Voltage-Sensitive PIP2 Phosphatase Shows PIP2 Requirements for KCNQ and Ca 2+ Channels

Abstract

The four talks in this symposium highlight the dependence of ion channel function on membrane phospholipids and fatty acids. Although of low abundance (1-2%), the phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) is a signature lipid of plasma membranes. The PIP2 lipid aids ion channel function there, and depleting PIP2 can decrease the activity of >80 types of channels (Logothetis et al, Pflugers 2010). A voltage sensitive 5-phosphatase (VSP) converts PIP2 to PI(4)P rapidly during strong depolarizing voltage steps (Okamura et al, J Physiol 2009). Exogenously expressed, this VSP serves to test the PIP2 dependence of plasma membrane ion channels and the kinetics of resynthesis of PIP2. Using the PLCdelta1 PH (pleckstrin homology) domain as a reporter, we measure the membrane PIP2 concentration as a change in FRET (Forster resonance energy transfer) between YFP-PH and CFP-PH. The PH-domain assay reports that strongly activated VSP depletes PIP2 with a time constant <150 ms. Activating VSP for 500 ms with a +100 mV depolarization reduces current in KCNQ channels by 90% (Falkenburger et al, JGP 2010) and reduces current in four subtypes of voltage-gated Ca2+ channels by 30-60% (Suh et al, Neuron 2010). The channel currents and the PH domain signals recover with time constants of 7-15 s. Such observations imply that: (1) KCNQ channels and several Ca2+ channels need PIP2 for best operation, (2) the PIP2 likely binds directly to the channel protein, (3) PH domains and channels form low-affinity complexes with PIP2 that last < 10 ms, and (4) resynthesis of PIP2 from plasma membrane PI(4)P takes only ∼10 s. This resynthesis from PI(4)P is much faster than the 150-s time seen for two-step resynthesis from PI after activation of receptors coupled to PLC. Support: NIH grants NS08174, GM83913.