A novel mitochondrial K(ATP) channel assay

Abstract

Rationale: The mitochondrial ATP sensitive potassium channel (mK(ATP)) is implicated in cardioprotection by ischemic preconditioning (IPC), but the molecular identity of the channel remains controversial. The validity of current methods to assay mK(ATP) activity is disputed.

Objective: We sought to develop novel methods to assay mK(ATP) activity and its regulation.

Methods and results: Using a thallium (Tl(+))-sensitive fluorophore, we developed a novel Tl(+) flux based assay for mK(ATP) activity, and used this assay probe several aspects of mK(ATP) function. The following key observations were made. (1) Time-dependent run down of mK(ATP) activity was reversed by phosphatidylinositol-4,5-bisphosphate (PIP(2)). (2) Dose responses of mK(ATP) to nucleotides revealed a UDP EC(50) of approximately 20 micromol/L and an ATP IC(50) of approximately 5 micromol/L. (3) The antidepressant fluoxetine (Prozac) inhibited mK(ATP) (IC(50)=2.4 micromol/L). Fluoxetine also blocked cardioprotection triggered by IPC, but did not block protection triggered by a mK(ATP)-independent stimulus. The related antidepressant zimelidine was without effect on either mK(ATP) or IPC.

Conclusions: The Tl(+) flux mK(ATP) assay was validated by correlation with a classical mK(ATP) channel osmotic swelling assay (R(2)=0.855). The pharmacological profile of mK(ATP) (response to ATP, UDP, PIP(2), and fluoxetine) is consistent with that of an inward rectifying K(+) channel (K(IR)) and is somewhat closer to that of the K(IR)6.2 than the K(IR)6.1 isoform. The effect of fluoxetine on mK(ATP)-dependent cardioprotection has implications for the growing use of antidepressants in patients who may benefit from preconditioning.

Figure 1. BTC-AM Tl⁺ fluorescence assay for mK_ATP activity in isolated heart mitochondria

(A): Representative traces of BTC-AM Tl⁺ fluorescence in isolated mitochondria. Fluorescence (λ_ex 488 nm, λ_em 525 nm) was normalized to the 10 s. of baseline prior to the injection of TlSO₄ (arrow). Where indicated, 1 mmol/L ATP (thick, black), 1 nmol/L AA5 plus ATP (thin, grey), or 300 μmol/L 5HD plus AA5 plus ATP (thick grey) were present from the beginning of incubations. (B): Magnitude of the change in BTC-AM fluorescence following Tl⁺ addition, relative to control. Change in fluorescence was determined by subtracting the average baseline fluorescence from the stabilized average fluorescence at 30–60 s. Delta fluorescence in controls was 26.4 ± 3.3 arbitrary units. Experimental conditions are listed below the x-axis. Data are mean ± SEM, N≥4. # P<0.05 versus control, * P<0.05 versus ATP, † P<0.05 versus the effect of ATP+AA5 or ATP+DZX. (C): Correlation between two assays for mK_ATP channel activity. Experiments using a variety of conditions that modulate the mK_ATP were performed using either the osmotic swelling assay and the BTC-AM-Tl⁺ assay. Both sets of results were expressed as a percent of their respective controls. Linear regression curve fit revealed an r² of 0.855.

Figure 2. PIP₂ modulation of mK_ATP channel activity using the swelling and BTC-AM-Tl⁺ assays

mK_ATP activity was monitored using the BTC-AM-Tl⁺ assay in fresh mitochondria (black bars), or mitochondria 5 hrs. post isolation (gray and white bars). Fresh mitochondria data were normalized to control (Δ fluorescence 26.4±3.3) while 5 hr. mitochondria were normalized to control + PIP₂ (3^rd gray bar, Δ fluorescence 29.3±6.4). Experimental conditions are listed below the x-axis. Data in both panels are means ± SEM, N≥4. Fresh mitochondria: # P<0.05 versus control, * P<0.05 versus ATP, † P<0.05 versus ATP+AA5. 5 hr. mitochondria: # P<0.05 versus control+PIP₂, * P<0.05 versus ATP, † P<0.05 versus ATP+AA5, like symbols are not significantly different.

Figure 3. Modulation of mK_ATP activity and IPC-mediated cardioprotection by fluoxetine

(A): FLX dose-response of mK_ATP activity. Activity was measured using the BTC-AM-Tl⁺ assay. Data were plotted as % mK_ATP inhibition, with 100% inhibition defined as the condition in the presence of 1 mmol/L ATP, and 0% closed (i.e. open) being the baseline (Ctrl.) condition without ATP. FLX experiments were measured in the absence of ATP. Curve fit using the Hill equation revealed the FLX IC₅₀ to be 2.39 ± 0.22 μmol/L. Data are means ± SEM, N≥4. (B): mK_ATP activity was measured by the BTC-AM-Tl⁺ assay. Where indicated, FLX or ZM were present. Data are means ± SEM, N≥4. *P<0.05 versus control. (C): Effect of FLX or ZM on IR injury and IPC. Hearts were subjected to Langendorff perfusion as detailed in the methods. Infarct size / area at risk was quantified from TTC staining, with representative stained hearts shown above each condition. Data are means ± SEM, N≥4, *P<0.05 vs. IR. (D): Rate pressure product (RPP, expressed as % of initial) in hearts subjected to each protocol. Data are split across two panels for clarity (IR data shown in both panels), and are means ± SEM, N≥4. *P<0.05 vs. IR, ^#P<0.05 vs. IPC+IR. The initial RPP (mmHg·min⁻¹, ×10³) for each group is listed in the legend.