Inositol lipid binding and membrane localization of isolated pleckstrin homology (PH) domains. Studies on the PH domains of phospholipase C delta 1 and p130.

Endocrinology and Reproduction Research Branch, NICHD/National Institutes of Health, 49 Convent Drive, Bldg. 49, Bethesda, MD 20892, USA.
Journal of Biological Chemistry (Impact Factor: 4.6). 08/2002; 277(30):27412-22. DOI: 10.1074/jbc.M109672200
Source: PubMed

ABSTRACT The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inositol lipids or soluble inositol phosphates in vitro and to localize to cellular membranes in live cells was examined by comparing the PH domains of phospholipase Cdelta(1) (PLCdelta(1)) and the recently cloned PLC-like protein p130 fused to the green fluorescent protein (GFP). The prominent membrane localization of PLCdelta(1)PH-GFP was paralleled with high affinity binding to inositol 1,4,5-trisphosphate (InsP(3)) as well as to phosphatidylinositol 4,5-bisphosphate-containing lipid vesicles or nitrocellulose membrane strips. In contrast, no membrane localization was observed with p130PH-GFP despite its InsP(3) and phosphatidylinositol 4,5-bisphosphate-binding properties being comparable with those of PLCdelta(1)PH-GFP. The N-terminal ligand binding domain of the type I InsP(3) receptor also failed to localize to the plasma membrane despite its 5-fold higher affinity to InsP(3) than the PH domains. By using a chimeric approach and cassette mutagenesis, the C-terminal alpha-helix and the short loop between the beta6-beta7 sheets of the PLCdelta(1)PH domain, in addition to its InsP(3)-binding region, were identified as critical components for membrane localization in intact cells. These data indicate that binding to the inositol phosphate head group is necessary but may not be sufficient for membrane localization of the PLCdelta(1)PH-GFP fusion protein, and motifs located within the C-terminal half of the PH domain provide auxiliary contacts with additional membrane components.

  • [Show abstract] [Hide abstract]
    ABSTRACT: All subtypes of KCNQ channel subunits (KCNQ1-5) require calmodulin as a co-factor for functional channels. It has been demonstrated that calmodulin plays a critical role in KCNQ channel trafficking as well as calcium-mediated current modulation. However, how calcium-bound calmodulin suppresses the M-current is not well understood. In this study, we investigated the molecular mechanism of KCNQ2 current suppression mediated by calcium-bound calmodulin. We show that calcium induced slow calmodulin dissociation from the KCNQ2 channel subunit. In contrast, in homomeric KCNQ3 channels, calcium facilitated calmodulin binding. We demonstrate that this difference in calmodulin binding was due to the unique cysteine residue in the KCNQ2 subunit at aa 527 in Helix B, which corresponds to an arginine residue in other KCNQ subunits including KCNQ3. In addition, a KCNQ2 channel associated protein AKAP79/150 (79 for human, 150 for rodent orthologs) also preferentially bound calcium-bound calmodulin. Therefore, the KCNQ2 channel complex was able to retain calcium-bound calmodulin either through the AKPA79/150 or KCNQ3 subunit. Functionally, increasing intracellular calcium by ionomycin suppressed currents generated by KCNQ2, KCNQ2(C527R) or heteromeric KCNQ2/KCNQ3 channels to an equivalent extent. This suggests that a change in the binding configuration, rather than dissociation of calmodulin, is responsible for KCNQ current suppression. Furthermore, we demonstrate that KCNQ current suppression was accompanied by reduced KCNQ affinity toward phosphatidylinositol 4,5-bisphosphate (PIP2) when assessed by a voltage-sensitive phosphatase, Ci-VSP. These results suggest that a rise in intracellular calcium induces a change in the configuration of CaM-KCNQ binding, which leads to the reduction of KCNQ affinity for PIP2 and subsequent current suppression.
    PLoS ONE 12/2013; 8(12):e82290. DOI:10.1371/journal.pone.0082290 · 3.53 Impact Factor
    This article is viewable in ResearchGate's enriched format
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the function of ion channels and transporters. Here, we demonstrate that PIP2 directly binds the human dopamine (DA) transporter (hDAT), a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). This binding occurs through electrostatic interactions with positively charged hDAT N-terminal residues and is shown to facilitate AMPH-induced, DAT-mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT-PIP2 interactions and AMPH-induced DA efflux without altering the hDAT physiological function of DA uptake. We evaluated the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH-induced locomotion without altering basal locomotion. We present what is to our knowledge the first demonstration of how PIP2 interactions with a membrane protein can regulate the behaviors of complex organisms.
    Nature Chemical Biology 06/2014; DOI:10.1038/nchembio.1545 · 13.22 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Many cellular functions are driven by variations in the intracellular Ca(2+) concentration ([Ca(2+)]i), which may appear as a single-event transient [Ca(2+)]i elevation, repetitive [Ca(2+)]i increases known as Ca(2+) oscillations, or [Ca(2+)]i increases propagating in the cytoplasm as Ca(2+) waves. Additionally, [Ca(2+)]i changes can be communicated between cells as intercellular Ca(2+) waves (ICWs). ICWs are mediated by two possible mechanisms acting in parallel: one involving gap junctions that form channels directly linking the cytoplasm of adjacent cells and one involving a paracrine messenger, in most cases ATP, that is released into the extracellular space, leading to [Ca(2+)]i changes in neighboring cells. The intracellular messenger inositol 1,4,5-trisphosphate (IP3) that triggers Ca(2+) release from Ca(2+) stores is crucial in these two ICW propagation scenarios, and is also a potent trigger to initiate ICWs. Loading inactive, "caged" IP3 into cells followed by photolytic "uncaging" with UV light, thereby liberating IP3, is a well-established method to trigger [Ca(2+)]i changes in single cells that is also effective in initiating ICWs. We here describe a method to load cells with caged IP3 by local electroporation of monolayer cell cultures and to apply flash photolysis to increase intracellular IP3 and induce [Ca(2+)]i changes, or initiate ICWs. Moreover, the electroporation method allows loading of membrane-impermeable agents that interfere with IP3 and Ca(2+) signaling. © 2015 Cold Spring Harbor Laboratory Press.
    Cold Spring Harbor Protocols 01/2015; 2015(3):pdb.top066068. DOI:10.1101/pdb.top066068 · 4.63 Impact Factor


Available from
May 28, 2014