PI4Kβ, PIPs and BKCa channel function

Sci. Bull. (2016) 61(23):1779–1782 DOI 10.1007/s11434-016-1186-8

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PI4Kb, PIPs and BKCa channel function Xiuli Cheng • Xiaoqiu Tan • Weixia Liu • Hui Li Li Yan • Yan Yang • Xiaorong Zeng • Jimin Cao

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Published online: 28 October 2016 Ó Science China Press and Springer-Verlag Berlin Heidelberg 2016

Large conductance calcium-activated potassium (BKCa) channels are broadly expressed in vascular smooth muscle cells (VSMCs) and play a crucial role in the regulation of vascular tone [1]. Activation of BKCa channel by elevation of the [Ca2?]i due to membrane depolarization increases the K? conductance of the membrane, and drives the VSMC membrane potential to hyperpolarization, which in turn, closes the L-type voltage-dependent Ca2? channels, decreases the global [Ca2?]i of VSMCs, and induces vascular relaxation [2]. Enhancing the BKCa current is a practicable strategy in treating diseases with increased vascular tone such as hypertension and coronary artery syndrome [2–4]. The synthesized subunits of BKCa channel need to be transferred and docked to the cell membrane to function. Xiuli Cheng and Xiaoqiu Tan contributed equally to this work. X. Cheng
X. Tan
L. Yan
J. Cao (&) Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing 100730, China e-mail: [email protected] Present Address: X. Cheng Clinical Laboratory, Tianjin Huanhu Hospital, Tianjin 300350, China X. Tan
H. Li
Y. Yang
X. Zeng Key Laboratory of Medical Electrophysiology, Ministry of Education, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease, Institute of Cardiovascular Research, Southwest Medical University, Luzhou 646000, China W. Liu Clinical Laboratory, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250355, China

Therefore, elucidation of channel protein trafficking mechanisms may help to develop strategies for channel manipulation therapy in vascular diseases. It is known that phosphoinositides (PIs) exert a role in the protein trafficking/function and their metabolism is controlled by phosphatidylinositol kinases (PIKs). The family of PIKs in mammalian cells has two type II PI4Ks (a and b) and two type III enzymes (a and b). At present, little is known about how PIKs regulate the trafficking and other activities of ion channels. The present study aimed to investigate the regulatory roles of PI4Kb on the activities of BKCa channels including expression, trafficking and electrophysiology. The study also investigated the underlying mechanisms with a focus on the interactions between BKCa channel and phospholipids. The results may help to reveal the role of PI4Kb in the regulation of BKCa channel function and to provide insight into the BKCa channel-related diseases. To observe the behaviors of BKCa channel, we transfected the human BKCa gene (hSlo1) to HEK293 cells with the reconstructed plasmid pcDNA3.1-Flag-hSlo1-EGFP, and confirmed that the Flag and GFP tags did not affect the electrophysiological property and expression of BKCa channels in the membrane. PI4KIIIb shRNA plasmid was also transfected to HEK293 cells to observe the effect of PI4KIIIb on the activities of BKCa channel. Western blotting and flow cytometry was performed to evaluate the subcellular localization of hSlo1. Protein-lipid overlay assay was performed using the PIP Strips (Echelon Biosciences, USA) to determine the protein-phospholipids interaction/binding [5]. Whole-cell and inside-out patch (single channel) recordings of BKCa currents (IBKCa) and channel open probability were performed to observe the effects of PI4Kb and PIs on the electrophysiological activities of BKCa channels. Data were expressed as mean ± SEM.

