Functional study of CLC-1 mutants expressed in Xenopus oocytes reveals that a C-terminal region Thr891-Ser892-Thr893 is responsible for the effects of protein kinase C activator.
ClC-1 plays an important part in the maintenance of membrane potential in the mammalian skeletal muscle. To investigate the phosphorylation sites responsible for the effect of PKC (protein kinase C) activator, we constructed 21 different ClC-1 mutants with mutations at predicted phosphorylation sites for PKC. The functional experiments were performed on both wild-type and mutant proteins (17 point mutants and 4 double mutants) expressed in Xenopus oocytes with two-electrode voltage-clamp recording. PMA (12-myristate 13-acetate), a PKC activator, caused a right shift of half-maximum activation potential (V(1/2)) significantly in the wild-type (from -42.9+/-4.4 to -13.7+/-1.7 mV; n = 8, P < 0.05) and most of the single mutants except the S892P (from -39.5+/-4.5 to -35.7+/-5.7 mV; n = 6) and S892D (from -10.2+/-4.9 to -9.6+/-3.5 mV; n = 4). S892D, a mutant mimicking PKC-mediated phosphorylation at position 892, can also mimic the effect of wild-type treated with PMA in V(1/2) value (-10.2+/-4.9 mV vs -13.7+/-1.7 mV, n = 4 - 8). However, S892A still had a significant response to PMA indicating that other sites responsible for PMA might exist. Thus double mutants are generated for the following analysis. The V(1/2) of double mutants, T891A/S892A, S892A/T893A and T891A/T893A, show no significant difference between before and after PMA treatment. We hypothesize that this structural modification results in the observed alteration of the gating properties of ClC-1 by PMA. In summary, our observations show that a C-terminal region Thr891-Ser892-Thr893, at least in part, responsible for the effect of PMA on ClC-1.
Hsiao
Kuang-Ming
KM
Department of Life Science and Institute of Molecular Biology, National Chung Cheng University, Chia-Yi, Taiwan.
Huang
Ren-Yu
RY
Tang
Pei-Hua
PH
Lin
Min-Jon
MJ
D000818:Animals; D018118:Chloride Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004594:Electrophysiology; D020536:Enzyme Activators / Q000378:metabolism*; D005260:Female; D006801:Humans; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives* / Q000494:pharmacology; D014982:Xenopus laevis / Q000235:genetics / Q000378:metabolism
D018118:Chloride Channels; D020536:Enzyme Activators; C016658:phorbolol myristate acetate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate