Control of Inward Rectifier K Channel Activity by Lipid Tethering of Cytoplasmic Domains

Abstract

Interactions between nontransmembrane domains and the lipid membrane are proposed to modulate activity of many ion channels. In Kir channels, the so-called “slide-helix” is proposed to interact with the lipid headgroups and control channel gating. We examined this possibility directly in a cell-free system consisting of KirBac1.1 reconstituted into pure lipid vesicles. Cysteine substitution of positively charged slide-helix residues (R49C and K57C) leads to loss of channel activity that is rescued by in situ restoration of charge following modification by MTSET+ or MTSEA+, but not MTSES− or neutral MMTS. Strikingly, activity is also rescued by modification with long-chain alkyl-MTS reagents. Such reagents are expected to partition into, and hence tether the side chain to, the membrane. Systematic scanning reveals additional slide-helix residues that are activated or inhibited following alkyl-MTS modification. A pattern emerges whereby lipid tethering of the N terminus, or C terminus, of the slide-helix, respectively inhibits, or activates, channel activity. This study establishes a critical role of the slide-helix in Kir channel gating, and directly demonstrates that physical interaction of soluble domains with the membrane can control ion channel activity.

Molecular mechanism underlying ethanol activation of G-protein–gated inwardly rectifying potassium channels

“…Here, we show that Dr-VSPmediated depletion of PIP 2 completely reversed MTS-activated and alcohol-induced GIRK currents, suggesting that alcohol activation of GIRK require PIP 2 . Importantly, alcohol or MTS reagents did not affect PIP 2 or PIP kinase (enzyme that converts PIP to PIP 2 ) (46), because MTS reagents did not affect PIP 2 -dependent basal current in GIRK2* or PIP 2 interaction with inward rectifiers (47). The reliance of Gβγ-activated (19,20) and ethanol-activated GIRK channels on PIP 2 suggests convergence of gating mechanisms at the PIP 2 binding site.…”

The C-Terminal PAL Motif and Transmembrane Domain 9 of Presenilin 1 Are Involved in the Formation of the Catalytic Pore of the -Secretase

“…Thus, we speculate that the C-terminal short helix is tethered to the lipid bilayer by virtue of the hydrophobic nature of the extreme C terminus. This structure is reminiscent of a "slide helix," an amphipathic feature that contributes to the control of the conformation and activity of Kir6.2 potassium channels (Antcliff et al, 2005;Enkvetchakul et al, 2007). Notably, sequential deletion of the C-terminal residues of PS1 gradually decreased the ␥-secretase activity, and the deletion of residues 451-467, which correspond to the amphipathic region identified here, totally abolished the activity (Tomita et al, 1999;Bergman et al, 2004), implicating the auxiliary role of the short C-terminal helix in the assembly and activity of ␥-secretase.…”

Direct Regulation of Prokaryotic Kir Channel by Cholesterol

“…Purified KirBac1.1 or KcsA proteins (2.5-10 g/mg of total lipid) was added to CHAPS (37 mM) solubilized mixture of phosphatidylethanolamine:phosphatidylglycerol (9:1, Avanti Polar Lipids, Inc., 10 mg of total lipid/ ml) in buffer A and incubated for 30 min. The choice of lipids was based on a well established protocol for assaying the activity of purified KirBac1.1 channels, as described in earlier studies (19,(21)(22)(23). The ratio of 9:1 PE/PG was also based on previous studies, in which it was established that this lipid composition yields the highest stability of the liposomes, as determined by the maximal uptake of 86 Rb ϩ (23).…”

“…Furthermore, using a system of purified proteins incorporated into liposomes, we have previously demonstrated that KirBac1.1 channels exhibit many of the same key properties as eukaryotic Kir channels (19,21,22). Specifically, KirBac1.1, like eukaryotic channels, is highly sensitive to membrane phosphatidylinositol 4,5-bisphosphate (22).…”