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We demonstrated that PI4KIIIb involved in the regulation of BKCa channel a subunit (hSlo1) expression in HEK293 cells. Transfection of HEK293 cells with the PI4KIIIb RNAi plasmid significantly decreased the total expression level of hSlo1 compared with the control cells (Fig. 1a, b). This result suggests that PI4KIIIb enhances the expression of BKCa channel a subunit. The pharmacological inhibition of PI4Kb with wortmannin and phenylarsine oxide (PAO) suggests that PI4Kb enhances the trafficking of BKCa channel protein possibly via catalyzing the production of PIP2. PI4Kb and PIP2 accelerated the trafficking of BKCa channel towards the cell membrane. Blocking PI4Kb with 10 lmol/L wortmannin (a type III PI4K-sensitive inhibitor of PI4Ks) [6] or 3 lmol/L PAO (a more specific inhibitor for PI4K) [7] inhibited the trafficking of BKCa channel protein, as indicated by the reduction of the MFIFlag/GFP ratio (normalized to control) (Fig. 1c, d), and the reduction of the Flag-positive cell percentage in the GFP-positive cell pool (Fig. 1e). RNA interference (shRNA) for PI4KIIIb also decreased the MFIFlag/GFP ratio (Fig. 1f). Furthermore, wortmannin or PAO significantly decreased the intracellular PIP2 level (Fig. 1g). These results suggest that PI4Kb enhances the trafficking of BKCa channel protein towards the plasma membrane potentially via catalyzing the production of PIP2. Although phospholipids have been found able to interact with BKCa channel and regulate their activities [8], it is still unclear what kinds of phospholipids could specifically interact with the BKCa channel. Taking the advantage of protein-lipid overlay assay (PIP strips), we identified seven phospholipids which could directly interact with the BKCa channel protein. Fifteen phospholipids were pre-spotted on a hydrophobic membrane and the location of each phospholipid is shown in Fig. 1h. The lysate of control HEK293 cells (without hSlo1 transfection) basically showed negative binding with these phospholipids, except that three kinds of PIPs (PI(3)P, PI(4)P and PI(5)P) showed weak binding with hSlo1 (Fig. 1i, gray spots in the left column of the strip). HEK293 cell lysate containing hSlo1 showed strong bindings with seven of the fifteen tested phospholipids, and these seven phospholipids were all phosphorylated PIs (i.e., phosphoinositide (mono/bis/tris) phosphates, namely PIPx) (Fig. 1j, the black spots), including three kinds of PIP (PI(3)P, PI(4)P and PI(5)P), three kinds of PIP2 (PI(3,4)P, PI(3,5)P and PI(4,5)P) and one kind of PIP3 (PI(3,4,5)P3). The remaining eight phospholipids, including lysophosphatidic acid (LPA), lysophosphocholine (LPC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingosine 1-phosphate (S1P), phosphatidic acid (PA) and phosphatidylserine (PS), did not show interactions with the BKCa protein (Fig. 1j). These results suggest that BKCa

