MEETING
1%
REPORT
Out ASW In
25mM KF k
k
I mM Cdl2
1
1OmM CaClp
k
1
_1
I C
3mM C&l2
I,
D
I!-
30”MCaC’2
1
t
FIG. 2. Effect of various concentrations of CaCI, solutions on the memrane potential when 25 mM KF was internally perfused previously. Sudden spike productions are shown during the time course of depolarization in record A and B and during the time course of hyperpolarization in record C and D.
The mechanism of spike production between them is supposed to be common basically. But, in the case of the internal Ca induced spike, the hyperpolarization comes first, and
then the spike occurs without any threshold potential. A further analysis will be done on the nature of the Ca induced spike.
REFERENCES 1. Begenisich, T. and C. Lynch. Effects of internal divalent cations on voltage clamped squid axons. J. gen. Physiol. 63: 675689, 1974. 2. Haaiwara, S. and S. Nakajima. Effects of the intracellular Ca ion-concentration upon the excitability of the muscle fiber membrane of a Barnacle. J. gen. Physio/. 49: 807-818, 1966. 3. Kostyuk, P. G. and 0. A. Krishtal. Effects of calcium and calcium chelating agents on the inward and outward current in the membrane of mollusc neurons. J. Physiol. 270: 569580, 1977. 4. Kmjevic, K. and A. Lisiewicz. Injections of calcium ions into spinal motoneurons. J. Physiol. 225: 363-390, 1972. 5. Kmjevic, K., E. Fuil and R. Werman. Evidence for Ca’+activated K+ conductance in cat spinal motoneurous from intracellular EGTA injections. Can J. Physio/. Pharmacol. 53: 1214-1218, 1975. 6 Meech, R. W. Intracellular calcium injection causes increased potassium conductance in Aplysia nerve cells. Comp. Biochem. Physiol. 42A: 493-499, 1972. 7 Meech, R. W. The sensitivity of Helix aspersa neurons to injected calcium ions. J. Physiol. 237: 259-277.
Chemical Modification
of Membrane
8. Meech, R. W. and F. Strumwasser. Intracellular calcium activates potassium conductance in Ap/ysicr nerve cells. Fedn. Proc. 29: 834, 1970. 9. Oikawa, T., C. S. Spyropoulos, I. Tasaki and T. Teorell. Methods for perfusing the giant axon of LoliRo peulii. Actu. physiol. scant.
53: 19%l%,-l%l.
10. Takahashi, K. and M. Yoshii. Effects of internal free calcium upon the sodium and calcium channels in the tunicate egg analysed by the internal perfusion technique. J. Ph.vsiol. 279: 519-549, 1978. 11. Takenaka, T. and S. Yamagishi. Morphology and electrophysiological properties of squid giant axons perfused intracellularly with protease solution. J. gen. Physiol. 53: 81-%, 1%9. 12. Tasaki, I., T. Takenaka and S. Yamagishi. Abrupt depolarization and bi-ionic potentials in internally perfused squid giant axons. Am. J. Physiol. 215: 152-159, 1968. 13. Tasaki, I., A. Watanabe and T. Takenaka. Resting and action potential of intracellulary perfused squid giant axon. Pot. Nut. Acud. Sci. 48: 1177-1184, 1%2.
Proteins in the Squid Giant Axon
TOHRU YOSHIOKA, TOSHIFUMI TAKENAKA, HIDENORE HORIE, HIROKO INOUE, KIMIE INOMATA AND HIDEAKI HORI Department
of Physiology,
Yokohama
THE membrane labelling techniques currently in use are derived from the chemical modification methodology developed for studying structure function relationships in proteins [3]. Since it was rarely possible to determine which protein is crucial to maintain excitability, several independent labelling
City University,
Yokohama,
Japan
methods have been examined. The major work in the analysis of membrane protein occurred with the application of SDS polyacrylamide electrophoresis to the fractionation of the membrane protein. The use of this method for determining the absolute
MEETING
197
REPORT
molecular weights of proteins can be justifiably criticized, but it still has utility for identifying changes in molecular weight distribution due to some perturbations of the membrane. One must recognize the molecular weight values obtained by this technique may be incorrect because of abnormal binding of proteins, particularly glycoproteins, to detergent [4]. Furthermore, we must consider the possibility that the protein modification could change the electrophoretic mobility of protein. Visualization of the proteins in SDS polyacrylamide gels can be achieved by staining with Coomassie Blue. Glycosylated proteins are usually detected by periodate-Schiff staining, but glycoproteins with low carbohydrate content and without sialic acid may not be detectable by this technique. Several radioactive labelling methods were performed and they are summarized in Table 1. SDS polyacrylamide gel was sliced transversely for gamma counting and scintillation counting. Iodination Reugent
of Nerve Membrune
MW
150 100
.
