Crosstalk between cholesterol and PIP2 in the regulation of Kv7.2/Kv7.3 channels

It is well known that Kv7 channels activation is critically dependent on the lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Furthermore, we have recently shown that Kv7.2/Kv7.3 channels are �nely modulated by cholesterol. In this study, we have investigated the crosstalk between cholesterol and PIP 2 in the regulation of Kv7.2/Kv7.3 channels transiently expressed in HEK-293 cells. To evaluate the effect of cholesterol on PIP 2 -mediated regulation of Kv7.2/Kv7.3 channels, methyl-β -cyclodextrin was acutely applied to decrease the plasma membrane cholesterol level. Currents passing through Kv7.2/Kv7.3 channels in cholesterol-depleted cells were less sensitive to PIP 2 dephosphorylation strategies, either via the voltage-sensitive phosphatase Dr-VSP or the rapamycin-translocatable lipid 5-phosphatase system, compared to control cells, suggesting that cholesterol depletion limits the action of these phosphatases, probably by strengthening the interaction between Kv7.2/Kv7.3 channels and PIP 2 . In contrast, the sensitivity of the Kv7.2/Kv7.3 channels to acute membrane cholesterol depletion by methyl-β -cyclodextrin was not modi�ed in channels with different apparent a�nities for PIP 2 . Taken together, our results reveal a novel functional crosstalk between cholesterol and PIP 2 in the regulation of Kv7.2/Kv7.3 channels.


Introduction
The heteromeric assembly of Kv7.2 and Kv7.3 subunits (encoded by the KCNQ2 and KCNQ3 genes, respectively) forms low-threshold, non-inactivating voltage-gated K + channels that underlie the M-current in neurons and play a crucial role in the regulation of neuronal excitability (Wang 1998).Typically de ned as voltage-gated channels, it has recently been suggested that Kv7.2/Kv7.3should also be considered as ligand-activated ion channels (Abbott 2020), as they are stimulated by a wide variety of exogenous and endogenous compounds acting on the pore, the voltage-sensing domain, or the coupling between them (Zaydman and Cui 2014; Manville et al. 2018;Larsson et al. 2020).In particular, the Kv7.2/Kv7.3channels are regulated by different lipids (Taylor and Sanders 2017), with phosphatidylinositol 4,5bisphosphate (PIP 2 ) being strictly required for their opening (Suh and Hille 2002;Zhang et al. 2003).
Recently, we have also shown that Kv7.2/Kv7.3channels are overly sensitive to acute changes in plasma membrane cholesterol content, both depletion and enrichment of plasma membrane cholesterol from the basal level inhibit Kv7.The present study aims to determine the functional interaction between cholesterol and PIP 2 in the regulation of human Kv7.2/Kv7.3channels transiently expressed in HEK-293 cells.

Reagents
Methyl-β-cyclodextrin (MβCD) and rapamycin (prepared solution, 2.4 mg/ml in DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA).In Dr-VSP and FKBP-Inp54p experiments (see below), MβCD was added to the external solution and applied to patch-clamped cells 5 minutes before current recording.
The nal DMSO concentration in the rapamycin experiments was < 0.05%, which did not modify the basal properties of Kv7.2/Kv7.3channels (data not shown).

Data and statistical analysis
Patch-clamp data were processed with Clamp t 10 software (Molecular Devices) and analyzed with Origin 8.6 software (OriginLab Corp., Northampton, MA, USA).Data are presented as the mean ± S.E.M.Where appropriate, Student's unpaired t-test or one-way ANOVA was used to assess statistical differences, normality of the residuals was evaluated by the Shapiro-Wilk test.Statistical signi cance was set at p < 0.05.
Previous studies have shown that both cholesterol and PIP 2 regulate Kv7.2/Kv7.3channels.However, a possible functional interplay between these two membrane lipids on Kv7.2/Kv7.3channels has not been explored.To address this question, we rst assessed the inhibition of Kv7.2/Kv7.3channels induced by PIP 2 dephosphorylation strategies under basal and reduced plasma membrane cholesterol levels.To this end, we co-expressed Kv7.2/Kv7.3channels with the Danio rerio voltage-dependent phosphatase (Dr-VSP), which catalyzes the removal of a phosphate group at position 5 of the inositol ring, resulting in a rapid and reversible depletion of PIP 2 (Hossain et al. 2008).We took advantage of the fact that Dr-VSP is inactive at low-and middle-depolarized potentials but is activated at high depolarizations.We rst depolarized the cells to -30 mV (from a holding potential of -80 mV) to measure basal Kv7.2/Kv7.3currents, then stepped to + 100 mV for 1.5 seconds to fully activate Dr-VSP, and nally returned to -30 mV to measure currents after evoked PIP 2 depletion (Fig. 1).In cells with basal cholesterol levels, the reduction of Kv7.2/Kv7.3currents after Dr-VSP activation was 73.2 ± 3.9 % (n = 12; Fig. 1A, ), whereas in cells pretreated with MbCD (5 mM for 5 minutes) it was only 37.3 ± 8.1 % (n = 12; Fig. 1B, C, indicating for the latter a signi cant lower depletion of PIP 2 by Dr-VSP action (p = 0.0005).As a rst possibility, these results suggest a strengthening of the PIP 2 -Kv7.2/Kv7.3channels interaction in cholesterol-depleted cells that hinders the action of Dr-VSP.However, an alternative explanation is that Dr-VSP, which is a transmembrane protein, was also affected by the reduction of plasma membrane cholesterol and its activity on PIP 2 was decreased.To de ne this issue, we decided to use a different experimental approach to dephosphorylate PIP 2 .We co-expressed Kv7.2/Kv7.3channels with a rapamycin-translocatable lipid 5phosphatase (FKBP-Inp54p) and the membrane anchor LDR-CFP.The FKBP and LDR domains dimerize when cells are exposed to rapamycin, leading to dephosphorylation of PIP 2 at position 5 of the inositol ring.Rapamycin (1 mM)-induced recruitment of FKBP-Inp54p to the plasma membrane reduced Kv7.2/Kv7.3currents by 50.7 ± 8.7 % (n = 8; Fig. 1D, F in cells with basal cholesterol levels, and just by 26.0 ± 5.9 % (n = 7; Fig. 1E, F in cells pretreated with MbCD (p = 0.04).Taken together, these results show that the reduction of cholesterol levels render Kv7.2/Kv7.3channels less sensitive to inhibition by PIP 2 depletion strategies.Inhibition of Kv7.2/Kv7.3channels by cholesterol depletion is not altered in channels with different a nities for PIP 2 .

