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.

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 \u3bcM). 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\u2019s 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 \u201cgating ring\u201d. 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

Specular reflection of matter waves from a rough mirror

We have made a high resolution study of the specularity of the atomic reflection from an evanescent wave mirror using velocity selective Raman transitions. We have observed a double structure in the velocity distribution after reflection: a peak consistent with specular reflection and a diffuse reflection pedestal, whose contribution decreases rapidly with increasing detuning. The diffuse reflection is due to two distinct effects: spontaneous emission in the evanescent wave and a roughness in the evanescent wave potential whose amplitude is smaller than the de Broglie wavelength of the reflected atoms

The RCK2 domain of the human BKCa channel is a calcium sensor

Large conductance voltage and Ca2+-dependent K+ channels (BKCa) are activated by both membrane depolarization and intracellular Ca2+. Recent studies on bacterial channels have proposed that a Ca2+-induced conformational change within specialized regulators of K+ conductance (RCK) domains is responsible for channel gating. Each pore-forming α subunit of the homotetrameric BKCa channel is expected to contain two intracellular RCK domains. The first RCK domain in BKCa channels (RCK1) has been shown to contain residues critical for Ca2+ sensitivity, possibly participating in the formation of a Ca2+-binding site. The location and structure of the second RCK domain in the BKCa channel (RCK2) is still being examined, and the presence of a high-affinity Ca2+-binding site within this region is not yet established. Here, we present a structure-based alignment of the C terminus of BKCa and prokaryotic RCK domains that reveal the location of a second RCK domain in human BKCa channels (hSloRCK2). hSloRCK2 includes a high-affinity Ca2+-binding site (Ca bowl) and contains similar secondary structural elements as the bacterial RCK domains. Using CD spectroscopy, we provide evidence that hSloRCK2 undergoes a Ca2+-induced change in conformation, associated with an α-to-β structural transition. We also show that the Ca bowl is an essential element for the Ca2+-induced rearrangement of hSloRCK2. We speculate that the molecular rearrangements of RCK2 likely underlie the Ca2+-dependent gating mechanism of BKCa channels. A structural model of the heterodimeric complex of hSloRCK1 and hSloRCK2 domains is discussed

Negative phenotypic and genetic associations between copulation duration and longevity in male seed beetles

Reproduction can be costly and is predicted to trade-off against other characters. However, while these trade-offs are well documented for females, there has been less focus on aspects of male reproduction. Furthermore, those studies that have looked at males typically only investigate phenotypic associations, with the underlying genetics often ignored. Here, we report on phenotypic and genetic trade-offs in male reproductive effort in the seed beetle, Callosobruchus maculatus. We find that the duration of a male's first copulation is negatively associated with subsequent male survival, phenotypically and genetically. Our results are consistent with life-history theory and suggest that like females, males trade-off reproductive effort against longevity

Non-destructive, dynamic detectors for Bose-Einstein condensates

We propose and analyze a series of non-destructive, dynamic detectors for Bose-Einstein condensates based on photo-detectors operating at the shot noise limit. These detectors are compatible with real time feedback to the condensate. The signal to noise ratio of different detection schemes are compared subject to the constraint of minimal heating due to photon absorption and spontaneous emission. This constraint leads to different optimal operating points for interference-based schemes. We find the somewhat counter-intuitive result that without the presence of a cavity, interferometry causes as much destruction as absorption for optically thin clouds. For optically thick clouds, cavity-free interferometry is superior to absorption, but it still cannot be made arbitrarily non-destructive . We propose a cavity-based measurement of atomic density which can in principle be made arbitrarily non-destructive for a given signal to noise ratio

Resolved diffraction patterns from a reflection grating for atoms

We have studied atomic diffraction at normal incidence from an evanescent standing wave with a high resolution using velocity selective Raman transitions. We have observed up to 3 resolved orders of diffraction, which are well accounted for by a scalar diffraction theory. In our experiment the transverse coherence length of the source is greater than the period of the diffraction grating.Comment: 8 pages, 4 figure

