| Abstract No.: | C-B3068 |
| Country: | Canada |
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| Title: | CONTRIBUTION OF KV1.1 AND 1.2 VOLTAGE-GATED POTASSIUM CHANNELS TO D2 AUTORECEPTOR REGULATION OF AXONAL DOPAMINE RELEASE. |
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| Authors/Affiliations: | 1 Stephanie Fulton*; 1 Alfredo Mendez; 1 Faiza Benaliouad; 1 Nicolas Lahaie; 1 Dominic Thibault; 3 Michel Bouvier; 2 Bruce Tempel; 1 Louis-Eric Trudeau;
1 University of Montreal, QC, Canada; 2 University of Wasington, Seattle, WA; 3 Universityof Montreal, QC, Canada
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| Content: | Objectives: The D2 dopamine autoreceptor (D2-AR) exerts tight regulatory control of dopamine (DA) release. In contrast to the somatodendritic receptor, axonal D2-AR does not appear to diminish DA release via the opening of G-protein gated inward rectifying potassium channels. We examined the contribution of voltage-gated potassium channels of the Shaker family (Kv1) and determined the role of Gα, Gβγ and PKA to D2-AR mediated release.
Methods: With the use of tyrosine hydroxylase (TH)-eGFP mice, we isolated DA neurons of the midbrain by FACS. By means of PCR amplification of cDNA derived from these neurons we assessed the presence of Kv1.1, 1.2, and 1.3 mRNA in DA neurons. In addition, we performed immunocytochemistry on cultures of DA neurons with the use of antibodies against the dopamine transporter (DAT), TH and Kv1.1 through 1.6. To examine the functional effects of Kv1 subtype blockade on axonal DA release we measured stimulation-evoked DA release via a carbon fiber electrode in the caudate of acute slices derived from male TH-eGFP mice. To determine which G proteins contribute to D2-AR regulated release, we assessed the effect of n-ethylmaleimide (NEM), a blocker of pertussis-toxin sensitive Gα proteins, and a Gβγ scavenger peptide (TAT-BARKct) to modulate the ability of quinpirole to inhibit evoked DA release. Results: Kv1.1 through 1.3 mRNA is expressed in DA neurons. In addition, we find that Kv1.1 through 1.6, but not Kv4.2, is colocalized with both TH and DAT in axonal-like processes of DA neurons. Using voltammetry we observed that bath application of the D2 agonist quinpirole (1µM) decreased DA release by 76.27% whereas application of a non-specific Kv1 blocker, 4-aminopyridine (4-AP) (100 μM), increased release by 65.2 %. Pre-application of 4-AP prevented 52.5% of quinpirole inhibition of release whereas pre-application of α-dendrotoxin (100nM), a blocker of kv1.1, 1.2 and 1.6 channels, reversed the effect of quinpirole by 44.1%. Pre-application of: (1) 20nM of dendrotoxin-k, a selective kv1.1 blocker, reversed the effect of quinpirole by 27%; (2) 10nM of maurotoxin, a selective kv1.2 blocker, inhibited the effect of quinpirole by 34.4%; (3) 2nM of Osk-1, a selective kv1.3 blocker, failed to alter quinpirole inhibition of DA release. In addition, pre-application of 200 μM of NEM blocked 82.7% of the effect of quinpirole whereas pre-application of 1μM of TAT-BARKct inhibited the effect of quinpirole by 23.6%. Pre-application of 20μM of H-89, a PKA inhibitor, did not alter the effect of quinpirole on evoked DA release.
Conclusion: These data suggest that the axonal D2-AR inhibits DA release via kv1.1 and kv1.2. The axonal D2-AR regulates evoked DA release by activating pertussis toxin senstiive Gα subunits and via Gβγ signaling. We are currently evaluating DA release in kv1.2 knockout mice. |
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