Dopamine modulation of neuronal Na(+) channels requires binding of A kinase-anchoring protein 15 and PKA by a modified leucine zipper motif - PubMed (original) (raw)

Dopamine modulation of neuronal Na(+) channels requires binding of A kinase-anchoring protein 15 and PKA by a modified leucine zipper motif

W Preston Few et al. Proc Natl Acad Sci U S A. 2007.

Abstract

In hippocampal pyramidal cells, dopamine acts at D1 receptors to reduce peak Na(+) currents by activation of phosphorylation by PKA anchored via an A kinase-anchoring protein (AKAP15). However, the mechanism by which AKAP15 anchors PKA to neuronal Na(+) channels is not known. By using a strategy of coimmunoprecipitation from transfected tsA-201 cells, we have found that AKAP15 directly interacts with Na(v)1.2a channels via the intracellular loop between domains I and II. This loop contains key functional phosphorylation sites. Mutagenesis indicated that this interaction occurs through a modified leucine zipper motif near the N terminus of the loop. Whole-cell patch clamp recordings of acutely dissociated hippocampal pyramidal cells revealed that the D1 dopamine receptor agonist SKF 81297 reduces peak Na(+) current amplitude by 20.5%, as reported previously. Disruption of the leucine zipper interaction between Na(v)1.2a and AKAP15 through the inclusion of a small competing peptide in the patch pipette inhibited the SKF 81297-induced reduction in peak Na(+) current, whereas a control peptide with mutations in amino acids important for the leucine zipper interaction did not. Our results define the molecular mechanism by which G protein-coupled signaling pathways can rapidly and efficiently modulate neuronal excitability through local protein phosphorylation of Na(+) channels by specifically anchored PKA.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Interaction of LI–II of Nav1.2a with AKAP15. (A) Schematic diagram of myc-tagged LI–II constructs with N-terminal or C-terminal lipid anchors. Numbers refer to amino acids. (B) (Upper) Blots show coimmunoprecipitation of AKAP15 detected by using anti-AKAP15 (25) with the whole LI–II loop (_p_428–753) and _p_428–519 immunoprecipitated by using an anti-myc Ab or nonimmune IgG, as indicated. (Lower) Blots reprobed with anti-myc Ab to confirm immunoprecipitation (IP) of constructs. The faint band in the bottom left blot (55 kDa) is the heavy chain of the Ab used for immunoprecipitation.

Fig. 2.

Fig. 2.

Interaction of AKAP15 with the N terminus of LI–II of Nav1.2a via a modified leucine zipper motif. (A) The amphipathic helix and modified leucine zipper motif of AKAP15. Underlined leucines at positions 42 and 49 were mutated to alanine. (B) (Upper) Blots show coimmunoprecipitation of WT AKAP15 or AKAP15(L42,49A) with myc-tagged _p_428–519, as indicated. (Lower) Blots reprobed with anti-myc Ab to confirm adequate immunoprecipitation (IP) of _p_428–519. (C) Lysates from transfected cells probed with AKAP15 Ab demonstrating approximately equal expression of WT and mutant (L42,49A) AKAP15.

Fig. 3.

Fig. 3.

Binding of AKAP15 to the WT or mutant N terminus (amino acids 428–519) of LI–II of Nav1.2a (A) The modified leucine zipper motif of Nav1.2a. (B) (Upper) Blots show coimmunoprecipitation of AKAP15 with myc-tagged WT and mutant _p_428–519 or IgG, from transfected tsA-201 cells, as indicated. (Lower) Blots reprobed with anti-myc Ab to confirm similar immunoprecipitation (IP) of _p_428–519, the double mutant, and the triple mutant.

Fig. 4.

Fig. 4.

Modulation of hippocampal Na+ current by a dopaminergic agonist. (A) Normalized peak Na+ current is reduced by SKF 81297 application. Currents were elicited by a 40-ms pulse to −20 mV from a holding potential of −70 mV once every 2 s. (B) Example Na+ currents from the same cell as in A before and after SKF 81297 perfusion. (C) Control experiment demonstrating stability of peak current amplitude in the absence of SKF 81297 perfusion. (D) Example Na+ currents from the same cell as in C.

Fig. 5.

Fig. 5.

Block of dopaminergic modulation of hippocampal Na+ current by a peptide inhibitor of the AKAP15/Nav1.2 leucine zipper interaction. (A) Effect of intracellular dialysis of AKAP15 LZ peptide (100 μM) on SKF 81297 reduction of Na+ current amplitude. Currents were elicited by a 40-ms pulse to −20 mV from a holding potential of −70 mV once every 2 s. (B) Example Na+ currents from the same cell as in A before and after SKF 81297 perfusion. (C) Efect of intracellular dialysis of AKAP15 LZM peptide (100 μM) on SKF 81297 reduction of Na+ current amplitude. (D) Example Na+ currents from the same cell as in C before and after SKF 81297 perfusion. (E) Bar graph summarizing data from control (no peptide) and peptide experiments.

Fig. 6.

Fig. 6.

Conserved leucine zipper-like motifs in the Nav1 family of voltage-gated Na+ channels. (A) Amino acid sequence alignment of the leucine zipper-like region identified in Nav1.2 with other members of the Nav1 family. (B) Model of the leucine zipper-like interaction between AKAP15 and LI–II of Nav1.2. AKAP15 directly interacts with the N terminus of LI–II in close proximity to the PKA phosphorylation sites.

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