- Email: [email protected]
Effects of PKC and PKA phosphorylation on desensitization of nicotinic acetylcholine receptors
Brain Research 812 Ž1998. 242–245
Short communication
Effects of PKC and PKA phosphorylation on desensitization of nicotinic acetylcholine receptors Tomoyuki Nishizaki a
a, )
, Katumi Sumikawa
b
Department of Physiology, Kobe UniÕersity School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan b Department of Psychobiology, UniÕersity of California, IrÕine, CA 92717-4550, USA
Abstract The present study was designed to assess the effect of protein kinase C ŽPKC. and cAMP-dependent protein kinase ŽPKA. on desensitization of Torpedo acetylcholine ŽACh. receptors by analyzing summated macroscopic currents in an outside-out patch–clamp configuration. Normal ACh receptors desensitized with a fast Ž6 ms. and slow time constant Ž104 ms.. There was no significant difference in the current decay time between normal ACh receptors and mutant ACh receptors that possibly mimics PKC phosphorylation of the receptors. The selective PKC inhibitor, PKCl, prolonged the rate of desensitization of normal ACh receptors, and the similar effect was obtained with mutant ACh receptors lacking PKC phosphorylation sites. Phosphorylation of normal ACh receptors by the catalytic subunit of PKA or mutant ACh receptors that possibly mimic PKA phosphorylation of the receptors increased the rate of desensitization, but, in contrast, the receptors lacking PKA phosphorylation sites prolonged the current decay time. The results of the present study demonstrate that PKC or PKA phosphorylation of ACh receptors accelerates the rate of desensitization. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Torpedo ACh receptor; Outside-out patch; Protein phosphorylation; Desensitization; Site-directed mutagenesis
Protein phosphorylation plays a role in modulation of the neurotransmitter receptors, and Torpedo acetylcholine ŽACh. receptors are recognized to have phosphorylation sites for at least three protein kinases such as protein kinase C ŽPKC., cAMP-dependent protein kinase ŽPKA., and tyrosine kinase ŽPTK. w5,6x. Although PKA or PTK phosphorylation of nicotinic ACh receptors is suggested to accelerate the rate of the current decay time, the effect of PKC phosphorylation of the receptors on desensitization still remains controversial. One reason is that phorbol esters, which are widely used to activate PKC, have a direct blocking effect on the receptor channels w7x, and therefore, the actual effect of PKC phosphorylation of ACh receptors on desensitization may be masked. The present study investigated the effect of PKC or PKA phosphorylation of ACh receptors on desensitization by analyzing macroscopic currents through normal and mutant Torpedo ACh receptors expressed in Xenopus oocyte. We show
) Corresponding author. [email protected]
Fax:
q 81-78-341-5732;
E-mail:
here that PKC and PKA phosphorylation of the receptors increases the rate of desensitization. mRNAs coding for the Torpedo ACh receptor a , b, g, and d subunits were synthesized by in vitro transcription as described previously w8x. The mutant a and d subunits were constructed by site-directed mutagenesis w4x: Ser 333 on the a subunit Žma q PKCrNA333. and Ser 377 on the d subunit Žmd q PKCrNA377. were replaced with the negatively charged amino acid residue, Asp. To construct mutant ACh receptors lacking PKC phosphorylation sites, Ser 333 on the a subunit Žma DPKCrSer333. and Ser 377 on the d subunit ŽmdDPKCrSer377. were replaced with Ala. For mutant ACh receptors linked to the PKA phosphorylation sites, Ser 353,354 on the g subunit and Ser 361,362 on the d subunit were replaced with aspartic acid residues Žmg q PKArNA353,354 and md q PKArNA361,362. or with Gly and Ala Ž m gD PKArSer353,354 and mdDPKArSer361,362.. Xenopus oocytes were manually separated from the ovary, and incubated overnight in Barth’s solution Žin mM: 88 NaCl, 1 KCl, 2.4 NaHCO 3 , 0.82 MgSO4 , 0.33 CaŽNO 2 . 2 , 0.41 CaCl 2 , and 7.5 Tris, pH 7.6. after collagenase Ž0.5 mgrml. treatment. Oocytes were injected with the normal or mutant ACh receptor
