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The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize,2007-2008 / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Chloride intracellular channel proteins have homology to ion channels and omega class of glutathione-S-transferases but channel activity is not well established, suggesting roles in other signaling pathways. Among the six CLICs, CLIC1 and CLIC4 are expressed in endothelial cells (EC) and act to promote EC proliferation, capillary-like networks, and lumen formation. We and others determined that Sphingosine-1-phosphate (S1P) signaling promotes transient CLIC4 membrane localization. We report that CLIC1 and CLIC4 have distinct roles in endothelial S1P signaling. In knockdown studies, CLIC1 and CLIC4 were independently required for S1PR1-mediated Rac1 activation, enhanced EC barrier integrity, and EC migration. CLIC1 was uniquely required for S1PR2/3-driven RhoA activation and actin stress fiber formation, while CLIC4 was uniquely required for thrombin/PAR-driven RhoA activation and endothelial permeability. CLICs were not required for other GPCR-mediated pathways measured, including S1PR1-mediated cAMP regulation downstream of Gαi, or Ras and ERK activation downstream of Gβγ. Endothelial β-adrenergic signaling, which uses Gαs, was unaltered by loss of CLICs. Further investigation of receptor tyrosine kinase signaling (VEGF, EGF) in endothelial cells reveals their signaling cascades do not depend on CLICs as well. We conclude that CLICs mediate S1PR-driven RhoA and Rac1 regulation, and thrombin/PAR-driven RhoA activation, and a possible mediator for endothelial GPCR by modulating Rac1 and RhoA. CLIC N-termini contain membrane insertion motifs and the putative ion channel domain, while the C-termini contain two predicted SH domains. Chimeric proteins generated by swapping N and C-termini of CLIC1 and CLIC4 were used in rescue experiments. The C-terminal domain was determined to confer S1PR1-CLIC-Rac1 mediated barrier function and migration. We further characterized N-termini of CLIC4 and membrane localization of by generating CLIC4 C-termini truncated protein, along with CLIC4 C-termini fusing with Lck-peptide for myristylation and plasma membrane re-localization. CLIC4 C-termini alone fails to rescue S1PR1-CLIC-Rac1 mediated barrier function, while membrane localization of the CLIC4 C-terminal domain functions in S1P signaling, suggesting the N-terminal domain confers membrane localization but not signaling function. Thus, we conclude S1P promotes cell localization of CLIC4 to the EC plasma membrane through N-termini, which then regulates Rac1 mediated events through C-termini. Through these findings, our work defines a molecular mechanism through which CLICs function in endothelium.

ClC-1 is the major skeletal muscle chloride channel and is essential for re-establishing the resting membrane potential of muscle cells after an action potential has occurred. Many mutations throughout the CLCN1 gene, which codes for the CIC-1 protein, have been demonstrated via characterisation in heterologous expression systems, to be causative mutations for either Dominant Myotonia Congenita or Recessive Generalised Myotonia. Recently, increasing numbers of myotonic mutations have been found in the carboxyl tail of CIC-1, which demonstrates its importance as a domain that is essential for the normal function of CIC-1 channels. Previous studies in our laboratory defined a region of 18 amino acids in the immediate post D13 segment of rat CIC-1, essential for the expression of functional channels. / thesis (PhDBiomedicalScience)--University of South Australia, 2002.

Glutamate-gated chloride channels (GluCls) are the proposed site of action for macrocyclic lactone anthelminthics, such as IVM, and the milbemycins, such as MOX. The objective of this thesis was to determine whether Haemonchus contortus GluCls are important targets for these anthelminthics. To begin to address this we cloned a full length GluCl alpha-subunit cDNA from H. contortus (HcGluCla). This subunit shares a high homology with GluCl subunits from Caenhorhabditis elegans that have been shown to be important targets for IVM, suggesting that HcGluCla is also an IVM target. However, if HcGluCla is an IVM receptor then it should contain an IVM binding site. To investigate this, the HcGluCla gene was expressed in COS-7 cells. The resulting subunit bound [3H]IVM and [ 3H]MOX with affinities sufficiently high enough to explain their high in vivo potency. Interestingly, glutamate was an allosteric modulator of [ 3H]MOX and [3H]IVM binding where it increased the affinity of these drugs to HcGluCla. To gain further insight into the potentiation of IVM, various glutamatergic and non-glutamatergic ligands were screened for their ability to enhance [3H]IVM binding to HcGluCla. Of the ligands tested, only the GluCl agonists glutamate and ibotenate potentiated [3H]IVM binding. It is possible therefore that if IVM interacts with GluCls in vivo then IVM efficacy may be enhanced by GluCl agonists. To examine this, we tested whether ibotenate could enhance IVM efficacy in gerbils infected with H. contortus. In in vivo efficacy studies, ibotenate (at 1 mg/kg) increased IVM efficacy by 15% (p = 0.048). The enhancement of IVM efficacy in vivo by a GluCl agonist suggests that one of the IVM targets in H. contortus is the GluCl. Finally, to determine the potential physiological response from an interaction between IVM and H. contortus GluCls, we expressed HcGluCla in Xenopus oocytes. HcGluCla expressed in oocytes formed a homomeric channel that responded to

ClC-1 is the major skeletal muscle chloride channel and is essential for re-establishing the resting membrane potential of muscle cells after an action potential has occurred. Many mutations throughout the CLCN1 gene, which codes for the CIC-1 protein, have been demonstrated via characterisation in heterologous expression systems, to be causative mutations for either Dominant Myotonia Congenita or Recessive Generalised Myotonia. Recently, increasing numbers of myotonic mutations have been found in the carboxyl tail of CIC-1, which demonstrates its importance as a domain that is essential for the normal function of CIC-1 channels. Previous studies in our laboratory defined a region of 18 amino acids in the immediate post D13 segment of rat CIC-1, essential for the expression of functional channels. / Thesis (PhDBiomedicalScience)--University of South Australia, 2002.

ClC-1 is the major skeletal muscle chloride channel and is essential for re-establishing the resting membrane potential of muscle cells after an action potential has occurred. Many mutations throughout the CLCN1 gene, which codes for the CIC-1 protein, have been demonstrated via characterisation in heterologous expression systems, to be causative mutations for either Dominant Myotonia Congenita or Recessive Generalised Myotonia. Recently, increasing numbers of myotonic mutations have been found in the carboxyl tail of CIC-1, which demonstrates its importance as a domain that is essential for the normal function of CIC-1 channels. Previous studies in our laboratory defined a region of 18 amino acids in the immediate post D13 segment of rat CIC-1, essential for the expression of functional channels. / thesis (PhDBiomedicalScience)--University of South Australia, 2002.

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / Typescript. Vita. Includes bibliographical references (leaves 178-187). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 1999. / "May 1999." Typescript. Vita. Includes bibliographical references (leaves 178-187). Also available on the Internet.

Thesis (Ph. D. in Neuroscience)--Vanderbilt University, Aug. 2007. / Title from title screen. Includes bibliographical references.