BK channels and a cGMP-dependent protein kinase (PKG) function through independent mechanisms to regulate the tolerance of synaptic transmission to acute oxidative stress at the Drosophila larval neuromuscular junction

Figures & data

Figure 1. Genetic elimination of BK channels increases synaptic transmission tolerance to acute oxidative stress. (A) Average time to synaptic failure is prolonged in Slo⁴ mutants compared to w¹¹¹⁸ control larvae during H₂O₂ exposure [one-way ANOVA, F(3, 16) = 17.76, p < .05]. Vertical bar chart is shown as mean ± SEM. Different letters on bar charts indicate statistical significance, whereas the same letter indicates a nonsignificant difference. (B) Average amplitude decline of evoked EJPs in w¹¹¹⁸ controls and Slo⁴ mutants in the presence and absence of 2.25 mM H₂O₂ (n = 4–6 preparations per group). Black arrows indicate time to synaptic failure of individual w¹¹¹⁸ control larval preparations in HL3 saline. Each data point is displayed as mean ± SEM. (C) Representative waveforms of evoked EJPs from w¹¹¹⁸ control larvae 1 min before H₂O₂ exposure (black line) and 10 min after H₂O₂ exposure (gray line). (D) Representative waveforms of evoked EJPs from Slo⁴ mutant larvae 1 min before H₂O₂ exposure (black line) and 10 min after H₂O₂ exposure (gray line). H₂O₂ does not acutely affect characteristic EJP shape in both w¹¹¹⁸ control and Slo⁴ mutant larvae.

Figure 2. Drosophila muscle resting membrane potential (RMP) and EJP amplitude decline during acute oxidative stress. (A) RMP from Drosophila larval muscle 6 was recorded in w¹¹¹⁸ control larvae and Slo⁴ mutants in the presence and absence of 2.25 mM H₂O₂ (n = 6 per group). RMP depolarization occurs more rapidly in w¹¹¹⁸ and Slo⁴ mutant larvae exposed to 2.25 mM H₂O₂ compared to HL3 control larvae. (B) In the same larval preparations, EJP amplitude was also recorded (n = 6 per group). Loss of RMP tracks EJP amplitude decline across all treatments.

Figure 3. PKG functions independently of BK channels to protect synaptic function from H₂O₂ exposure. PKG activation using 40 µM 8-bromo-cGMP [one-way ANOVA, F(9, 53) = 13.24, p < .001] and 40 µM sildenafil citrate [p < .001] in Slo⁴ mutant larvae significantly decreases time to synaptic failure during acute oxidative stress. Activation of PKG with 40 µM 8-bromo-cGMP [p = .651] and 40 µM sildenafil citrate [p = .313] does not alter time to synaptic failure in w¹¹¹⁸ control larvae. Activation of voltage-gated K⁺ channels using 0.25 mM DCA in w¹¹¹⁸ control larvae does not alter time to synaptic failure during acute oxidative stress [p = .561]. Application of 0.25 mM DCA to Slo⁴ mutant larval preparations decreases time to synaptic failure during oxidative stress [p < .05]. Vertical bar chart is shown as mean ± SEM. n = 5–9 preparations per group.

Figure 4. Pharmacological blockade of BK channel conductance protects synaptic transmission from acute stress. Pharmacological inhibition of BK channel conductance using 500 pM iberiotoxin significantly increases time to synaptic failure in w¹¹¹⁸ control larvae exposed to 2.25 mM H₂O₂ compared to w¹¹¹⁸ H₂O₂ controls [one-way ANOVA, F(3, 19) = 9.449, p < .05]. Nonselective K⁺ channel inhibition using 0.25 mM TEA also increases synaptic transmission tolerance to acute oxidative stress in w¹¹¹⁸ control larvae [p < .05]. Vertical bar chart is shown as mean ± SEM. n = 5–6 preparations per group.

Figure 5. Iberiotoxin does not possess major off-target effects using this experimental model. Administration of BK channel inhibitor iberiotoxin does not significantly alter time to synaptic failure in Slo⁴ mutant larvae [one-way ANOVA, F(2, 16), p = .736], suggesting that iberiotoxin does not possess any major off-target effects at the Drosophila larval NMJ. Vertical bar chart is shown as mean ± SEM. n = 4–6 preparations per group.