.
.I......
x 1o-3 60
20 -
40
’
I
- 1.2
0
. 0.8 E L= 0 . 0.6 = d.
Fraction by Bolton-Hunter
. 0.4 o
Two membrane fractions were isolated from homogenates of the giant axons of squid, Doryteuthis bleekeri, by a sequence of differential, discontinuous and gradient centrifugation in sucrose solution, developed by Villegas [2]. According to the explanation of Villegas, Fraction I, which has a density of 1.00 g/ml, was identified as the axolemma and Fraction II, 1.14 g/ml, as the Schwann cell plasma membrane. These two fractions were labelled with ‘ZsI-labelled Bolton-Hunter reagent [ 11. Both of them showed very similar patterns and their peaks were 250, 145,99,84,66,54,34 and 17x 103 daltons.
RELATIVE
MOBILITY
Iodination of Inner und Outer Surface of Squid Giant Axon Membrane by Y-diazotized Iodosulfanilic Acid [6] A non-permeant reagent was used to determine which components existed at the surface of the nerve membrane and which span the membrane. The most advanced reagent of this type is diazotized lZsI-iodosulfanilic acid (DISA) which is commercially available. Labelling by this reagent is limited to a small number of proteins which have tyrosine and histidine residues as reacting groups. External application of the radioactive reagent was carried out by mounting the axon in a paraffin coated chamber, in which axons were incubated in 50 ~1 of bathing solution containing 50 &i of DISA. Internal labelling was performed by perfusion. The solid lines in Fig. 1 (a) and (b) show the densitometric scan of the Coomassie Blue stained 7.5% polyacrylamide gel pattern of axoplasm extruded axon (a) and perfused axon (b). The distribution of ‘2sI-1abe11ed proteins in squeezed axon and perfused axon are presented by the dotted line. The molecular weight of transmembrane proteins determined from these two figures are determined as (250), 145, 66, 54 and 17x 10” daltons.
Photouffinity Lube/l@ of TTX Receptor 3H-Arylazido-PAlanine-TTX
in Squid Axon by
Arylazido-/3-alanine was prepared as described by Guillory et al. [5] and it was coupled to “H-tetrodotoxin by the method described for its coupling to ATP. The reagent was applied to squid giant axon extracellulary, and the preparation was photoirradiated by 500 W tungsten lamp for 5 min.
- 0.6 E c 0 - 0.4 = . =. - 0.2 O
0.5
0 RELATIVE
1.0
MOBILITY
FIG. 1. (a) Molecular weight distribution on 7.5% SDS-polyacrylamide gels of proteins in axoplasm extruded axon (solid line) and (““I)-labelled protein (dotted line), after the giant axon had been incubated for 30 min in ASW containing 50 $Zi of DISA. (b) Molecular weight distribution on 7.5% SDS gels of proteins in perfused axon (solid line) and (‘251)-labelled protein (dotted line).
MEETING REPORT
198 TABLE LISTINGOFMEMBRANE-LABELLING
Primary Functional Groups Reacted
Bolton-Hunter
B-H
NH,
2‘2.q
Diazotized Sulfanilic acid
DSA
his. tyr, SH
I3.5 I
Thiamine
V.B,
Adenosine Triphosphate
ATP
ser, thr
:!2P
LPO-I
his, tyr
I?.?,
AAA-TTX
-CH
“H
Enzymatic Iodination Arylazido-flalanine-TTX complex
Mw x 145 I
3
$
99 I
84 66 54 III I
DSA
V.01
i;
$$
to-3 34
17
I1
*3
D
,\
n
.,
w
n
n
vv
iI n
nn
w
u
&
FIG. 2. Schematic summary of ious techniques. Branches on parallel lines means membrane spond to exterior and
Radio Isotopes
’ ‘C
73
B-H
ANDTHEIRREAcTIvITY
Abbreviations
Reagent Name
250 1
I
REAGENTS
About 20 axons were minced and homogenized to submit to the membrane fraction differentiation. Membrane proteins were analyzed by SDS polyacrylamide gel. Dominant labeiled peak was found at 250x IO” daltons. Membrane proteins labelled by various techniques are summarized in Fig. 2 schematically, in which parallel line means membrane. Upper part and lower part of the membrane stand for exterior and interior of the membrane, respectively. Hence, channel proteins might be contained in the Iabelled membrane proteins shown in Fig. 2.
membrane proteins labelled by varthe protein represents sugar. Two and its upper and lower part correinterior of it, respectively.
REFERENCES 4. Grefrath, S. P. and J. A. Reynolds. The molecular weight of the 1. Bolton, A. E. and W. M. Hunter. The labelling of proteins to major glycoprotein from the human erythrocyte membrane. high specific radioactivities by conjugation to a ““I-containing Proc. Note. Awtl. Sci. U.S.A. 71: 3913-3916, 1974. acylating agent. Biocherrr. J. 133: 529-539, 1973. 5. Guillory, R. J., M. D. Rayner and J. S. D’Arrigo. Covalent label2. Camejo, G., G. M. Villegas, F. V. Barnola and R. Villegas. ing of the tetrodotoxin receptor in excitable membranes. Sciet7cr Characterization of two different membrane fractions isolated 1%: 883-885, 1977. from the first stellar nerves of the squid Dosiciicus gigcts. 6. Yoshioka, T., T. Takenaka, H. Horie, H. Inoue and K. fnomata. 3i~~~~zil~l.Bioplr,v&cr Acrtt 193: 247-259, 1%9. Dete~inatjon of transmembrane protein with diazotized (‘y”I)3. Caraway, K. L. Covalent labeling of membranes. ~j~~~.~7;~??. iodosuifanilic acid in the giant squid axon. Proc. Jrip. Accra!. 54B: BiophyGcct Actcc 415: 37910, 1975. 310-315, 1978.
1%
REPORT
Out ASW In
25mM KF k
k
I mM Cdl2
1
1OmM CaClp
k
1
_1
I C
3mM C&l2
I,
D
I!-
30”MCaC’2
1
t
FIG. 2. Effect of various concentrations of CaCI, solutions on the memrane potential when 25 mM KF was internally perfused previously. Sudden spike productions are shown during the time course of depolarization in record A and B and during the time course of hyperpolarization in record C and D.
The mechanism of spike production between them is supposed to be common basically. But, in the case of the internal Ca induced spike, the hyperpolarization comes first, and
then the spike occurs without any threshold potential. A further analysis will be done on the nature of the Ca induced spike.
REFERENCES 1. Begenisich, T. and C. Lynch. Effects of internal divalent cations on voltage clamped squid axons. J. gen. Physiol. 63: 675689, 1974. 2. Haaiwara, S. and S. Nakajima. Effects of the intracellular Ca ion-concentration upon the excitability of the muscle fiber membrane of a Barnacle. J. gen. Physio/. 49: 807-818, 1966. 3. Kostyuk, P. G. and 0. A. Krishtal. Effects of calcium and calcium chelating agents on the inward and outward current in the membrane of mollusc neurons. J. Physiol. 270: 569580, 1977. 4. Kmjevic, K. and A. Lisiewicz. Injections of calcium ions into spinal motoneurons. J. Physiol. 225: 363-390, 1972. 5. Kmjevic, K., E. Fuil and R. Werman. Evidence for Ca’+activated K+ conductance in cat spinal motoneurous from intracellular EGTA injections. Can J. Physio/. Pharmacol. 53: 1214-1218, 1975. 6 Meech, R. W. Intracellular calcium injection causes increased potassium conductance in Aplysia nerve cells. Comp. Biochem. Physiol. 42A: 493-499, 1972. 7 Meech, R. W. The sensitivity of Helix aspersa neurons to injected calcium ions. J. Physiol. 237: 259-277.
