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. 2001 Feb 2;890(2):222-32.
doi: 10.1016/s0006-8993(00)03163-2.

Potassium chloride depolarization mediates CREB phosphorylation in striatal neurons in an NMDA receptor-dependent manner

W Macías  1 , R Carlson, A Rajadhyaksha, A Barczak, C Konradi
Affiliations

Potassium chloride depolarization mediates CREB phosphorylation in striatal neurons in an NMDA receptor-dependent manner

W Macías et al. Brain Res. .

Abstract

Potassium chloride (KCl)-depolarization has been used to study the properties of L-type Ca2+ channel-mediated signal transduction in hippocampal neurons. Calcium influx through L-type Ca2+ channels stimulates a second messenger pathway that transactivates genes under the regulatory control of the Ca2+-and cyclic AMP-responsive element (CRE). Here, we show that in striatal neurons, but not in hippocampal neurons, CRE binding protein (CREB) phosphorylation and CRE-mediated gene expression after KCl-depolarization depends on functional NMDA receptors. This difference in NMDA receptor dependence is not due to different properties of L-type Ca2+ channels in either neuronal type, but rather to different neuron-intrinsic properties. Despite this variation, the second messenger pathway activated by KCl requires Ca2+/calmodulin (CaM) kinase for CREB phosphorylation in both neuronal types. We conclude that depolarization by KCl works differently in striatal and hippocampal neurons.

