Abstract
U0126 (1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene), a widely used mitogen-activated protein kinase kinase (MEK) inhibitor, was found to accelerate voltage-gated K+ channel (KV) inactivation in heterologous cells expressing several types of KV. The goal of this study was to examine whether U0126 at a concentration thought to specifically inhibit MEK signaling also inhibits KV in native neurons of primary cultures or brain slices. U0126 caused a dose-dependent inhibition of both the transient (IA) and sustained (IDR) components of K+ currents in hippocampal neurons. U0126 also exhibited much higher potency on the IA and IDR than the classical KV blockers 4-aminopyridine (4-AP) and tetraethylammonium (TEA). Consistent with its inhibitory effect on KV, U0126 broadened action potential duration, profoundly affected the repolarizing phase, and dramatically reduced firing frequency in response to current pulse injections. Despite the potent and reversible action of U0126 on Kv channels, PD98059, a structurally-unrelated MEK inhibitor, did not induce such an effect, suggesting U0126 may act independently of MEK inhibition. Together, these results raise cautions for using U0126 as a specific inhibitor for studying MEK signaling in neurons; on the other hand, further studies on the blocking mechanisms of U0126 as a potent inhibitor of KV may provide useful insights into the structure-function relationship of KV in general.
Highlights
The mitogen-activated protein kinase (MAPK), known as extracellular signal-regulated kinase (ERK1/2) is activated by the dual phosphorylation catalyzed by MAPK kinase (MAPKK, known as mitogenactivated protein kinase kinase (MEK))
At 10 μM, a concentration commonly used in neuronal culture studies, U0126 produced a significant inhibition of the inactivating K+ current (IA) (65 ± 11%, n = 20; p < 0.05) (Fig. 1D) but had no significant inhibition of the IDR (6 ± 3%, n = 20; p > 0.05) (Fig. 1E)
At 20 μM, U0126 significantly reduced both the IA (82 ± 4%, n = 33; p < 0.05) (Fig. 1D) and IDR (38 ± 6%, n = 33; p < 0.05) (Fig. 1E). Both IA and IDR can be inhibited by U0126, with the IA being more sensitive to the inhibition of U0126
Summary
The mitogen-activated protein kinase (MAPK), known as extracellular signal-regulated kinase (ERK1/2) is activated by the dual phosphorylation catalyzed by MAPK kinase (MAPKK, known as MEK). The concentration of U0126 used to block the MAPK/ERK signaling pathway is typically 10 μM in cultured neurons[7,8], and 20 μM in brain slices[9,10,11]. It has been demonstrated in various cell types that the ERK signaling pathway plays important roles in modulating KV12–14, synaptic plasticity[10,15,16,17,18,19,20], and learning and memory[21,22,23] (reviewed by[19,24,25]). This inhibiting effect of U0126 appeared to be of much higher potency (100- to 1000-fold) on the IA and IDR than the classical KV blockers 4-AP or TEA
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