Chemical Modification
of Membrane
8. Meech, R. W. and F. Strumwasser. Intracellular calcium activates potassium conductance in Ap/ysicr nerve cells. Fedn. Proc. 29: 834, 1970. 9. Oikawa, T., C. S. Spyropoulos, I. Tasaki and T. Teorell. Methods for perfusing the giant axon of LoliRo peulii. Actu. physiol. scant.
53: 19%l%,-l%l.
10. Takahashi, K. and M. Yoshii. Effects of internal free calcium upon the sodium and calcium channels in the tunicate egg analysed by the internal perfusion technique. J. Ph.vsiol. 279: 519-549, 1978. 11. Takenaka, T. and S. Yamagishi. Morphology and electrophysiological properties of squid giant axons perfused intracellularly with protease solution. J. gen. Physiol. 53: 81-%, 1%9. 12. Tasaki, I., T. Takenaka and S. Yamagishi. Abrupt depolarization and bi-ionic potentials in internally perfused squid giant axons. Am. J. Physiol. 215: 152-159, 1968. 13. Tasaki, I., A. Watanabe and T. Takenaka. Resting and action potential of intracellulary perfused squid giant axon. Pot. Nut. Acud. Sci. 48: 1177-1184, 1%2.
Proteins in the Squid Giant Axon
TOHRU YOSHIOKA, TOSHIFUMI TAKENAKA, HIDENORE HORIE, HIROKO INOUE, KIMIE INOMATA AND HIDEAKI HORI Department
of Physiology,
Yokohama
THE membrane labelling techniques currently in use are derived from the chemical modification methodology developed for studying structure function relationships in proteins [3]. Since it was rarely possible to determine which protein is crucial to maintain excitability, several independent labelling
City University,
Yokohama,
Japan
methods have been examined. The major work in the analysis of membrane protein occurred with the application of SDS polyacrylamide electrophoresis to the fractionation of the membrane protein. The use of this method for determining the absolute
MEETING
197
REPORT
molecular weights of proteins can be justifiably criticized, but it still has utility for identifying changes in molecular weight distribution due to some perturbations of the membrane. One must recognize the molecular weight values obtained by this technique may be incorrect because of abnormal binding of proteins, particularly glycoproteins, to detergent [4]. Furthermore, we must consider the possibility that the protein modification could change the electrophoretic mobility of protein. Visualization of the proteins in SDS polyacrylamide gels can be achieved by staining with Coomassie Blue. Glycosylated proteins are usually detected by periodate-Schiff staining, but glycoproteins with low carbohydrate content and without sialic acid may not be detectable by this technique. Several radioactive labelling methods were performed and they are summarized in Table 1. SDS polyacrylamide gel was sliced transversely for gamma counting and scintillation counting. Iodination Reugent
of Nerve Membrune
MW
150 100
.
.
.I......
x 1o-3 60
20 -
40
’
I
- 1.2
0
. 0.8 E L= 0 . 0.6 = d.
Fraction by Bolton-Hunter
. 0.4 o
Two membrane fractions were isolated from homogenates of the giant axons of squid, Doryteuthis bleekeri, by a sequence of differential, discontinuous and gradient centrifugation in sucrose solution, developed by Villegas [2]. According to the explanation of Villegas, Fraction I, which has a density of 1.00 g/ml, was identified as the axolemma and Fraction II, 1.14 g/ml, as the Schwann cell plasma membrane. These two fractions were labelled with ‘ZsI-labelled Bolton-Hunter reagent [ 11. Both of them showed very similar patterns and their peaks were 250, 145,99,84,66,54,34 and 17x 103 daltons.
RELATIVE
MOBILITY
Iodination of Inner und Outer Surface of Squid Giant Axon Membrane by Y-diazotized Iodosulfanilic Acid [6] A non-permeant reagent was used to determine which components existed at the surface of the nerve membrane and which span the membrane. The most advanced reagent of this type is diazotized lZsI-iodosulfanilic acid (DISA) which is commercially available. Labelling by this reagent is limited to a small number of proteins which have tyrosine and histidine residues as reacting groups. External application of the radioactive reagent was carried out by mounting the axon in a paraffin coated chamber, in which axons were incubated in 50 ~1 of bathing solution containing 50 &i of DISA. Internal labelling was performed by perfusion. The solid lines in Fig. 1 (a) and (b) show the densitometric scan of the Coomassie Blue stained 7.5% polyacrylamide gel pattern of axoplasm extruded axon (a) and perfused axon (b). The distribution of ‘2sI-1abe11ed proteins in squeezed axon and perfused axon are presented by the dotted line. The molecular weight of transmembrane proteins determined from these two figures are determined as (250), 145, 66, 54 and 17x 10” daltons.
