Activation of M-Type Potassium Channels by Different Membrane Phospholipids and Analogs

Abstract

M-type (Kv7.2/7.3) channels are activated by the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) [Li et al., 2005: J.Neurosci., 25,9825], through interaction with a cluster of basic residues in the C-terminus [Hernandez et al, 2008: J.Gen.Physiol., 132,361]. However, little is known of the phospholipid specificity and requirements for this activation. We have explored this using inside-out membrane patches from CHO cells stably-expressing Kv7.2 and Kv7.3 subunits and held at a constant voltage ca. −20 mV. The dioctanoyl mono-, di- and tri-phosphatidylinositides DiC8-PI(4)P, DiC8-PI(4,5)P2 and DiC8-PI(3,4,5)P3 all produced biphasic Popen-concentration curves, maximizing at Popen ∼0.8. EC50s for the ‘high-affinity’ component 1 (maximum Popen ∼0.2) were similar at ∼1 μM; ‘low-affinity’ EC50s were inversely proportional to the number of phosphates (DiC8-PI(4)P ∼100 μM, DiC8-PI(4,5)P2 ∼50 μM, DiC8-PI(3,4,5)P3 ∼35 μM). In contrast, the inositol phosphates I(1,4,5)P3 and I(4,5)P2 neither activated nor inhibited the channels up to 300 μM, suggesting a crucial role for the lipophilic moiety. This was tested further using sphingosine-1-phosphate (S-1-P), fingolimod phosphate (FTY720-P) and 1-oleoyl lysophosphatidic acid (LPA). All three activated the Kv7.2/7.3 channels, to Popen values at 100 μM of ∼0.8 (LPA), 0.15 (S-1-P) and 0.022 (FTY720-P). In each case ‘high’ and ‘low’ affinity components to the activation curves could be discerned, with EC50s of 1.5 and 40 μM (LPA), 3 and 160 μM (S-1-P) and 0.5 and 61 μM (FTY720-P). No channel activation was observed using membrane lipids devoid of phosphate groups (D-erythrosphingosine, fingolimod, phosphatidylglycerol and phosphatidylcholine, all at 100 μM). Thus, M-channels can be activated in a rather similar manner by a range of membrane lipids, the minimal requirements at concentrations tested being one or more terminal phosphates and a lipophilic domain.Supported by Wellcome Trust grant 085419.