ION CHANNELS PATHOPHYSIOLOGY AND BIOPHYSICS:1. THE ROLE OF THE EXPRESSION OF CLIC1 ION CHANNELS IN CANCER STEM CELLS FROM HUMAN GLIOBLASTOMA; 2. CA2+ AND VOLTAGE DEPENDENT STRUCTURAL CHANGES IN THE HUMAN BK CHANNEL DURING OPERATION REVEALED BY VOLTAGE CLAMP FLUOROMETRY.
Creators: name: SAVALLI, NICOLETTA
Publisher: name: Universit� degli Studi di Milano
Year: 2013

description: Ion channels are transmembrane proteins that create a pathway for charged ions (sodium, potassium, calcium, and chloride) to pass through the otherwise impermeant lipid membranes. They are present both at the plasma membrane level and in the membrane of intracellular organelles. Ion channels engage in fundamental functions, such as muscle contraction, cell excitability, hormone secretion, mechanosensitivity, just to mention a few. Thus, the understanding of their physiology and pathophysiology remains an important task for science. This thesis embraces two projects involving two different ion channels: the Chloride Intracellular Channel 1 (CLIC1) and the large conductance Ca2+-voltage activated K+ channel (BK).
The goal of the first project aimed to understand the role of CLIC1 channels in glioblastoma cancer stem cells. Among brain tumors, glioblastomas are very frequent and have the worst outcome. They are composed of two cell types: a small population of cells able to self-renew and generate progeny (cancer stem cells, CSCs) and a larger population possessing a limited division capacity and committed to a precise fate (bulk cells). Glioblastomas are very aggressive tumors because of CSC brain infiltration efficiency and their resistance to chemotherapies. Therefore, CSCs are the most tumorigenic component of glioblastomas and we have focused our efforts on this small population of cells. Several forms of glioblastomas show a high level of expression of CLIC1 compared to normal brains. CLIC1 is a protein mainly localized to the cytoplasm and nucleoplasm that is able to translocate to the plasma membrane and to the nuclear membrane where it acts as a Cl- channel. The soluble form of CLIC1 belongs to the glutathione S-transferase superfamily. Upon oxidation, the protein forms a dimer that translocates to the membrane and operates as an ion channel. Four human glioblastomas (all expressing CLIC1) have been studied. Human glioblastoma biopsies were cultured in a medium selecting for CSCs. By knocking down CLIC1 protein using siRNA viral infection (siCLIC1), we found that CLIC1-deficient cells migrated about 50% less efficiently than control cells treated with siRNA for luciferase (siLUC). Is this phenotype the result of CLIC1 absence in plasma membranes? To answer this question, we performed electrophysiological experiments from perforated patches for both siLUC and siCLIC1 cells. Cl- currents mediated by CLIC1 were isolated using a specific inhibitor (IAA94 100 ?M). The results showed that siCLIC1 cells did not display IAA94-sensitive currents, while siLUC cells presented the CLIC1-mediated chloride current. Interestingly, in the four glioblastomas analyzed, there is a direct correlation between tumor aggressiveness and the relative abundance of IAA94 sensitive current: the more aggressive the tumor, the greater the relative abundance of CLIC1 current. These results point to the view that CLIC1 is involved in glioblastoma CSCs migration. However, the mechanism has yet to be elucidated.
The second project investigated the Ca2+-voltage dependent structural rearrangements of the human BK channel during its activation. BK channels are Ca2+-voltage-activated K+ channels. They are potent regulators of cellular excitability involved in processes such as neuronal firing, synaptic transmission, cochlear hair cell tuning and smooth muscle tone. The BK channel?s unique activation pathway is a consequence of its structurally distinct regulatory domains, including four transmembrane voltage sensors (VSD) and four pairs of intracellular Ca2+ sensors, RCK1 and RCK2 (Regulation of K+ Conductance). RCK1 includes residues D362/D367 involved in high-affinity Ca2+ sensing, while RCK2 encompasses a stretch of five Asps (D894-898 or Ca bowl) that coordinate Ca2+. In the functional tetrameric channel, the two RCK domains from each subunit assemble into a superstructure called the ?gating ring?. To understand the allosteric interplay between these sensing apparata (VSD and RCK1/RCK2 in the gating ring), we have simultaneously tracked the conformational status of the VSD and the pore while activating the Ca2+ sensors in the gating ring by combining voltage clamp fluorometry with UV-photolysis of caged Ca2+. In WT channels, we found that the VSD conformational changes were triggered not only by voltage but also by [Ca2+] increase, demonstrating that Ca2+-induced rearrangements of the BK intracellular gating ring allosterically propagate to the transmembrane VSD. The impairment of the Ca bowl in the RCK2 domain (D894-898N mutations) abolished the VSD facilitation induced by the rapid increase of [Ca2+]. However, the neutralization of the Ca2+ sensor in RCK1 (D362A/D367A mutations) did not prevent VSD facilitation by Ca2+ (as in WT channels, but to a lesser extent). Thus, RCK1 and RCK2 domains play different functional roles in the Ca2+-dependent activation of the human BK channel. A statistical-mechanical model has been implemented to quantify the thermodynamics of the functional coupling between intracellular and transmembrane regulatory domains in BK channels. This model includes one pore, four VSDs, four RCK1 Ca2+ sensors and four RCK2 Ca2+ sensors. All these domains are regulated by equilibrium constants and linked by allosteric factors.