Photouffinity Lube/l@ of TTX Receptor 3H-Arylazido-PAlanine-TTX
in Squid Axon by
Arylazido-/3-alanine was prepared as described by Guillory et al. [5] and it was coupled to “H-tetrodotoxin by the method described for its coupling to ATP. The reagent was applied to squid giant axon extracellulary, and the preparation was photoirradiated by 500 W tungsten lamp for 5 min.
- 0.6 E c 0 - 0.4 = . =. - 0.2 O
0.5
0 RELATIVE
1.0
MOBILITY
FIG. 1. (a) Molecular weight distribution on 7.5% SDS-polyacrylamide gels of proteins in axoplasm extruded axon (solid line) and (““I)-labelled protein (dotted line), after the giant axon had been incubated for 30 min in ASW containing 50 $Zi of DISA. (b) Molecular weight distribution on 7.5% SDS gels of proteins in perfused axon (solid line) and (‘251)-labelled protein (dotted line).
MEETING REPORT
198 TABLE LISTINGOFMEMBRANE-LABELLING
Primary Functional Groups Reacted
Bolton-Hunter
B-H
NH,
2‘2.q
Diazotized Sulfanilic acid
DSA
his. tyr, SH
I3.5 I
Thiamine
V.B,
Adenosine Triphosphate
ATP
ser, thr
:!2P
LPO-I
his, tyr
I?.?,
AAA-TTX
-CH
“H
Enzymatic Iodination Arylazido-flalanine-TTX complex
Mw x 145 I
3
$
99 I
84 66 54 III I
DSA
V.01
i;
$$
to-3 34
17
I1
*3
D
,\
n
.,
w
n
n
vv
iI n
nn
w
u
&
FIG. 2. Schematic summary of ious techniques. Branches on parallel lines means membrane spond to exterior and
Radio Isotopes
’ ‘C
73
B-H
ANDTHEIRREAcTIvITY
Abbreviations
Reagent Name
250 1
I
REAGENTS
About 20 axons were minced and homogenized to submit to the membrane fraction differentiation. Membrane proteins were analyzed by SDS polyacrylamide gel. Dominant labeiled peak was found at 250x IO” daltons. Membrane proteins labelled by various techniques are summarized in Fig. 2 schematically, in which parallel line means membrane. Upper part and lower part of the membrane stand for exterior and interior of the membrane, respectively. Hence, channel proteins might be contained in the Iabelled membrane proteins shown in Fig. 2.
membrane proteins labelled by varthe protein represents sugar. Two and its upper and lower part correinterior of it, respectively.
REFERENCES 4. Grefrath, S. P. and J. A. Reynolds. The molecular weight of the 1. Bolton, A. E. and W. M. Hunter. The labelling of proteins to major glycoprotein from the human erythrocyte membrane. high specific radioactivities by conjugation to a ““I-containing Proc. Note. Awtl. Sci. U.S.A. 71: 3913-3916, 1974. acylating agent. Biocherrr. J. 133: 529-539, 1973. 5. Guillory, R. J., M. D. Rayner and J. S. D’Arrigo. Covalent label2. Camejo, G., G. M. Villegas, F. V. Barnola and R. Villegas. ing of the tetrodotoxin receptor in excitable membranes. Sciet7cr Characterization of two different membrane fractions isolated 1%: 883-885, 1977. from the first stellar nerves of the squid Dosiciicus gigcts. 6. Yoshioka, T., T. Takenaka, H. Horie, H. Inoue and K. fnomata. 3i~~~~zil~l.Bioplr,v&cr Acrtt 193: 247-259, 1%9. Dete~inatjon of transmembrane protein with diazotized (‘y”I)3. Caraway, K. L. Covalent labeling of membranes. ~j~~~.~7;~??. iodosuifanilic acid in the giant squid axon. Proc. Jrip. Accra!. 54B: BiophyGcct Actcc 415: 37910, 1975. 310-315, 1978.
