MEETING
REPORT
Physiological
187
Role of Histidine Residues in the Inner Surface Membrane Squid Giant Axon
TOSHIFUMI TAKENAKA, Depurtment
of the
TOHRU YOSHIOKA, HIDENORI HORIE, HIROKO INOUE AND KIMIE INOMATA of Physiology,
Yokohama
THE phenomenon of membrane excitability was studied by modifying the imidazole group through the exposure of the interior of squid axons to potent photo-oxidizing agents. The giant axons of squid, Dorytheuthis bleekeri, were intracellularly perfused with 400 mM KF solution and voltage-clamped with an axial wire system. For the photooxidation experiments, the photo-sensitive reagent, rose bengal, was passed through an axon which was illuminated by a 100 W quartz-iodine lamp mounted 15 cm above the axon. A heat cut filter was mounted between the axon and the lamp to reduce the direct thermal effect of the light. After 0.1-0.2 PM rose bengal was introduced into the perfusion solution intracellularly, the axon was illuminated with the lamp. The resting potential was depolarized about 4 mV, but the excitability was completely maintained. Then the rose bengal was washed out with dye free perfusion solution before conducting the voltage-clamp experiments. After photo-oxidation the amplitudes of the delayed outward currents were reduced 40-50%, while the peak early currents were decreased 10%. Figure 1 shows the effects of the photo-oxidation on the potassium currents after complete separation of sodium and potassium currents by application of TTX. The amplitudes of the potassium currents were reduced 40-45% after photo-oxidation. These results indicate that the histidine residue could be associated with the membrane components involved in the control of the potassium current [l, 2, 5, 61. The diazotized (““I)-iodosulfanilic acid (DISA) is a nonpermeant reagent for the membrane protein and labelling by this reagent is limited to proteins which have histidine and tyrosine residues as reacting groups. It is also known that the imidazole group in histidine is selectively oxidized by rose bengal at pH=7.2 [3]. Therefore the experiments using iodosulfanilic acid in combination with rose bengal should give useful information about the molecular structure of potassium channels. Labelling the internal surface of the membrane protein by perfusion was carried out in the following manner. The solution containing radioactive DISA was perfused slowly for 30 min through the axon which had been cleaned of the surrounding connective tissue. Then the axon was quickly transferred to a glass homogenizer tube and homogenized. The samples were dissolved in a 5% SDS solution and then the protein in this solution was precipitated by the addition of cold 10% trichloroacetic acid (TCA). To remove nonreacting TCA soluble radioactive material, the mixture was centrifuged at 12,000 g for 10 min. The TCA pellet was then washed twice with a mixture of ethanol-ether (1:l) and once with ether to remove lipids. The pellet was dissolved in a mixture containing 2% sodium carbonate and 5% SDS and was reduced with 2% 2-P-mercaptethanol for
City University,
Yokohama,
11X
CONTROL +;: +&4
J-7 J-----I
+55
J---Y
+25
-j------
+ -
9
-f----
TlX+RBtLISNT
-r_ J---T
r._
-
-
7-
r-
Y-
-_
1 -r-
-31
Japan
-_
v-
14mA/cm?
zsec
IbmA/cmz-
x*cc
1 bmA/cm? Get
FIG. 1. Effects of photo-oxidation on potassium currents. Depolarization to levels given V,=-75 mV. subsequent gel analysis. SDS gel electrophoresis on 7.5% polyacrylamide gels were performed according to Neville’s method [4]. At the end of the electrophoretic run, the gel was stained with Coomassie blue and destained. The destained gels were routinely scanned at 560 nm with a Beckman Model 35 gel scanner. The gel was sliced into 1.3 mm thick sections using a Canalco gel slicer and the radioactivity of each slice of gel was counted using a Packard auto gamma spectrometer. Figure 2 shows the molecular weight distribution on 7.5% SDS gel of proteins in the perfused axon. The solid line in Fig. 2 shows the desitometric scan and the major peaks are 145,99,84,66,54 and 17x lo3 daltons. The dotted line shows the ‘““I-labelled proteins have molecular weights of 73000 and 66000 daltons and also smaller peaks at 145,54 and 17x 10” daltons. The possible contamination of axoplasm was examined carefully by in vitro labelling of axoplasm and it was concluded that axoplasm contributed only slightly to the result. The effects of the photo-oxidation on the release of labelled imidazole into the perfusate are shown in Fig. 3. After 30 min labelling period, the perfusion solution was switched to one containing 5 PM of rose bengal. The axon was perfused with this solution for 10 min in the dark, then illuminated with a 100 W quartz-iodine lamp for 5 min. After irradiation, the washing was continued for a further 2 min in the dark. Drops of perfusate were collected continuously and their radioactivity was counted. An increment in the radioactivity of the perfusate was observed during the irradiation period (Fig. 3). This means that ‘*“I-labelled imidazole group of the membrane protein was destroyed by photo-oxidation and left the axon. In the perfusate, no
MEETING
188
IN THEOARK
YI m
K
ILLUMINATED
uf m
REPORT
‘~A~~ ii
- 0.8
= 0.6 E = 0
I
0
RELATIVE
115 MO6lllTY
1.0
FIG. 2. Molecular weight distribution on 7.5% SDS gel of proteins in perfused axon (solid line) and (‘251)-labelled protein (dotted line), after introducing the radioactive reagent intracellularly. Ordinate: Percent of total ~dioactivity on the SDS gel. Lower abscissa: Mobility relative to cytochrome C (= 1.0). Upper abscissa: Molecular weight determined by standard protein.
PERFUSATE radio-labelled proteins were found. About 50-7@‘%of the covalently labelled proteins was released from the inner surface of the membrane by the photo-oxidation. Rose bengal treated axons were submitted to SDS polyacrylamide gel electrophoresis and their labelled patterns were compared with the control pattern shown in Fig. 2. If the reduction in radioactivity of the membrane protein occurred in the special molecular weight protein, the change in the gel pattern of the radioactivity should be noticeable. But the photo-oxidation did not change the molecular weight distribution pattern obtained from SDS electrophoresis of radioactive membrane proteins. These results could be explained if the potassium channel becomes one protein which is composed of four
NUMBER
FIG. 3. Effects of photo-oxidation in releasing labeiled imidazole into the per&ate. Abscissa is the perfusate number. Its volume was approximately SO~1 and was collected for every 1 min.
peaks by dissolving action of SDS and mercaptethanol, or if the channel proteins become several proteins having the same concentration of histidine residues in each peak. These experimental results have shown that the covalent labelling method used in combination with the photo-oxidation technique is an effective means of elucidating the configuration of imidazole in a transmembrane protein of the squid giant axon [7].
REFERENCES 1. Baumgold, J., G. Matsumoto and I. Tasaki. Biochemical studies of nerve excitability: The use of protein m~~ying reagents for characterizing sites involved in nerve excitation. J. Neurochem. 30: 91-100, 1978. 2. Clark, H. R. and A. Strickholm. Evidence for a conformational change in nerve membrane with depolarization. Narure 234: 470-471, 1971. 3. Means, G. E. and R. E. Feeney. Chemical Modification qf Pmrein. New York: Holden-Day, Inc., 1971, p. 206. 4. Neville, D. M. Molecular weight dete~ination of proteindodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. 1. Biol. Chem. 246: 6328-6334, 1971.
5. Shraget, P. Ionic conductance changes in voltage clamped crayfish axons at low pH. J. gerr. Physiof. 6& 6f6-690, 1974. 6. Shrager, P. Specific chemical groups involved in the control of ionic conductance in nerve. Ann. N. Y. Acad. 5%. 264: 293-303, 1975. 7. Yoshioka, T., T. Takenaka, H. Horie, II. Inoue and K. Inomata. Determination of transmembrane protein with diazotized (‘““I)iodosulfanilic acid in the squid giant axon. Proc. Jup. Acud. 54B: 310-315, 1978.
