Regulation of Kv7 (KCNQ) K⁺Channel Open Probability by Phosphatidylinositol 4,5-Bisphosphate

<jats:p>Voltage-gated Kv7 (KCNQ) channels underlie important K<jats:sup>+</jats:sup>currents, including the neuronal M current, and are thought to be sensitive to membrane phosphatidylinositol 4,5-bisphosphate (PIP<jats:sub>2</jats:sub>) and PIP<jats:sub>2</jats:sub>depletion to underlie muscarinic receptor inhibition. We studied regulation of Kv7.2-7.4 channels by PIP<jats:sub>2</jats:sub>in Chinese hamster ovary (CHO) cells using single-channel and whole-cell patch clamp and biochemical analysis. Maximal open probabilities (<jats:italic>P</jats:italic><jats:sub>o</jats:sub>) of Kv7.2-Kv7.4 homomultimers and of Kv7.2/7.3 heteromultimers were found to be strongly dependent on the [diC8-PIP<jats:sub>2</jats:sub>] applied to inside-out patches, with differential apparent affinities that correlate with their maximal<jats:italic>P</jats:italic><jats:sub>o</jats:sub>in on-cell mode. Unitary conductance was not affected by PIP<jats:sub>2</jats:sub>. Raising tonic [PIP<jats:sub>2</jats:sub>] by coexpression of phosphatidylinositol (4)5-kinase increased the maximal<jats:italic>P</jats:italic><jats:sub>o</jats:sub>of both Kv7.2 and Kv7.2/7.3 channels studied in on-cell patches and increased whole-cell Kv7.2, but not Kv7.3, current amplitudes. In cells coexpressed with muscarinic M<jats:sub>1</jats:sub>receptors, bath application of muscarinic agonist reduced the maximal<jats:italic>P</jats:italic><jats:sub>o</jats:sub>of Kv7.2/7.3 channels isolated in on-cell patches. Coexpression of a PIP<jats:sub>2</jats:sub>sequestering construct moderately reduced whole-cell Kv7.2/7.3 currents, and coexpression of a construct containing a PIP<jats:sub>2</jats:sub>phosphatase nearly abolished them. Finally, biochemical analysis of anionic phospholipids in CHO cells stably expressing M<jats:sub>1</jats:sub>receptors shows that PIP<jats:sub>2</jats:sub>and PIP are nearly depleted 1 min after muscarinic stimulation, with an unexpected rebound after 10 min. These results strongly support the direct regulation of Kv7 channels by PIP<jats:sub>2</jats:sub>and its depletion as the mechanism of muscarinic suppression of M channels. Divergent apparent affinities of Kv7.2-7.4 channels for PIP<jats:sub>2</jats:sub>may underlie their highly differential maximal<jats:italic>P</jats:italic><jats:sub>o</jats:sub>observed in cell-attached patches.</jats:p>