Abstract
Phosphoinositides, in particular, phosphatidylinositol 4,5-bisphosphate (PIP2), are known regulators of numerous ion channels and have been implicated in cellular processes such modulation of the actin cytoskeleton, cell migration and vesicle trafficking. Recently, it has been shown that PIP2 modulates BK channel activity through a direct interaction with its cytoplasmic domain (Vaithianathan et al., 2008). To understand the molecular nature of this modulation, we expressed and purified the region of the human BK channel C terminus (322IIE...ALK1005) corresponding to the Gating Ring (GR) apparatus, which includes the Regulators of K+ Conductance (RCK1 and RCK2) domains, and the RCK1-S6 linker. Using steady-state and time-resolved fluorescence spectroscopy, we have investigated the effects of the soluble PIP2 form (PIP2-diC4 and PIP2-diC8) on the conformational properties of GR in physiologically-relevant conditions. These lipids quenched the Tryptophan (Trp) fluorescence of GR in a dose-dependent and saturable fashion (Khalf = 21±0.88 μM; n = 2.3±0.094), suggesting PIP2-mediated conformational changes. PIP2 did not cause appreciable changes in the fluorescence intensity of a Trp analog (NATA, 5 μM). Similarly, a solution containing the purified RCK2 domain (665HDP...ALK1005) was largely insensitive to PIP2 up to 62 μM. The fluorescence lifetime of Trp residues was reduced by the addition of PIP2-diC8 (τavg from 2.3 ns to 1.5 ns, PIP2 62 μM), revealing the dynamic nature of PIP2 quenching of the Trp residues. Moreover, PIP2-diC8 significantly reduced the fluorescence intensity of GR complexed with ANS (8-Anilinonaphthalene-1-sulfonate), suggesting that the structural rearrangements caused by PIP2 may correlate with a decrease in the surface hydrophobicity of the GR. In summary, the BK GR appears to be sensitive to micromolar PIP2. The corresponding structural changes in this sensor may represent the elementary steps of the BK channel lipid modulation pathway.
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