REPORT
Physiological
187
Role of Histidine Residues in the Inner Surface Membrane Squid Giant Axon
TOSHIFUMI TAKENAKA, Depurtment
of the
TOHRU YOSHIOKA, HIDENORI HORIE, HIROKO INOUE AND KIMIE INOMATA of Physiology,
Yokohama
THE phenomenon of membrane excitability was studied by modifying the imidazole group through the exposure of the interior of squid axons to potent photo-oxidizing agents. The giant axons of squid, Dorytheuthis bleekeri, were intracellularly perfused with 400 mM KF solution and voltage-clamped with an axial wire system. For the photooxidation experiments, the photo-sensitive reagent, rose bengal, was passed through an axon which was illuminated by a 100 W quartz-iodine lamp mounted 15 cm above the axon. A heat cut filter was mounted between the axon and the lamp to reduce the direct thermal effect of the light. After 0.1-0.2 PM rose bengal was introduced into the perfusion solution intracellularly, the axon was illuminated with the lamp. The resting potential was depolarized about 4 mV, but the excitability was completely maintained. Then the rose bengal was washed out with dye free perfusion solution before conducting the voltage-clamp experiments. After photo-oxidation the amplitudes of the delayed outward currents were reduced 40-50%, while the peak early currents were decreased 10%. Figure 1 shows the effects of the photo-oxidation on the potassium currents after complete separation of sodium and potassium currents by application of TTX. The amplitudes of the potassium currents were reduced 40-45% after photo-oxidation. These results indicate that the histidine residue could be associated with the membrane components involved in the control of the potassium current [l, 2, 5, 61. The diazotized (““I)-iodosulfanilic acid (DISA) is a nonpermeant reagent for the membrane protein and labelling by this reagent is limited to proteins which have histidine and tyrosine residues as reacting groups. It is also known that the imidazole group in histidine is selectively oxidized by rose bengal at pH=7.2 [3]. Therefore the experiments using iodosulfanilic acid in combination with rose bengal should give useful information about the molecular structure of potassium channels. Labelling the internal surface of the membrane protein by perfusion was carried out in the following manner. The solution containing radioactive DISA was perfused slowly for 30 min through the axon which had been cleaned of the surrounding connective tissue. Then the axon was quickly transferred to a glass homogenizer tube and homogenized. The samples were dissolved in a 5% SDS solution and then the protein in this solution was precipitated by the addition of cold 10% trichloroacetic acid (TCA). To remove nonreacting TCA soluble radioactive material, the mixture was centrifuged at 12,000 g for 10 min. The TCA pellet was then washed twice with a mixture of ethanol-ether (1:l) and once with ether to remove lipids. The pellet was dissolved in a mixture containing 2% sodium carbonate and 5% SDS and was reduced with 2% 2-P-mercaptethanol for
City University,
Yokohama,
11X
CONTROL +;: +&4
J-7 J-----I
+55
J---Y
+25
-j------
+ -
9
-f----
TlX+RBtLISNT
-r_ J---T
r._
-
-
7-
r-
Y-
-_
1 -r-
-31
Japan
-_
v-
14mA/cm?
