Role of Kv1 potassium channels in regulating dopamine release and presynaptic D2 receptor function

Abstract

Dopamine (DA) release in the CNS is critical for motor control and motivated behaviors. Dysfunction of its regulation is thought to be implicated in drug abuse and in diseases such as schizophrenia and Parkinson's. Although various potassium channels located in the somatodendritic compartment of DA neurons such as G-protein-gated inward rectifying potassium channels (GIRK) have been shown to regulate cell firing and DA release, little is presently known about the role of potassium channels localized in the axon terminals of these neurons. Here we used fast-scan cyclic voltammetry to study electrically-evoked DA release in rat dorsal striatal brain slices. We find that although G-protein-gated inward rectifying (GIRK) and ATP-gated (K(ATP)) potassium channels play only a minor role, voltage-gated potassium channels of the Kv1 family play a major role in regulating DA release. The use of Kv subtype-selective blockers confirmed a role for Kv1.2, 1.3 and 1.6, but not Kv1.1, 3.1, 3.2, 3.4 and 4.2. Interestingly, Kv1 blockers also reduced the ability of quinpirole, a D2 receptor agonist, to inhibit evoked DA overflow, thus suggesting that Kv1 channels also regulate presynaptic D2 receptor function. Our work identifies Kv1 potassium channels as key regulators of DA release in the striatum.

Figure 1. Fast-scan cyclic voltammetry in the rat dorsolateral striatum.

(A) DA release was electrically-evoked with a bipolar stimulating electrode and recorded with FSCV using a 5 µm carbon-fiber recording electrode placed in the dorsolateral striatum. (B) Background-subtracted traces showing oxydo-reduction of DA recorded with a triangular voltage ramp (bottom trace). In vitro calibration with 1 µM DA (top recording) was used to convert the signal to DA concentrations. DA overflow was electrically-evoked with a 400 µA/1 ms single stimulation (middle trace). Quinpirole (1 µM) reduced DA overflow more than 75%. (C) Example of DA overflow kinetics. Each dot represents the peak amplitude of each oxidation traces obtained at intervals of 100 ms after a 400 µA/1 ms single stimulation. Abbreviations: aca, anterior commissure anterior part; CPu, Caudate putamen; Quinp, Quinpirole.

Figure 2. Effect of broad-spectrum potassium channel blockers on evoked DA overflow.

(A) A 30 min perfusion of the broad-spectrum potassium channels blocker 4-AP (100 µM) (n = 10;4) increased DA overflow kinetics compared to control (n = 8;6). Data were normalized to the first 6 recordings (10 min) of their respective control period (means ± SEM). (B) A 30 min perfusion of the broad-spectrum potassium channels blocker TEA (10 mM) (n = 10;3) drastically increased DA overflow kinetics compared to control (n = 8;6). Data were normalized to the first 6 recordings (10 min) of their respective control period (means ± SEM). (C) Time-course of the effect of a 30 min application of 100 µM 4-AP (n = 10;4) in comparison to control (n = 8;6). DA was electrically evoked at intervals of 2 min. Each group is normalized to the first 6 recordings (10 min) of its respective control period and graphically plotted against time (means ± SEM). After 10 min of stable recordings, 4-AP was superfused for 30 min. (D) Time-course of the effect of a 30 min application of 10 mM TEA (n = 10;3) in comparison to control (n = 8;6). (E) After a 30 min application of 100 µM 4-AP, 1 µM ConoTX was applied and caused a ∼ 90% decrease in evoked DA release. (F) After a 30 min application of 10 mM TEA, 1 µM ConoTX was applied and failed to inhibit DA overflow. Abbreviations: 4-AP, 4-aminopyridine; TEA, tetraethylammonium; ConoTX, ω-conotoxin GVIA.

Figure 3. Effect of selective Kv potassium channel subunit blockers on evoked DA overflow.

(A) Summary traces showing the average electrically-evoked DA overflow in response to single electrical pulses after 30 min perfusion with 100 nM MgTX (n = 5;3) or a combination of both 100 nM MgTX and 100 nM α-DTX (n = 4;2), in comparison to control (n = 8;6). Data were normalized to the first 6 recordings (10 min) of their respective control period (means ± SEM). The effect of other Kv subunits blockers is omitted for clarity. (B) Time-course of the experiment shown in A. DA was electrically evoked at intervals of 2 min. Each group is normalized to the first 6 recordings (10 min) of its respective control period and graphically plotted against time (means ± SEM). After 10 min of stable recordings, drugs were superfused for 30 min. Apparent equilibrium was reached for all Kv channel blockers after 20 min of application. The effect of others Kv channel blockers is omitted for clarity. (C) Histogram showing the mean increase in electrically-evoked DA overflow during the last 10 min of application of several Kv potassium channel blockers: control (n = 8;6), 100 µM 4-AP (n = 10;4), 100 nM MgTX (n = 5;3), 50 nM TiTX (n = 6;2), 100 nM DTX-K (n = 6;3), 100 nM α-DTX (n = 6;4), 50 nM TiTX + 100 nM MgTX (n = 4;3), 100 nM α-DTX + 100 nM MgTX (n = 4;2), 500 nM BDS-I (n = 6;2) and 100 nM HetTX (n = 6;2). * p<0.05, ** p<0.01, *** p<0.001. Abbreviations: 4-AP, 4-aminopyridine; α-DTX, α-dendrotoxin; MgTX, rMargatoxin; TiTX, rTityustoxin Kα; DTX-K, dendrotoxin-K; BDS-I, blood depressing substance I; HetTX, rHeteropodatoxin-2.

