PIP 2 controls voltage-sensor movement and pore opening of Kv channels through the S4–S5 linker

Voltage-gated K + (Kv) channels couple the movement of a voltage sensor to the channel gate(s) via a helical intracellular region, the S4–S5 linker. A number of studies link voltage sensitivity to interactions of S4 charges with membrane phospholipids in the outer leaflet of the bilayer. Although the phospholipid phosphatidylinositol-4,5-bisphosphate (PIP 2 ) in the inner membrane leaflet has emerged as a universal activator of ion channels, no such role has been established for mammalian Kv channels. Here we show that PIP 2 depletion induced two kinetically distinct effects on Kv channels: an increase in voltage sensitivity and a concomitant decrease in current amplitude. These effects are reversible, exhibiting distinct molecular determinants and sensitivities to PIP 2 . Gating current measurements revealed that PIP 2 constrains the movement of the sensor through interactions with the S4–S5 linker. Thus, PIP 2 controls both the movement of the voltage sensor and the stability of the open pore through interactions with the linker that connects them. Note: By “distinct molecular determinants,” we mean that the two effects of PIP 2 (i.e., on voltage sensitivity and open probability) showed dependence on distinct amino acids. Thus, although mutation of K322 affected both voltage sensitivity and open probability, mutation of the N-terminal residue R147 controlled voltage sensitivity but not open probability, whereas mutation of R326 controlled open probability but not voltage sensitivity. Our data support a model in which electropositive residues in the N terminus and the S4–S5 linker interact with PIP 2 and exert two distinct effects. First, PIP 2 stabilizes the voltage sensor of Kv channels in a state of decreased sensitivity [charge–voltage (Q–V) and conductance–voltage (G–V) curves are shifted to the right], favoring the closed state of the channel. Second, PIP 2 stabilizes the Kv channel pore in the conducting state. These two apparently contradictory effects could be distinguished by differences in kinetics, sensitivities to PIP 2 , and molecular determinants. This study, which provides molecular evidence linking PIP 2 to the activation mechanism of Kv channels, does not explain why PIP 2 has the dual effect we have described in certain Kv channels (e.g., Kv1, CaV, HCN) but not in others (e.g., KCNQ, HERG, TRP, K2P). The molecular and physiological reasons for this differential design in voltage-gated channel regulation remain to be clarified. We identi