Structural Determinants of Phosphatidylinositol 4,5-Bisphosphate (PIP2) Regulation of BK Channel Activity through the RCK1 Ca2+ Coordination Site

Abstract

Background: PIP 2 has been reported to enhance Ca 2ϩ -driven gating, but the molecular determinants of this interplay are not known. Results: PIP 2 interacts with specific basic residues and enhances Ca 2ϩ gating through the ␣A-KDRDD-␣B structural elements. Conclusion: The RCK1 Ca 2ϩ -binding site is coupled to PIP 2 . Significance: PIP 2 is a key element in the regulation of BK channel activity.

Phosphoinositide Control of Membrane Protein Function: A Frontier Led by Studies on Ion Channels

“…Another study explored the relationship between Ca 2+ sensitivity of the Slo1 subunit of big-conductance K + (BK) channels and PI(4,5)P 2 regulation (80). BK channels show little to no rundown in inside-out patch recordings.…”

“…Yet, rundown can be observed under conditions of low [Ca 2+ ] i . The PI(4,5)P 2 sensitivity of these channels (81) was tightly coupled to the RCK1 Ca 2+ coordination site (80). In this case, increases in [Ca 2+ ] i allosterically enhance channel-PI(4,5)P 2 interactions and thus channel activity.…”

Mutations in Nature Conferred a High Affinity Phosphatidylinositol 4,5-Bisphosphate-binding Site in Vertebrate Inwardly Rectifying Potassium Channels

“…Electrophysiology-Potassium currents were recorded from oocytes using standard electrophysiological methods (22)(23)(24)(25)(26). For two-electrode voltage clamp (TEVC), we used a Geneclamp 500B amplifier (Axon Instruments, Foster City, CA) or an OC-725C amplifier (Warner Instruments, Hamden, CT).…”

“…diC 8 PIP 2 was purchased from Avanti Polar Lipids or Echelon Biosciences (Salt Lake City, UT). The lipids were prepared as described previously (23,25,27). All other chemicals were purchased from Sigma or VWR.…”

“…Oocytes were released and defolliculated by gentle agitation in 0.5 mg/ml collagenase A (Sigma) dissolved in a Ca 2ϩ -free solution containing (in mM) 96 NaCl, 2 KCl, 1 MgCl 2 , 5 HEPES, pH 7.4. Oocytes were then washed and selected, and stage V/VI oocytes were microinjected with 36 -50 nl of cRNA dissolved in diethyl pyrocarbonate-treated water (0.05-15 ng of cRNA/oocyte), depending on functional expression levels (23)(24)(25). Oocytes were maintained for 2-12 days at 14 -17°C in a solution containing (in mM) 96 NaCl, 1 KCl, 1 CaCl 2 , 2 MgCl 2 , 10 HEPES, 0 -5 sucrose, and 2 sodium pyruvate, pH 7.4, with 50 units/ml penicillin G and 50 g/ml streptomycin.…”