Potential Sites of Action for Phloretin on BK Channels

Abstract

Activation of large-conductance (BK) potassium channels by voltage and/or Ca2+ plays an important role in maintaining homeostasis. Reduced BK channel activity has pathophysiological consequences, suggesting BK channel openers may have therapeutic potential. Here, we focus on the mechanism of action of the BK opener phloretin. Previously, we reported the predominant effect of phloretin (100 μM) was to increase Po with greater efficacy at negative voltages (∼100-fold increase in 0 Ca2+), an effect consistent with actions on the gate and/or its coupling to voltage-sensors. Phloretin has effects on other Slo family members. Interestingly, phloretin inhibited unitary currents from heterologously-expressed mSlo3 channels. Thus, we used a chimeric approach between Slo1 (BK) and Slo3 to identify potential sites of action for phloretin. Domain exchange studies revealed the intracellular tail does not play a significant role in mediating phloretin's effects as an opener. However, chimeras with S6 domains from Slo3 were inhibited by phloretin. Activation was partially restored when only the distal end of S6 was exchanged, suggesting the proximal end of S6 is important in mediating phloretin's effects. Sequence alignment of this region revealed two residues of interest-G311 and G310. The former is the proposed gating hinge and is not conserved in Slo3. G310 provides added flexibility to S6 during gating of Slo1 and is conserved in Slo3. The effects of phloretin were tested on Slo1 G310A and G311A. Although phloretin increased nPo for both mutants, the effects were significantly less at G311A and similar to wild-type values for G310A, highlighting the importance of the gating hinge in mediating the effects of phloretin. These findings suggest the ability of phloretin to act as an opener at BK channels may require the structural arrangement and flexibility provided by the gating hinge.