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Figures

Fig. 1
Fig. 1
In primary striatal culture, Ser133 CREB phosphorylation mediated by KCl-depolarization and by an L-type Ca2+ channel agonist is differently affected by the NMDA antagonist MK 801. (A) Pretreatment of primary striatal cultures with the NMDA antagonist, MK 801 (1 μM) does block CREB phosphorylation induced by KCl-depolarization (20 mM), but does not block CREB phosphorylation induced by treatment with FPL 64176 (20 μM). Levels of CREB protein were unchanged (lower panel). (B) Pretreatment of primary striatal cultures with nifedipine (20 μM) blocks depolarization-mediated CREB phosphorylation after treatment with KCl (20 mM) and L-type Ca2+ channel-mediated CREB phosphorylation after treatment with FPL 64176 (20 μM). Levels of CREB protein were unchanged (lower panel). All data are shown in duplicates. (C,D) Statistical analysis of the effect of MK 801 on Ser133 CREB phosphorylation by KCl or FPL 64176 in primary striatal neurons demonstrates that MK 801 blocks KCl-mediated (20 mM or 90 mM) Ser133 CREB phosphorylation (C), but not FPL 64176-mediated (0.5 μM, 5 μM or 20 μM) Ser133 CREB phosphorylation (D). n=6 samples for each treatment, level of induction normalized to control levels. Average fold induction±S.E.M. Asterisks mark significant inhibitions of the induction of CREB phosphorylation.
Fig. 2
Fig. 2
In hippocampal culture, Ser133 CREB phosphorylation after KCl-depolarization is not blocked by the NMDA antagonist MK 801. (A) Pretreatment of primary hippocampal cultures with the NMDA antagonist, MK 801 (1 μM) did not block CREB phosphorylation induced by KCl-depolarization (20 mM or 90 mM). Levels of CREB protein were unchanged. (B) Pretreatment of primary hippocampal cultures with nifedipine (20 μM) blocked KCl-depolarization-mediated CREB phosphorylation (20 mM). Levels of CREB protein were unchanged. All data are shown in duplicate. (C,D) Statistical analysis of the effect of MK 801 on Ser133 CREB phosphorylation by KCl (C) or FPL 64176 (D) shows that MK 801 blocks neither KCl-, nor FPL 64176-mediated Ser133 CREB phosphorylation in primary hippocampal neurons. n=6 samples for each treatment, level of induction normalized to an internal control. Average fold induction±S.E.M.
Fig. 3
Fig. 3
Potassium chloride depolarization-induced c-fos gene expression in striatal neurons is dependent on functional NMDA receptors. (Upper) Treatment with MK 801 (1 μM) blocks c-fos induction mediated by 10 mM or 20 mM KCl. (Lower) Cyclophilin was used as internal loading control. Data are shown in duplicate and were repeated twice.
Fig. 4
Fig. 4
Striatal neurons regulate a 33CRE-luciferase construct in a pattern similar to CREB phosphorylation. Neurons were transfected with the 33CRE-luciferase construct and treated with KCl (20 mM, left) or FPL 64176 (20 μM, right). KCl-mediated 3×CRE-luciferase induction was blocked by nifedipine (20 μM), MK 801 (1 μM) and GYKI 52466 (50 μM), while FPL 64176-mediated 3×CRE-luciferase induction was blocked by nifedipine only. Average fold induction±S.E.M., n=9 for all conditions. Asterisks mark statistically significant inhibitions of the induction of the 3×CRE-luciferase construct.
Fig. 5
Fig. 5
Calcium ions are important for KCl and FPL 64176-mediated CREB phosphorylation. (A) In Ca2+-free medium, neither KCl nor FPL 64176 induce Ser133 CREB phosphorylation (upper panel). CREB protein levels were unchanged (lower panel). (B) Statistical analysis confirms the block of CREB phosphorylation in Ca2+-free medium (n=4). Average fold induction±S.E.M. is shown.
Fig. 6
Fig. 6
Antagonists of the GABA receptor do not eliminate the dependency of KCl-mediated Ser133 CREB phosphorylation on NMDA receptors in striatal neurons. Primary striatal cultures were treated with Ro 05-3663 (100 μM) or phaclofen (100 μM) before treatment with KCl (20 mM; top) or FPL 64176 (20 μM; bottom). Neither GABA antagonist, alone or in combination with other drugs, increased CREB phosphorylation. On the contrary, a modest reduction in CREB phosphorylation was observed. P-CREB, immunoblots developed with the Ser133 antiserum; CREB, immunoblots developed with the CREB antiserum. Some control treatments shown in single lanes, FPL 64176- and KCl treatments in duplicates. See Table 1 for statistical analysis.
Fig. 7
Fig. 7
The SRE enhancer is not involved in KCl or FPL 64176-mediated gene expression in primary striatal culture. Primary striatal cultures were transfected with an SRE-luciferase construct and treated with KCl (20 mM), FPL 64176 (20 μM) or BDNF (50 ng/ml). Neither KCl nor FPL 64176 induced the SRE-luciferase construct, while BDNF led to a 15-fold induction. Asterisk marks significant induction. Average fold induction±S.E.M. is shown. n=6 for KCl and FPL 64176, n=3 for BDNF.
Fig. 8
Fig. 8
The CaM kinase inhibitor KN62 (30 μM) blocks KCl-, FPL 64176-, and glutamate-mediated Ser133 CREB phosphorylation in striatal culture. (A) Ser133 CREB phosphorylation (P-CREB) and CREB levels (CREB) in KCl (20 mM)-treated primary striatal cultures. (B) Ser133 CREB phosphorylation (P-CREB) and CREB levels (CREB) in FPL 64176 (20 μM)-treated primary striatal cultures. (C) Ser133 CREB phosphorylation in glutamate (50 μM)-treated primary striatal cultures. All treatments are shown in duplicate. All experiments were repeated at least once.
Fig. 9
Fig. 9
The proposed signal transduction pathway induced by L-type Ca2+ channels and by KCl-depolarization in primary striatal culture. L-Type Ca2+ channels, stimulated by FPL 64176, activate an independent signal transduction pathway that translocates to the nucleus to stimulate Ser133 CREB phosphorylation (left diagram). While FPL 64176 may aid NMDA receptor function, this is not needed for signal transduction. In contrast, KCl depends on the combined activation of the NMDA receptor channel and of the L-type Ca2+ channel to successfully transduce a Ca2+ signal to the nucleus (right diagram). We suggest a sequential activation, i.e. that KCl depolarizes the neuron which (1a) leads to the removal of Mg2+ ions from the NMDA receptor channel (2), and the activation of NMDA receptors with the aid of glutamate and glycine in the medium. AMPA/kainate receptors may help to facilitate the removal of the Mg2+ block (1b). Ion influx through the NMDA receptor channel amplifies the depolarization and causes the opening of L-type Ca2+ channels along the dendrites (3a–c). Ca2+ entering through L-type Ca2+ channels at the cell body stimulates second messenger pathways to activate CREB phosphorylation and gene expression in the nucleus (4). While this hypothesis is in agreement with the interaction of NMDA receptors and L-type Ca2+ channels in primary striatal culture [37], the evidence does not exclude the need for a simultaneous activation of NMDA receptors and L-type Ca2+ channels to allow for enough Ca2+-entry to stimulate second messenger pathways. However, orthodromic inputs that engage the activation of synaptic receptors have been shown to be strong stimulators of L-type Ca2+ channels, which in turn are effective activators of CREB phosphorylation [29].
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References

    1. Bading H, Ginty DD, Greenberg ME. Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways. Science. 1993;260:181–186. - PubMed
    1. Berkowitz LA, Gilman MZ. Two distinct forms of active tran scription factor CREB (cAMP response element binding protein) Proc. Natl. Acad. Sci. USA. 1990;87:5258–5262. - PMC - PubMed
    1. Berninger B, Marty S, Zafra F, da Penha Berzaghi M, Thoenen H, Lindholm D. GABAergic stimulation switches from enhancing to repressing BDNF expression in rat hippocampal neurons during maturation in vitro. Development. 1995;121:2327–2335. - PubMed
    1. Bito H, Deisseroth K, Tsien RW. CREB phosphorylation and dephosphorylation: A Ca2+- and stimulus duration-dependent switch for hippocampal gene expression. Cell. 1996;87:1203–1214. - PubMed
    1. Bravi D, Mouradian MM, Roberts JW, Davis TL, Sohn YH, Chase TN. Wearing-off fluctuations in Parkinson's disease: contri bution of postsynaptic mechanisms. Ann. Neurol. 1994;36:27–31. - PubMed

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