The CamKKβ Inhibitor STO609 Causes Artefacts in Calcium Imaging and Selectively Inhibits BK_Ca in Mouse Carotid Body Type I Cells

Abstract

It has previously been reported that AMP-activated protein kinase (AMPK) may be critical for hypoxic chemotransduction in carotid body type I cells. This study sought to determine the importance of the regulatory upstream kinase of AMPK, CamKKβ, in the acute response to hypoxia in isolated mouse type I cells.

Initial data indicated several previously unreported artefacts associated with using the CamKKβ inhibitor STO609 and Ca²⁺ imaging techniques. Most importantly Fura-2 and X-Rhod1 imaging revealed that STO609 quenched emission fluorescence even in the absence of intracellular Ca²⁺ ([Ca²⁺]_i). Furthermore, STO609 (100 μM) rapidly inhibited outward macroscopic currents and this inhibition was abolished in the presence of the selective BK_Ca inhibitor paxilline.

Taken together these data suggest that ST0609 should be used with caution during Ca²⁺ imaging studies as it can directly interact with Ca²⁺ binding dyes. The rapid inhibitory effect of STO609 on BK_Ca was unexpected as the majority of studies using this compound required an incubation of approximately 10 min to inhibit the kinase. Furthermore, as AMPK activation inhibits BK_Ca, inhibiting AMPK’s upstream kinases would, if anything, be predicted to have the opposite effect on BK_Ca. Future work will determine if the inhibition of BK_Ca is via CamKKβ or via an off target action of STO609 on the channel itself.