0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 8 3 6 - 1
T. Nishizaki, K. Sumikawar Brain Research 812 (1998) 242–245
subunit mRNAs, and incubated at 188C. The injected oocytes were transferred to the recording chamber a day after incubation and continuously superfused at room temperature Ž20 to 228C. in standard from Ringer’s solution Žin mM: 115 NaCl, 2 KCl, 1.8 CaCl 2 , and 5 HEPES, pH 7.0.. Outside-out patches were made from oocytes using the patch electrode-filling solution Žin mM: 80 KF, 20 KCl, 10 K-EGTA, 10 HEPES, and 0.5 ATP, pH 7.2.. ACh was applied by 1-s pulses using a piezo-driven liquid filament switch w2,3x. Macroscopic currents from excised patches were recorded with an Axopatch-1D amplifier ŽAxon Instruments, USA. ŽFig. 1A.. Summated macroscopic currents ŽFig. 1B. were fitted by two exponential curves and each time constant Žt f and ts . of the current decay was calculated using a program provided by H. Chang. ACh Ž100 mM. produced currents at a holding potential of y60 mV. Normal ACh receptors desensitized with a fast time constant Žt f . of 6 " 3 ms and a slow time constant Žts . of 104 " 63 ms Žcontrol. ŽFig. 2A and Table 1.. Currents through either ma q PKCrNA333bgd or abgmd q PKCrNA377, which possibly mimics PKC phosphorylation of the receptors, decayed with t f and ts of 5 " 0 and 213 " 115 ms or 6 " 0 and 201 " 155 ms, respectively ŽFig. 3A and Table 1., but there was no
243
Fig. 2. Effects of PKC or PKA on desensitization of ACh receptors. Outside-out patches were made from oocytes expressing normal Torpedo ACh receptors. PKCl Ž ; 500 mgrml final concentration. or PKA Ž ;1 mgrml final concentration. was added to the patch electrode-filing solution. Summated currents obtained from 20 macroscopic currents elicited by ACh Ž100 mM. are illustrated and fitted by two exponential curves. The holding potential was y60 mV.
significant difference in time constant between these mutant ACh receptors and normal receptors. In contrast, mutant ACh receptors lacking PKC phosphorylation sites on both the a and d subunits Žt f s 9 " 4 ms and ts s 352 " 117 ms. or on the d subunit alone Žt f s 14 " 2 ms and ts s 398 " 180 ms. desensitized more slowly than normal receptors, although the decay of currents through ma DPKCrSer333bgd Žt f s 6 " 2 ms and ts s 260 " 127 ms. was not affected ŽFig. 3A and Table 1., suggesting the possibility that PKC dephosphorylation of the receptors employs a decrease in the rate of desensitization and that the responsible site is the d subunit. Then, one would assume that mutant ACh receptors mimicking PKC phosphorylation accelerates the rate of desensitization as compared with normal ACh receptors. However, this is not the case here. A possible explanation is that ACh receptors are phosphorylated by PKC activated via an ACh receptormediated signaling pathway and that therefore, normal ACh receptors desensitize rapidly as same as ma q PKCrNA333bg and md q PKCrNA377. In support of
Table 1 Effects of protein phosphorylation on desensitization of ACh receptors Time constant Žms.
Fig. 1. ACh receptor currents induced by a piezo-driven liquid filament switch. Outside-out patches were made from oocytes expressing Torpedo ACh receptors. ACh Ž100 mM. was applied by 1-s pulses using a piezo-driven liquid filament switch. The arrow indicates switching artifact. Five macroscopic currents evoked at a holding potential of y60 mV are illustrated in ŽA. and summated current obtained from five recordings are shown in ŽB..
N
tf
tS
abgd Control qPKCl qPKA
6"3 10"7 ) ) 4"1)
104"63 188"41 33"1) )
7 6 7
ma qPKCrNA333 mdqPKCrNA377 ma DPKCrSer333 mdDPKCrSer377 ma DPKCrSer333mdDPKCrSer377
5"0 6"0 6"2 14"2 ) ) 9"4 ) )
213"115 201"155 260"127 398"180 ) 352"117 )
5 4 12 6 5
3"1) ) 3"1) ) 5"1 8"3
37"24 ) ) 53"15 ) ) 411"94 ) ) 230"63 ) )
11 3 10 9
mgqPKArNA353,354 mdqPKArNA361,362 mgDPKArSer353,354 mdDPKArSer361,362 )
P - 0.1, ence test.