Discussion
Previous studies have demonstrated a key role for cholesterol and PIP 2 in modulation of ion channels Cholesterol and PIP 2 antagonistically modulate Kir2.1 channels, the former acting as an inhibitor (Romanenko et al. 2004), while PIP 2 is essential for its opening (Zhang et al. 1999 (Glaaser and Slesinger 2017).
Different residues are known to be important for PIP 2 -dependent activation of Kv7.2/Kv7.3channels (Hernandez et al. 2008), and mutations in these residues change the apparent a nity for PIP 2 (Hernandez et al. 2009).Similarly, the apparent a nity of Kv7.2 channels for PIP 2 is modulated by phosphorylation (Salzer et al. 2017) or methylation (Kim et al. 2016) events of the channels.On the other hand, although the key residues involved in cholesterol regulation of Kv7.2/Kv7.3channels have not yet been identi ed, our present results suggest that the presence of cholesterol modulates the apparent a nity of Kv7.2/Kv7.3 for PIP 2 .Thus, cholesterol depletion by MbCD pretreatment would strengthen PIP 2 -channel interactions, making the channels less prone to PIP 2 depletion strategies (Fig. 1).
We have previously shown that at basal levels plasma membrane cholesterol promotes the activity of Kv7.2/Kv7.3channels (Delgado-Ramírez et al. 2018).Thus, cholesterol depletion partially inhibits Kv7.2/Kv7.3currents (Fig. 2A, D).Interestingly, our present data show that the cholesterol sensitivity of the Kv7.2/Kv7.3channels is not modi ed in channels with different a nities for PIP 2 (Fig. 2), suggesting that the functional interaction between cholesterol and PIP 2 only interferes with channels modulation by PIP 2 .
Taken together, our data show that, under basal conditions, cholesterol promotes the activity of Kv7.2/Kv7.3channels and, in turn, modulates the apparent a nity of the channels for PIP 2 .Thus, the dynamic state of membrane cholesterol levels could make the channels more or less sensitive to PIP 2 depletion in response to different physiological and pharmacological challenges.Therefore, there is a complex and delicate modulation of Kv7.2/Kv7.3channels activity as a result of the dynamic interaction between cholesterol and PIP 2 that deserves further investigation.

Declarations Figures
2/Kv7.3 currents (Delgado-Ramírez et al. 2018).PIP 2 and cholesterol are two prominent plasma membrane lipids involved in several cellular processes, such as protein regulation, membrane organization and cell signaling (Mandal 2020; Schade et al. 2020).PIP 2 plays a key role as a precursor molecule in multiple receptors signaling pathways.Its hydrolysis by phospholipase C (PLC) produces inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG), both important intracellular second messengers involved in Ca 2+ mobilization from the endoplasmic reticulum and PKC activation, respectively (Berridge 1984).PIP 2 itself acts as a signaling molecule interacting with different proteins, including ion channels and transporters (Hille et al. 2015; Wills and Hammond 2022).Cholesterol is an essential component of the mammalian plasma membrane that in uences its structural and biophysical properties, such as thickness, uidity, compressibility, water penetration, and intrinsic curvature (By eld et al. 2004; Khatibzadeh et al. 2012; Subczynski et al. 2017).Therefore, slight variations in the cholesterol content of the plasma membrane can affect the functions of membrane proteins (Sabine 1983; Meza et al. 2020).Moreover, cholesterol can also directly interact with membrane proteins and regulate their function (Rosenhouse-Dantsker 2019; Kumar et al. 2021).An interplay between PIP 2 and cholesterol in the regulation of membrane proteins (Rosenhouse-Dantsker et al. 2014; Bukiya and Rosenhouse-Dantsker 2017) and cellular processes (Kwik et al. 2003; Hao and Bogan 2009) has been documented.It has been shown, for example, that depletion of membrane cholesterol strengthens the interaction between Kir2 channels and PIP 2 (Rosenhouse-Dantsker et al. 2014).In cultured b (INS-1) cells, cholesterol regulates the level of plasma membrane PIP 2 (depletion of membrane cholesterol promotes PIP 2 hydrolysis, while cholesterol overload increases PIP 2 accumulation), in turn affecting glucose-induced actin reorganization, membrane depolarization, and insulin secretion (Hao and Bogan 2009).