Phosphatidylinositol-4,5-bisphosphate is required for KCNQ1/KCNE1 channel function but not anterograde trafficking

Abstract

The slow delayed-rectifier potassium current (IKs) is crucial for human cardiac action potential repolarization. The formation of IKs requires co-assembly of the KCNQ1 α-subunit and KCNE1 β-subunit, and mutations in either of these subunits can lead to hereditary long QT syndrome types 1 and 5, respectively. It is widely recognised that the KCNQ1/KCNE1 (Q1/E1) channel requires phosphatidylinositol-4,5-bisphosphate (PIP2) binding for function. We previously identified a cluster of basic residues in the proximal C-terminus of KCNQ1 that form a PIP2/phosphoinositide binding site. Upon charge neutralisation of these residues we found that the channel became more retained in the endoplasmic reticulum, which raised the possibility that channel–phosphoinositide interactions could play a role in channel trafficking. To explore this further we used a chemically induced dimerization (CID) system to selectively deplete PIP2 and/or phosphatidylinositol-4-phosphate (PI(4)P) at the plasma membrane (PM) or Golgi, and we subsequently monitored the effects on both channel trafficking and function. The depletion of PIP2 and/or PI(4)P at either the PM or Golgi did not alter channel cell-surface expression levels. However, channel function was extremely sensitive to the depletion of PIP2 at the PM, which is in contrast to the response of other cardiac potassium channels tested (Kir2.1 and Kv11.1). Surprisingly, when using the CID system IKs was dramatically reduced even before dimerization was induced, highlighting limitations regarding the utility of this system when studying processes highly sensitive to PIP2 depletion. In conclusion, we identify that the Q1/E1 channel does not require PIP2 or PI(4)P for anterograde trafficking, but is heavily reliant on PIP2 for channel function once at the PM.

A conserved arginine/lysine-based motif promotes ER export of KCNE1 and KCNE2 to regulate KCNQ1 channel activity

“…Positively charged amino acids have also been reported to facilitate ER export [42,43]. Thus the positively charged amino acids (K and R) in KSKRR or KSKR may be responsible for the forward trafficking of KCNE2.…”

“…Therefore, we speculate that KCNE2 and KCNE1 are very likely to interact directly with the Sar1 through the [R/K](S)[R/K][R/K] motif presented at the C-terminus for ER export. It has also been suggested that the positively charged amino acids may associate with phospholipid components, which are present on the cellular membrane and involved in vesicle formation and trafficking [47], thus modulating ER export or plasmamembrane localization of membrane proteins [42,48]. Since the residues R67, K69, K70, and H73 in KCNE1 have been reported to be a key structural determinant of I Ks phosphatidylinositol 4,5-bisphosphate (PIP2) sensitivity [49], so it cannot be ruled out that the positively charged amino acids in the proximal C-terminus promote the ER export of KCNE1 and KCNE2 through binding to PIP2 or other phosphoinositide species.…”

Membrane pools of phosphatidylinositol-4-phosphate regulate KCNQ1/KCNE1 membrane expression

“…Residues in the cytoplasmic domains of KCNQ1 have been shown to bind to other phospholipids 12 , 22 , 29 , 30 , including PI4P 12 , although consequences of the interactions with PI4P have not been explored. Recently, acute depletion of PI4P from the plasma membrane was shown not to affect KCNQ1/KCNE1 function 31 . In contrast to our results, the same study showed that prolonged depletion of membrane PI4P did not affect membrane expression using an N-terminus KCNE1 tagged construct fused to the KCNQ1 subunits.…”

“…GFP-tagged constructs were fused with GFP at the C-terminus. Additional constructs used were Pseudojanin (PJ), a fusion protein of Sac and INPP5E phosphatase domains with FKBP (Addgene, #37999); PJ-Sac, a PJ based construct containing an inactivating mutation (Asp1263Ala) in the INPP5E domain (Addgene, #38000); PJ-INPP5E, a PJ based construct with an inactivating mutation (Cys779Ser) in the SAC1 domain (Addgene, #38001); PJ-DEAD, a PJ based construct containing an inactivating mutation (Asp1263Ala) in the INPP5E domain and an inactivating mutation (Cys779Ser) in the SAC1 domain (Addgene, #38002) 10 , 31 ; PH-PLCδ1-GFP (Addgene, #51407); LYN11-FRB-mcherry (Addgene, #38004); GFP-P4M-SidM (Addgene, #51469), hKCNH2-EGFP (VectorBuilder); EGFP-Kir2.1 (Addgene, #107184); Kv7.4-EGFP (Addgene, #111453); Kv2.1-EGFP (Addgene, #111531); constitutively active PI4K (Addgene, #139311); dominant negative PI4K-dead (Addgene, #139312); KCNE1-KCNQ1 tandem 33 . The adenoviral constructs hKCNQ1-GFP, hKCNE1 (Vector Biolabs) were used to infect rat ventricular cardiomyocytes.…”

Imipramine as an alternative to formamide to detubulate rat ventricular cardiomyocytes

“…Therefore it constitutes an alternative to the drastic osmotic shock produced by formamide to detubulate cells, interfering with the binding of the phospholipid to BAR domain-containing proteins (Suetsugu, Kurisu, & Takenawa, 2014). However, it is worth noticing that it may interfere with many cellular functions since PI(4,5)P 2 not only is crucial for membrane tubulation (Hofhuis et al, 2017;Inaba et al, 2016), but is an actor in endocytosis and exocytosis (Suetsugu et al, 2014), regulates ion channels (Royal, Tinker, & Harmer, 2017), and is involved in Ca 2+ signalling and intracellular pathways (Berridge, 2009). In ventricular myocytes, bridging integrator 1 (BIN1) is likely to interact with PI(4,5)P 2 because it contains a BAR domain and thus may allow the correct folding of the T-tubules and formation of the dyad (Fu et al, 2016;Hong et al, 2014).…”