))
P - 0.01, ANOVA with Fisher’s least-significance differ-
244
T. Nishizaki, K. Sumikawar Brain Research 812 (1998) 242–245
Fig. 3. Effects of PKC ŽA. or PKA ŽB. phosphorylation of ACh receptors on desensitization. Outside-out patches were made from oocytes expressing mutant Torpedo ACh receptors as indicated. Summated currents obtained from 20 macroscopic currents elicited by ACh Ž100 mM. are illustrated and fitted by two exponential curves. The holding potential was y60 mV.
this idea, the selective PKC inhibitor, PKC inhibitor peptide ŽPKCl., prolonged the current decay time Žt f s 10 " 7 ms and ts s 188 " 41 ms. of normal ACh receptors ŽFig. 2 and Table 1.. There is presently no direct evidence that the ligandgated receptor, nicotinic ACh receptor, interacts with a PKC pathway. A study demonstrates that activation of nicotinic ACh receptors in chick embryo myotubes increases inositol phosphates w1x, suggesting that PKC can be activated by nicotinic ACh receptors. In addition, we have obtained further evidence for this idea: In outside-out patches from Torpedo ACh receptors expressed in oocytes, activation of PKC via an endogenous phosphatidyl inositol signaling pathway elevated the slope conductance of single-channel currents elicited by 1 mM ACh; the same effect was observed by pretreatment with a higher concentration of ACh Ž100 mM.; these effects were blocked by the selective PKC inhibitor, staurosporine or PKCl; the responses of ma q PKCrNA333bgmd q PKCrNA377 mimicked those induced by either endogenous PKC activation or pretreatment with 100 mM ACh; and the control
responses were decreased by staurosporine or PKCl in a fashion that mimics the effect of ma DPKCrSer333bg and mdDPKCrSer377 Žunpublished data.. There, PKCl or staurosporine did not cause additive decrease in the responses of ma DPKCrSer333bg mdDPKCrSer377, indicating that the effect of these inhibitors on ACh receptors was not due to non-specific action. This also indicates that the effect of PKCl on desensitization of normal ACh receptors observed in the present study was caused by blocking PKC activity. Thus, it appears that normal ACh receptors are constantly under the influence of PKC phosphorylation. We next examined the effect of PKA on desensitization of ACh receptors. Phosphorylation of normal ACh receptors by the catalytic subunit of PKA shortened the decay time of currents elicited by 100 mM ACh Žt f s 4 " 1 ms and ts s 33 " 1 ms. ŽFig. 2 and Table 1., suggesting that PKA phosphorylation of ACh receptors accelerates the rate of desensitization. To gain further insight into the effect of PKA phosphorylation on desensitization, we expressed mutant ACh receptors linked to the PKA phosphorylation sites. Currents through abmg q PKArNA353,354d or abgd q PKArNA361,362, which possibly mimic PKA phosphorylation of the receptors, desensitized more rapidly Žt f s 3 " 1 ms and ts s 37 " 24 ms or t f s 3 " 1 ms and ts s 53 " 15 ms, respectively. than normal ACh receptors ŽFig. 3B and Table 1.. In contrast, mutant ACh receptors lacking PKA phosphorylation sites prolonged the slow component of the current decay Žts s 411 " 94 ms for abmgDPKArSer353, 354d or ts s 230 " 63 ms for abgmdDPKArSer361, 362., while the fast component was not significantly affected Žt f s 5 " 1 ms for abmgDPKArSer353,354d or t f s 8 " 3 ms for abgmdDPKArSer361,362. ŽFig. 3B and Table 1.. One would wonder why the effect of mutant ACh receptors lacking PKA phosphorylation sites on desensitization was observed only in the slow component, although both fast and slow components were affected by mutation mimicking PKA phosphorylation. When currents were induced by a lower concentration Ž1 mM. of ACh, abmgDPKArSer353,354d desensitized more slowly in the fast component Ž26 " 12 ms; significant, P - 0.1; n s 6. than normal ACh receptors Ž11 " 4 ms, n s 5., with no significant change in the slow component Ž519 " 24 ms for mutant receptors vs. 590 " 31 ms for normal receptors.. This may imply that on which component of the current decay PKA dephosphorylation has an effect depends on the basal activity of ACh receptors. To address this item, further experiments need to be carried out. Anyway, PKA phosphorylation of ACh receptors are likely to accelerate the rate of desensitization. In conclusion, the results presented here indicate that PKC or PKA phosphorylation of ACh receptors accelerates the rate of desensitization. The most striking finding in the present study is that normal ACh receptors may interact with a PKC pathway, thereby phosphorylating own
T. Nishizaki, K. Sumikawar Brain Research 812 (1998) 242–245
receptors, and then leading to facilitation of desensitization. Acknowledgements We are grateful to Drs. S. Heinemann ŽSalk Institute. and T. Claudio ŽYale University. for providing us with mouse and Torpedo ACh receptor cDNA clones. This work was supported by grants from the Muscular Dystrophy Association. References w1x F. Eusebi, F. Grassi, C. Nervi, C. Caporale, S. Adamo, B.M. Zani, M. Molinaro, Acetylcholine may regulate its own nicotinic receptorchannel through the C-kinase system, Proc. R. Soc. Lond. B 230 Ž1987. 355–365.
