954) Metabolism and function of the nerve cell. Harvey Lectures, 19531954, Academic Press, New York. pp. 57-99. Rawlins, F.A. and J. Villegas (1978) Autoradiographic localization of acetylcholine receptors in the Schwann cell membrane of the squid nerve fiber. J. Cell. Biol. 77, 371-376. Relter, M.J., D.A. Cowhurn, J.M. Prives and A. Karlin (1972) Affinity labeling of the acetylcholine receptor in the electroplax: Electrophoretic separation in sodium dodecyl sulfate. Proc. Nat. Acad. Sci. USA 69, 1168-1172.
60
H.G.
Mautner, J. E. Jumblatt and J. K. Marquis
Ritchie, J.M. (1967) On the role of acetylcholine in conduction in mammalian non-myelinated nerve fibers. Ann. N. Y. Acad. Sci. 144, 504-516. Rosenberg, P. and II.G. Mautner (1967) Acetylcholine rece tot: Similarity in axons and junctions. Science 155, 1569-1571. Villegas, G. M. and J. Villegas (1974) Acetylcholinesterase localization in the giant nerve fiber of the squid. J. Ultrastruct. Res. 46, 149-163. Villegas, J. and D.J. Jenden (1979) Acetylcholine content of the Schwann cell and axon in the giant nerve fibre of the squid. J. Neurochem. 32, 761-766.