Abstract
All subtypes of KCNQ channel subunits (KCNQ1-5) require calmodulin as a co-factor for functional channels. It has been demonstrated that calmodulin plays a critical role in KCNQ channel trafficking as well as calcium-mediated current modulation. However, how calcium-bound calmodulin suppresses the M-current is not well understood. In this study, we investigated the molecular mechanism of KCNQ2 current suppression mediated by calcium-bound calmodulin. We show that calcium induced slow calmodulin dissociation from the KCNQ2 channel subunit. In contrast, in homomeric KCNQ3 channels, calcium facilitated calmodulin binding. We demonstrate that this difference in calmodulin binding was due to the unique cysteine residue in the KCNQ2 subunit at aa 527 in Helix B, which corresponds to an arginine residue in other KCNQ subunits including KCNQ3. In addition, a KCNQ2 channel associated protein AKAP79/150 (79 for human, 150 for rodent orthologs) also preferentially bound calcium-bound calmodulin. Therefore, the KCNQ2 channel complex was able to retain calcium-bound calmodulin either through the AKPA79/150 or KCNQ3 subunit. Functionally, increasing intracellular calcium by ionomycin suppressed currents generated by KCNQ2, KCNQ2(C527R) or heteromeric KCNQ2/KCNQ3 channels to an equivalent extent. This suggests that a change in the binding configuration, rather than dissociation of calmodulin, is responsible for KCNQ current suppression. Furthermore, we demonstrate that KCNQ current suppression was accompanied by reduced KCNQ affinity toward phosphatidylinositol 4,5-bisphosphate (PIP2) when assessed by a voltage-sensitive phosphatase, Ci-VSP. These results suggest that a rise in intracellular calcium induces a change in the configuration of CaM-KCNQ binding, which leads to the reduction of KCNQ affinity for PIP2 and subsequent current suppression.
Highlights
The M-current is a non-inactivating sub-threshold potassium current that regulates spike frequency adaptation and interspike frequency [1,2,3]
Whether calcium disturbs the interaction between calmodulin (CaM) and KCNQ channels has been controversial among several labs [4,5,8,11]
We demonstrated that calcium-mediated KCNQ current suppression involves CaM and the reduction in the affinity of the KCNQ2 channel for PIP2
Summary
The M-current is a non-inactivating sub-threshold potassium current that regulates spike frequency adaptation and interspike frequency [1,2,3]. Calcium-bound CaM, holoCaM, has been demonstrated to mediate bradykinin-induced suppression of KCNQ2/3 currents [8]. A scaffold protein AKAP79/150, anchored to KCNQ2 [18], selectively bound holoCaM, functioning as an acceptor for CaM that dissociated from the KCNQ2 subunit after calcium increase. We demonstrate that elevation of intracellular calcium reduced KCNQ2 affinity toward PIP2, as evaluated by a voltage sensitive PIP2 depleting phosphatase, CiVSP [19]. These results suggest that an increase in intracellular calcium suppresses KCNQ2 current by reducing PIP2 affinity due to the change in CaM-KCNQ2 configuration
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