245
w2x C. Franke, H. Hatt, J. Dudel, Steep concentration dependence and fast desensitization of nicotinic channel currents elicited by acetylcholine pulses, studied in adult vertebrate muscle, Pfugers Arch. 417 Ž1991. ¨ 509–516. w3x C. Franke, D. Koltgen, H. Hatt, J. Dudel, Activation and desensitization of embryonic-like receptor channels in mouse muscle by acetylcholine concentration steps, J. Physiol. 451 Ž1992. 145–158. w4x V.M. Gehle, K. Sumikawa, Site-directed mutagenesis of the conserved N-glycosylation site on the nicotinic acetylcholine receptor subunits, Mol. Brain Res. 11 Ž1991. 17–25. w5x R.L. Huganir, Regulation of the nicotinic acetylcholine receptor by protein phosphorylation, J. Rec. Res. 7 Ž1987. 241–256. w6x R.L. Huganir, P. Greengard, Regulation of neurotransmitter receptor desensitization by protein phosphorylation, Neuron 5 Ž1990. 555–567. w7x T. Nishizaki, K. Sumikawa, Direct action of 4-b-phorbol-12,13-dibutyrate ŽPDBu. on nicotinic acetylcholine receptor channel independent of protein kinase C activation, Neurosci. Lett. 188 Ž1995. 129–131. w8x K. Sumikawa, R. Miledi, Assembly and N-glycosylation of all ACh receptor subunits are required for their efficient insertion into plasma membranes, Mol. Brain Res. 5 Ž1989. 183–192.
Short communication
Effects of PKC and PKA phosphorylation on desensitization of nicotinic acetylcholine receptors Tomoyuki Nishizaki a
a, )
, Katumi Sumikawa
b
Department of Physiology, Kobe UniÕersity School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan b Department of Psychobiology, UniÕersity of California, IrÕine, CA 92717-4550, USA
Abstract The present study was designed to assess the effect of protein kinase C ŽPKC. and cAMP-dependent protein kinase ŽPKA. on desensitization of Torpedo acetylcholine ŽACh. receptors by analyzing summated macroscopic currents in an outside-out patch–clamp configuration. Normal ACh receptors desensitized with a fast Ž6 ms. and slow time constant Ž104 ms.. There was no significant difference in the current decay time between normal ACh receptors and mutant ACh receptors that possibly mimics PKC phosphorylation of the receptors. The selective PKC inhibitor, PKCl, prolonged the rate of desensitization of normal ACh receptors, and the similar effect was obtained with mutant ACh receptors lacking PKC phosphorylation sites. Phosphorylation of normal ACh receptors by the catalytic subunit of PKA or mutant ACh receptors that possibly mimic PKA phosphorylation of the receptors increased the rate of desensitization, but, in contrast, the receptors lacking PKA phosphorylation sites prolonged the current decay time. The results of the present study demonstrate that PKC or PKA phosphorylation of ACh receptors accelerates the rate of desensitization. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Torpedo ACh receptor; Outside-out patch; Protein phosphorylation; Desensitization; Site-directed mutagenesis
Protein phosphorylation plays a role in modulation of the neurotransmitter receptors, and Torpedo acetylcholine ŽACh. receptors are recognized to have phosphorylation sites for at least three protein kinases such as protein kinase C ŽPKC., cAMP-dependent protein kinase ŽPKA., and tyrosine kinase ŽPTK. w5,6x. Although PKA or PTK phosphorylation of nicotinic ACh receptors is suggested to accelerate the rate of the current decay time, the effect of PKC phosphorylation of the receptors on desensitization still remains controversial. One reason is that phorbol esters, which are widely used to activate PKC, have a direct blocking effect on the receptor channels w7x, and therefore, the actual effect of PKC phosphorylation of ACh receptors on desensitization may be masked. The present study investigated the effect of PKC or PKA phosphorylation of ACh receptors on desensitization by analyzing macroscopic currents through normal and mutant Torpedo ACh receptors expressed in Xenopus oocyte. We show