Figure 4. D2 receptor activation dose-dependently inhibits dopamine overflow.

A) DA was electrically-evoked at intervals of 2 min. The peak amplitude of DA overflow is normalized to the average amplitude of the first 6 recordings (10 min) of the control period and graphically plotted against time (means ± SEM). The control trace (n = 8;6) shows that evoked DA overflow was stable over time. Quinpirole was applied for 4 min (represented by the gray bar) at 3 different doses. Drug effects were measured at their peak (rectangle at 16 min). Sulpiride (5 µM) blocked entirely the effect of quinpirole. (B) Histogram showing the peak effect of quinpirole on DA overflow. A one-way ANOVA was used to compare groups. The effect of quinpirole at 0.1 µM (n = 5;4), 0.5 µM (n = 7;4) and 1 µM (n = 27;18) was significant. The D3 receptor subtype blocker, GR103,691, only reduced quinpirole effect at the non-selective dose of 1 µM (n = 4;2), while its effect was non-significant at 100 nM (n = 6;2). Used at 1 µM, L741,626, a D2 subtype selective blocker, significantly reduced the effect of quinpirole (n = 6;2). Sulpiride (5 µM), a broad-spectrum D2-family receptor antagonist, complety blocked the effect of quinpirole (n = 3;2). *** p<0.001 Abbreviations: Q, Quinpirole; Quinp, Quinpirole; GR, GR103,691; L, L741,626, Sulp, Sulpiride.

Figure 5. Regulation of D2-mediated inhibition of DA release by Kv1 potassium channels.

(A) Applied alone for 4 min, 1 µM quinpirole (n = 27;18) induced a large inhibition of evoked DA overflow compared to control (n = 8;6). Pre-application (30 min) of 100 nM MgTX (n = 5;3) or a combination of both 100 nM MgTX and 100 nM α-DTX (n = 4;2) reduced the effect of quinpirole. Data were normalized to the first 6 recordings (10 min) of their respective control period (means ± SEM). The effect of others Kv subunits blockers is omitted for clarity. (B) Time-course of the experiment shown in A. DA was electrically-evoked at intervals of 2 min. Each group was normalized to the first 6 recordings (10 min) of its respective control period (means ± SEM). The maximal effect of quinpirole was detected after 6 min (represented by the rectangle). (C) Histogram showing the ability of potassium channel blockers selective for various Kv subtypes to reduce the effect of quinpirole (1 µM) on evoked DA overflow: quinpirole alone (n = 27;18, represented by the black doted bar), 4-AP (n = 6;2), 100 nM α-DTX (n = 6;4), 100 nM MgTX (n = 5;3), 50 nM TiTX (n = 6;2), 100 nM DTX-K (n = 6;3), 50 nM TiTX + 100 nM MgTX (n = 6;4), 100 nM α-DTX + 100 nM MgTX (n = 6;3), 500 nM BDS-I (n = 6;2), 100 nM HetTX (n = 6;2) and high concentration of potassium (n = 4;2). * p<0.05, ** p<0.01, *** p<0.001. Abbreviations: K⁺, potassium; 4-AP, 4-aminopyridine; α-DTX, α-dendrotoxin; MgTX, rMargatoxin; TiTX, rTityustoxin Kα; DTX-K, dendrotoxin-K; BDS-I, blood depressing substance I; HetTX, rHeteropodatoxin-2; quinp, quinpirole.

Figure 6. Evaluation of the role of GIRK and KATP channels in regulating DA release.

(A) Effect of 30 min perfusion of the GIRK blocker barium (1 mM) (n = 7;3) or the KATP blocker glibenclamide (3 µM) (n = 7;4) on DA overflow kinetics compared to control (n = 8;6). Data were normalized to the first 6 recordings (10 min) of their respective control period (means ± SEM). No significant effect was noted. (B) Time-course of the experiment shown in A. DA was electrically evoked at intervals of 2 min. Each group was normalized to the first 6 recordings (10 min) of its respective control period (means ± SEM). After a 10 min stabilization period, blockers were applied for 30 min. No significant effect of these two antagonists was noted. Abbreviations: Glib, glibenclamide; Diazo, diazoxide.