zsec
IbmA/cmz-
x*cc
1 bmA/cm? Get
FIG. 1. Effects of photo-oxidation on potassium currents. Depolarization to levels given V,=-75 mV. subsequent gel analysis. SDS gel electrophoresis on 7.5% polyacrylamide gels were performed according to Neville’s method [4]. At the end of the electrophoretic run, the gel was stained with Coomassie blue and destained. The destained gels were routinely scanned at 560 nm with a Beckman Model 35 gel scanner. The gel was sliced into 1.3 mm thick sections using a Canalco gel slicer and the radioactivity of each slice of gel was counted using a Packard auto gamma spectrometer. Figure 2 shows the molecular weight distribution on 7.5% SDS gel of proteins in the perfused axon. The solid line in Fig. 2 shows the desitometric scan and the major peaks are 145,99,84,66,54 and 17x lo3 daltons. The dotted line shows the ‘““I-labelled proteins have molecular weights of 73000 and 66000 daltons and also smaller peaks at 145,54 and 17x 10” daltons. The possible contamination of axoplasm was examined carefully by in vitro labelling of axoplasm and it was concluded that axoplasm contributed only slightly to the result. The effects of the photo-oxidation on the release of labelled imidazole into the perfusate are shown in Fig. 3. After 30 min labelling period, the perfusion solution was switched to one containing 5 PM of rose bengal. The axon was perfused with this solution for 10 min in the dark, then illuminated with a 100 W quartz-iodine lamp for 5 min. After irradiation, the washing was continued for a further 2 min in the dark. Drops of perfusate were collected continuously and their radioactivity was counted. An increment in the radioactivity of the perfusate was observed during the irradiation period (Fig. 3). This means that ‘*“I-labelled imidazole group of the membrane protein was destroyed by photo-oxidation and left the axon. In the perfusate, no
MEETING
188
IN THEOARK
YI m
K
ILLUMINATED
uf m
REPORT
‘~A~~ ii
- 0.8
= 0.6 E = 0
I
0
RELATIVE
115 MO6lllTY
1.0
FIG. 2. Molecular weight distribution on 7.5% SDS gel of proteins in perfused axon (solid line) and (‘251)-labelled protein (dotted line), after introducing the radioactive reagent intracellularly. Ordinate: Percent of total ~dioactivity on the SDS gel. Lower abscissa: Mobility relative to cytochrome C (= 1.0). Upper abscissa: Molecular weight determined by standard protein.
PERFUSATE radio-labelled proteins were found. About 50-7@‘%of the covalently labelled proteins was released from the inner surface of the membrane by the photo-oxidation. Rose bengal treated axons were submitted to SDS polyacrylamide gel electrophoresis and their labelled patterns were compared with the control pattern shown in Fig. 2. If the reduction in radioactivity of the membrane protein occurred in the special molecular weight protein, the change in the gel pattern of the radioactivity should be noticeable. But the photo-oxidation did not change the molecular weight distribution pattern obtained from SDS electrophoresis of radioactive membrane proteins. These results could be explained if the potassium channel becomes one protein which is composed of four
NUMBER
FIG. 3. Effects of photo-oxidation in releasing labeiled imidazole into the per&ate. Abscissa is the perfusate number. Its volume was approximately SO~1 and was collected for every 1 min.
peaks by dissolving action of SDS and mercaptethanol, or if the channel proteins become several proteins having the same concentration of histidine residues in each peak. These experimental results have shown that the covalent labelling method used in combination with the photo-oxidation technique is an effective means of elucidating the configuration of imidazole in a transmembrane protein of the squid giant axon [7].
REFERENCES 1. Baumgold, J., G. Matsumoto and I. Tasaki. Biochemical studies of nerve excitability: The use of protein m~~ying reagents for characterizing sites involved in nerve excitation. J. Neurochem. 30: 91-100, 1978. 2. Clark, H. R. and A. Strickholm. Evidence for a conformational change in nerve membrane with depolarization. Narure 234: 470-471, 1971. 3. Means, G. E. and R. E. Feeney. Chemical Modification qf Pmrein. New York: Holden-Day, Inc., 1971, p. 206. 4. Neville, D. M. Molecular weight dete~ination of proteindodecyl sulfate complexes by gel electrophoresis in a discontinuous buffer system. 1. Biol. Chem. 246: 6328-6334, 1971.
5. Shraget, P. Ionic conductance changes in voltage clamped crayfish axons at low pH. J. gerr. Physiof. 6& 6f6-690, 1974. 6. Shrager, P. Specific chemical groups involved in the control of ionic conductance in nerve. Ann. N. Y. Acad. 5%. 264: 293-303, 1975. 7. Yoshioka, T., T. Takenaka, H. Horie, II. Inoue and K. Inomata. Determination of transmembrane protein with diazotized (‘““I)iodosulfanilic acid in the squid giant axon. Proc. Jup. Acud. 54B: 310-315, 1978.