) Corresponding author. [email protected]
Fax:
q 81-78-341-5732;
E-mail:
here that PKC and PKA phosphorylation of the receptors increases the rate of desensitization. mRNAs coding for the Torpedo ACh receptor a , b, g, and d subunits were synthesized by in vitro transcription as described previously w8x. The mutant a and d subunits were constructed by site-directed mutagenesis w4x: Ser 333 on the a subunit Žma q PKCrNA333. and Ser 377 on the d subunit Žmd q PKCrNA377. were replaced with the negatively charged amino acid residue, Asp. To construct mutant ACh receptors lacking PKC phosphorylation sites, Ser 333 on the a subunit Žma DPKCrSer333. and Ser 377 on the d subunit ŽmdDPKCrSer377. were replaced with Ala. For mutant ACh receptors linked to the PKA phosphorylation sites, Ser 353,354 on the g subunit and Ser 361,362 on the d subunit were replaced with aspartic acid residues Žmg q PKArNA353,354 and md q PKArNA361,362. or with Gly and Ala Ž m gD PKArSer353,354 and mdDPKArSer361,362.. Xenopus oocytes were manually separated from the ovary, and incubated overnight in Barth’s solution Žin mM: 88 NaCl, 1 KCl, 2.4 NaHCO 3 , 0.82 MgSO4 , 0.33 CaŽNO 2 . 2 , 0.41 CaCl 2 , and 7.5 Tris, pH 7.6. after collagenase Ž0.5 mgrml. treatment. Oocytes were injected with the normal or mutant ACh receptor
0006-8993r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 8 3 6 - 1
T. Nishizaki, K. Sumikawar Brain Research 812 (1998) 242–245
subunit mRNAs, and incubated at 188C. The injected oocytes were transferred to the recording chamber a day after incubation and continuously superfused at room temperature Ž20 to 228C. in standard from Ringer’s solution Žin mM: 115 NaCl, 2 KCl, 1.8 CaCl 2 , and 5 HEPES, pH 7.0.. Outside-out patches were made from oocytes using the patch electrode-filling solution Žin mM: 80 KF, 20 KCl, 10 K-EGTA, 10 HEPES, and 0.5 ATP, pH 7.2.. ACh was applied by 1-s pulses using a piezo-driven liquid filament switch w2,3x. Macroscopic currents from excised patches were recorded with an Axopatch-1D amplifier ŽAxon Instruments, USA. ŽFig. 1A.. Summated macroscopic currents ŽFig. 1B. were fitted by two exponential curves and each time constant Žt f and ts . of the current decay was calculated using a program provided by H. Chang. ACh Ž100 mM. produced currents at a holding potential of y60 mV. Normal ACh receptors desensitized with a fast time constant Žt f . of 6 " 3 ms and a slow time constant Žts . of 104 " 63 ms Žcontrol. ŽFig. 2A and Table 1.. Currents through either ma q PKCrNA333bgd or abgmd q PKCrNA377, which possibly mimics PKC phosphorylation of the receptors, decayed with t f and ts of 5 " 0 and 213 " 115 ms or 6 " 0 and 201 " 155 ms, respectively ŽFig. 3A and Table 1., but there was no
243
Fig. 2. Effects of PKC or PKA on desensitization of ACh receptors. Outside-out patches were made from oocytes expressing normal Torpedo ACh receptors. PKCl Ž ; 500 mgrml final concentration. or PKA Ž ;1 mgrml final concentration. was added to the patch electrode-filing solution. Summated currents obtained from 20 macroscopic currents elicited by ACh Ž100 mM. are illustrated and fitted by two exponential curves. The holding potential was y60 mV.
