Use of Voltage-Sensitive Phosphatase to Investigate the Location of the PIP2-Binding Site on KCNQ2 Channels

Abstract

Phosphatidylinositol 4,5-bisphosphate (PIP2) is known to be necessary for the activation of KCNQ K+ channels. Many studies have investigated the location of the PIP2-binding site on those channels, which have strongly implicated binding to the C-terminus. Our lab has suggested the linker between helices A and B in the C-terminus of KCNQ2 to be the primary site of PIP2-binding, with K452, R459 and R461 shown as critical (Hernandez et al., 2008, JGP, 132). However, other labs have reported PIP2 interactions to be dependent on basic residues at the start of the C-terminus (Zhang et al., 2003, Neuron, 37; Thomas et al., 2011, JBC, 286). A histidine at 328 position in KCNQ2 and four residues (K354, K358, R360, and K362) in KCNQ1 have been suggested to interact with PIP2. Since three of those four residues are conserved in KCNQ2-5 channels, we investigated a potential role of those residues in PIP2-channel interactions for KCNQ2. We found mutations at analogous positions in KCNQ2 (K319A, R325A and K327A) to decrease whole-cell current amplitudes. However, use of a voltage-sensitive phosphatase from danio rerio, which dephosphorylates almost all PIP2 upon strong membrane depolarizations, revealed the PIP2 affinity of the K319A and K327A mutants to be increased, rather than decreased, as assayed by the rates of current decay upon depolarization to 120 mV, and subsequent current recovery at 30 mV. These results indicate that the decrease of KCNQ2 currents for all the mutants is likely not due to a lower PIP2 affinity for the channels. Indeed, unlike KCNQ1, charge neutralization mutations at the start of the C-terminus seem to augment PIP2-binding to channels, consistent with the previously-indentified linker between helices A and B to be the primary PIP2-binding site in KCNQ2 channels.