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channel tends to be regulated by phosphorylated PIs, and at least four phosphorylated PIs (three kinds of PIP2 and PIP3) showed specific interaction/binding with hSlo1 (Fig. 1j) compared with the control (only the three PIPs showed weak interaction, Fig. 1i). Lipid regulation of BKCa channel function has gained attention for about two decades. The lipids that have been reported to modulate BKCa current include fatty acids and their derivatives (such as prostanoids, epoxyeicosatrienoic acids and leukotrienes), phospholipids and their derivatives (such as glycerophospholipids, phosphoinositides, lysophospholipids and sphingolipids), and steroids and their derivatives (such as cholesterol and vitamin D) [9]. Because lipids especially phospholipids are the major components of cell membrane, they may form an immediate lipid environment for membrane proteins including ion channels, and even directly interact with these membrane proteins to modulate their function by the ‘‘first come, first served’’ logic in evolution. Among the membranous phospholipids, PI4P, the direct metabolic product of PIs catalyzed by PI4Ks, can be further phosphorylated into PIP2 by PIP5Ks. Previously, PI4P has mainly been regarded as a precursor of PIPs. In recent years, PI4P has been proved as a fundamental signaling molecule in various cell activities, such as membrane protein trafficking and membrane charge, not merely acts as a precursor of PIPs [9]. Thus, PI4Ks may gain an important identity based on the special importance of PI4P. Generated from PI4P, the molecule PIP2 is further cleaved by phospholipase C (PLC) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), and thus initiate downstream signal cascades respectively through intracellular Ca2? stores and protein kinase C (PKC) and thereby regulate cell function. We further identified that PI4Kb interacts with certain PIPs and affects the PIP2 level. At the electrophysiological level, blockade of PI4Ks either with wortmannin (type III PI4K-sensitive) (10 lmol/ L) or with PAO (PI4K-specific) (3 lmol/L) significantly suppressed the BKCa macrocurrent (Fig. 1k, l). In the insideout patch recording, PIP2 (10 lmol/L) given at the ‘‘intracellular’’ side increased the open probability (NPo) of BKCa channel by over threefold (Fig. 1m, n). These results suggest PI4Kb (possibly including PI4KIIIb) and PIP2 promote the opening of BKCa channel and increased the BKCa currents. A previous study showed that PIP2 could directly activate the BKCa channel of VSMCs, skeletal myocytes and HEK cells under an inside-out configuration of patch clamp [10]. Here, we confirmed that PIP2 increased the total open probability (NPo) of single BKCa channel in HEK293 cells, and further showed that inhibition of PI4Ks by wortmannin or PAO suppressed the BKCa macro-current under the whole-cell configuration. These results are in consistent with the previous report [9], and provide further evidence

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1782 b Fig. 1 Serial assays illustrating the regulatory role of PI4Kb on the

activities of BKCa channel in HEK293 cells expressing human BKCa channel a subunits (hSlo1). a and b RNAi of PI4KIIIb decreased the expression of hSlo1 evaluated by Western blotting. Here ‘‘Control’’ means HEK293 cells transfected with a control shRNA (the same bellow for RNAi). c-f flow cytometry showing that blocking PI4Kb with wortmannin (10 lmol/L) or phenylarsine oxide (PAO) (3 lmol/ L), or RNAi of PI4KIIIb, reduced the MFIFlag/GFP ratio, decreased the Flag-positive cell percentage in the GFP-positive cell pool and membranous hSlo1 level, suggesting an enhancing effect of PI4Kb on the trafficking of BKCa protein. Here ‘‘negative’’ means HEK293 cells without BKCa channel transfection, ‘‘control’’ means HEK293 cells transfected with BKCa channel but without drug treatment. g blocking PI4Ks by wortamannin or PAO decreased the PIP2 level. h-j, proteinlipid overlay assay (PIP strip) showing the interactions of phospholipids with BKCa channel protein (hSlo1). ‘‘Control’’ indicates HEK293 cells without hSlo1 transfection. The full names of the abbreviations for the phospholipids can be found in the main text. k– n, whole-cell recording and inside-out patch (single channel) recording showing the enhancing effects of PI4Kb and PIP2 on the BKCa macrocurrents and channel open probability (NPo). *P \ 0.05, **P \ 0.01 vs. control

demonstrating the impact of PI4Kb on the electrophysiological activity of BKCa channels. In summary, we demonstrated that PI4Kb, possibly the PI4KIIIb or at least including the PI4KIIIb, promotes the expression, trafficking and electrophysiological activity of BKCa channels via facilitating the production of PIP2 and the interactions of PIPx (at least the PIP2 and PIP3) with BKCa channels. The study suggests that PI4Kb may be a target for the controlling of BKCa channel function. Acknowledgments This work was supported by the National Natural Science Foundation of China (31171088 and 31471126 to JMC., and 31300948 and 81670310 to XQT). We thank Prof. Ahring PK (NeuroSearch A/S, Denmark) for kindly providing the template plasmid pcDNA3.1-hSlo1.

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Sci. Bull. (2016) 61(23):1779–1782 Conflict of interest The authors declare that they have no conflict of interest.

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