significant difference in time constant between these mutant ACh receptors and normal receptors. In contrast, mutant ACh receptors lacking PKC phosphorylation sites on both the a and d subunits Žt f s 9 " 4 ms and ts s 352 " 117 ms. or on the d subunit alone Žt f s 14 " 2 ms and ts s 398 " 180 ms. desensitized more slowly than normal receptors, although the decay of currents through ma DPKCrSer333bgd Žt f s 6 " 2 ms and ts s 260 " 127 ms. was not affected ŽFig. 3A and Table 1., suggesting the possibility that PKC dephosphorylation of the receptors employs a decrease in the rate of desensitization and that the responsible site is the d subunit. Then, one would assume that mutant ACh receptors mimicking PKC phosphorylation accelerates the rate of desensitization as compared with normal ACh receptors. However, this is not the case here. A possible explanation is that ACh receptors are phosphorylated by PKC activated via an ACh receptormediated signaling pathway and that therefore, normal ACh receptors desensitize rapidly as same as ma q PKCrNA333bg and md q PKCrNA377. In support of
Table 1 Effects of protein phosphorylation on desensitization of ACh receptors Time constant Žms.
Fig. 1. ACh receptor currents induced by a piezo-driven liquid filament switch. Outside-out patches were made from oocytes expressing Torpedo ACh receptors. ACh Ž100 mM. was applied by 1-s pulses using a piezo-driven liquid filament switch. The arrow indicates switching artifact. Five macroscopic currents evoked at a holding potential of y60 mV are illustrated in ŽA. and summated current obtained from five recordings are shown in ŽB..
N
tf
tS
abgd Control qPKCl qPKA
6"3 10"7 ) ) 4"1)
104"63 188"41 33"1) )
7 6 7
ma qPKCrNA333 mdqPKCrNA377 ma DPKCrSer333 mdDPKCrSer377 ma DPKCrSer333mdDPKCrSer377
5"0 6"0 6"2 14"2 ) ) 9"4 ) )
213"115 201"155 260"127 398"180 ) 352"117 )
5 4 12 6 5
3"1) ) 3"1) ) 5"1 8"3
37"24 ) ) 53"15 ) ) 411"94 ) ) 230"63 ) )
11 3 10 9
mgqPKArNA353,354 mdqPKArNA361,362 mgDPKArSer353,354 mdDPKArSer361,362 )
P - 0.1, ence test.
))
P - 0.01, ANOVA with Fisher’s least-significance differ-
244
T. Nishizaki, K. Sumikawar Brain Research 812 (1998) 242–245
Fig. 3. Effects of PKC ŽA. or PKA ŽB. phosphorylation of ACh receptors on desensitization. Outside-out patches were made from oocytes expressing mutant Torpedo ACh receptors as indicated. Summated currents obtained from 20 macroscopic currents elicited by ACh Ž100 mM. are illustrated and fitted by two exponential curves. The holding potential was y60 mV.
this idea, the selective PKC inhibitor, PKC inhibitor peptide ŽPKCl., prolonged the current decay time Žt f s 10 " 7 ms and ts s 188 " 41 ms. of normal ACh receptors ŽFig. 2 and Table 1.. There is presently no direct evidence that the ligandgated receptor, nicotinic ACh receptor, interacts with a PKC pathway. A study demonstrates that activation of nicotinic ACh receptors in chick embryo myotubes increases inositol phosphates w1x, suggesting that PKC can be activated by nicotinic ACh receptors. In addition, we have obtained further evidence for this idea: In outside-out patches from Torpedo ACh receptors expressed in oocytes, activation of PKC via an endogenous phosphatidyl inositol signaling pathway elevated the slope conductance of single-channel currents elicited by 1 mM ACh; the same effect was observed by pretreatment with a higher concentration of ACh Ž100 mM.; these effects were blocked by the selective PKC inhibitor, staurosporine or PKCl; the responses of ma q PKCrNA333bgmd q PKCrNA377 mimicked those induced by either endogenous PKC activation or pretreatment with 100 mM ACh; and the control
