Use of a Voltage-Sensitive Phosphatase Highlights Two PiP2-Binding Sites in the C-Terminus of KCNQ3 Channels

Phosphatidylinositol 4,5-bisphosphate (PIP2) is necessary for the activation of KCNQ K+ channels. Many studies have investigated the location of the PIP2-binding site on those channels, which have implicated PIP2 interactions with the C-terminus. Our lab has suggested PIP2 to bind to the linker between helices A and B in the C-terminus of KCNQ3 (Hernandez et al., 2008, JGP, 132). Here, we find that deletion of this linker in well-expressing KCNQ3 A315T (KCNQ3T) channels decreased current amplitudes. Use of a voltage-sensitive phosphatase from danio rerio (Dr-VSP), which dephosphorylates PIP2 upon strong membrane depolarizations, suggests this decrease of current to be due to a lower PIP2 affinity for the channels, as assayed by the rates of current decay at 120 mV, and of current recovery at 30 mV. However, other labs have reported PIP2 to interact with the initial (pre-A helix) domain of the C-terminus (Zhang et al., 2003, Neuron, 37; Thomas et al., 2011, JBC, 286; Telezhkin et al., 2013, Pflugers Arch, 465). Residues (K354, K358, R360, and K362) in KCNQ1 and (H328 and R325) in KCNQ2 have been suggested to interact with PIP2. Since these residues are conserved in KCNQ2-5 channels, those residues may play a role in PIP2-channel interactions for KCNQ3. We found the R364A (corresponding to R360 in KCNQ1 and R325 in KCNQ2) and the H367C (corresponding to H328 in KCNQ2) mutations in KCNQ3T decreased current amplitudes. Dr-VSP showed the PIP2 affinity of these mutants to be decreased, suggesting the decrease of current for these mutants is also due to a lower PIP2 affinity for the channels. Our results suggest that PIP2 likely binds both to the linker between helices A and B and to the initial C-terminal domain in KCNQ3 channels.