responses were decreased by staurosporine or PKCl in a fashion that mimics the effect of ma DPKCrSer333bg and mdDPKCrSer377 Žunpublished data.. There, PKCl or staurosporine did not cause additive decrease in the responses of ma DPKCrSer333bg mdDPKCrSer377, indicating that the effect of these inhibitors on ACh receptors was not due to non-specific action. This also indicates that the effect of PKCl on desensitization of normal ACh receptors observed in the present study was caused by blocking PKC activity. Thus, it appears that normal ACh receptors are constantly under the influence of PKC phosphorylation. We next examined the effect of PKA on desensitization of ACh receptors. Phosphorylation of normal ACh receptors by the catalytic subunit of PKA shortened the decay time of currents elicited by 100 mM ACh Žt f s 4 " 1 ms and ts s 33 " 1 ms. ŽFig. 2 and Table 1., suggesting that PKA phosphorylation of ACh receptors accelerates the rate of desensitization. To gain further insight into the effect of PKA phosphorylation on desensitization, we expressed mutant ACh receptors linked to the PKA phosphorylation sites. Currents through abmg q PKArNA353,354d or abgd q PKArNA361,362, which possibly mimic PKA phosphorylation of the receptors, desensitized more rapidly Žt f s 3 " 1 ms and ts s 37 " 24 ms or t f s 3 " 1 ms and ts s 53 " 15 ms, respectively. than normal ACh receptors ŽFig. 3B and Table 1.. In contrast, mutant ACh receptors lacking PKA phosphorylation sites prolonged the slow component of the current decay Žts s 411 " 94 ms for abmgDPKArSer353, 354d or ts s 230 " 63 ms for abgmdDPKArSer361, 362., while the fast component was not significantly affected Žt f s 5 " 1 ms for abmgDPKArSer353,354d or t f s 8 " 3 ms for abgmdDPKArSer361,362. ŽFig. 3B and Table 1.. One would wonder why the effect of mutant ACh receptors lacking PKA phosphorylation sites on desensitization was observed only in the slow component, although both fast and slow components were affected by mutation mimicking PKA phosphorylation. When currents were induced by a lower concentration Ž1 mM. of ACh, abmgDPKArSer353,354d desensitized more slowly in the fast component Ž26 " 12 ms; significant, P - 0.1; n s 6. than normal ACh receptors Ž11 " 4 ms, n s 5., with no significant change in the slow component Ž519 " 24 ms for mutant receptors vs. 590 " 31 ms for normal receptors.. This may imply that on which component of the current decay PKA dephosphorylation has an effect depends on the basal activity of ACh receptors. To address this item, further experiments need to be carried out. Anyway, PKA phosphorylation of ACh receptors are likely to accelerate the rate of desensitization. In conclusion, the results presented here indicate that PKC or PKA phosphorylation of ACh receptors accelerates the rate of desensitization. The most striking finding in the present study is that normal ACh receptors may interact with a PKC pathway, thereby phosphorylating own
T. Nishizaki, K. Sumikawar Brain Research 812 (1998) 242–245
receptors, and then leading to facilitation of desensitization. Acknowledgements We are grateful to Drs. S. Heinemann ŽSalk Institute. and T. Claudio ŽYale University. for providing us with mouse and Torpedo ACh receptor cDNA clones. This work was supported by grants from the Muscular Dystrophy Association. References w1x F. Eusebi, F. Grassi, C. Nervi, C. Caporale, S. Adamo, B.M. Zani, M. Molinaro, Acetylcholine may regulate its own nicotinic receptorchannel through the C-kinase system, Proc. R. Soc. Lond. B 230 Ž1987. 355–365.
245
w2x C. Franke, H. Hatt, J. Dudel, Steep concentration dependence and fast desensitization of nicotinic channel currents elicited by acetylcholine pulses, studied in adult vertebrate muscle, Pfugers Arch. 417 Ž1991. ¨ 509–516. w3x C. Franke, D. Koltgen, H. Hatt, J. Dudel, Activation and desensitization of embryonic-like receptor channels in mouse muscle by acetylcholine concentration steps, J. Physiol. 451 Ž1992. 145–158. w4x V.M. Gehle, K. Sumikawa, Site-directed mutagenesis of the conserved N-glycosylation site on the nicotinic acetylcholine receptor subunits, Mol. Brain Res. 11 Ž1991. 17–25. w5x R.L. Huganir, Regulation of the nicotinic acetylcholine receptor by protein phosphorylation, J. Rec. Res. 7 Ž1987. 241–256. w6x R.L. Huganir, P. Greengard, Regulation of neurotransmitter receptor desensitization by protein phosphorylation, Neuron 5 Ž1990. 555–567. w7x T. Nishizaki, K. Sumikawa, Direct action of 4-b-phorbol-12,13-dibutyrate ŽPDBu. on nicotinic acetylcholine receptor channel independent of protein kinase C activation, Neurosci. Lett. 188 Ž1995. 129–131. w8x K. Sumikawa, R. Miledi, Assembly and N-glycosylation of all ACh receptor subunits are required for their efficient insertion into plasma membranes, Mol. Brain Res. 5 Ž1989. 183–192.