Properties of Microelectromagnet Mirrors as Reflectors of Cold Rb Atoms

Cryogenically cooled microelectromagnet mirrors were used to reflect a cloud of free-falling laser-cooled 85Rb atoms at normal incidence. The mirrors consisted of microfabricated current-carrying Au wires in a periodic serpentine pattern on a sapphire substrate. The fluorescence from the atomic cloud was imaged after it had bounced off a mirror. The transverse width of the cloud reached a local minimum at an optimal current corresponding to minimum mirror roughness. A distinct increase in roughness was found for mirror configurations with even versus odd number of lines. These observations confirm theoretical predictions.Comment: Physical Review A, in print; 11 pages, 4 figure

The role of cat eye narrowing movements in cat–human communication

Domestic animals are sensitive to human cues that facilitate inter-specific communication, including cues to emotional state. The eyes are important in signalling emotions, with the act of narrowing the eyes appearing to be associated with positive emotional communication in a range of species. This study examines the communicatory significance of a widely reported cat behaviour that involves eye narrowing, referred to as the slow blink sequence. Slow blink sequences typically involve a series of half-blinks followed by either a prolonged eye narrow or an eye closure. Our first experiment revealed that cat half-blinks and eye narrowing occurred more frequently in response to owners’ slow blink stimuli towards their cats (compared to no owner–cat interaction). In a second experiment, this time where an experimenter provided the slow blink stimulus, cats had a higher propensity to approach the experimenter after a slow blink interaction than when they had adopted a neutral expression. Collectively, our results suggest that slow blink sequences may function as a form of positive emotional communication between cats and humans

Incorporation of DPP6a and DPP6K Variants in Ternary Kv4 Channel Complex Reconstitutes Properties of A-type K Current in Rat Cerebellar Granule Cells

Dipeptidyl peptidase-like protein 6 (DPP6) proteins co-assemble with Kv4 channel α-subunits and Kv channel-interacting proteins (KChIPs) to form channel protein complexes underlying neuronal somatodendritic A-type potassium current (ISA). DPP6 proteins are expressed as N-terminal variants (DPP6a, DPP6K, DPP6S, DPP6L) that result from alternative mRNA initiation and exhibit overlapping expression patterns. Here, we study the role DPP6 variants play in shaping the functional properties of ISA found in cerebellar granule (CG) cells using quantitative RT-PCR and voltage-clamp recordings of whole-cell currents from reconstituted channel complexes and native ISA channels. Differential expression of DPP6 variants was detected in rat CG cells, with DPP6K (41±3%)>DPP6a (33±3%)>>DPP6S (18±2%)>DPP6L (8±3%). To better understand how DPP6 variants shape native neuronal ISA, we focused on studying interactions between the two dominant variants, DPP6K and DPP6a. Although previous studies did not identify unique functional effects of DPP6K, we find that the unique N-terminus of DPP6K modulates the effects of KChIP proteins, slowing recovery and producing a negative shift in the steady-state inactivation curve. By contrast, DPP6a uses its distinct N-terminus to directly confer rapid N-type inactivation independently of KChIP3a. When DPP6a and DPP6K are co-expressed in ratios similar to those found in CG cells, their distinct effects compete in modulating channel function. The more rapid inactivation from DPP6a dominates during strong depolarization; however, DPP6K produces a negative shift in the steady-state inactivation curve and introduces a slow phase of recovery from inactivation. A direct comparison to the native CG cell ISA shows that these mixed effects are present in the native channels. Our results support the hypothesis that the precise expression and co-assembly of different auxiliary subunit variants are important factors in shaping the ISA functional properties in specific neuronal populations

Time-Course of Changes in Inflammatory Response after Whole-Body Cryotherapy Multi Exposures following Severe Exercise

The objectives of the present investigation was to analyze the effect of two different recovery modalities on classical markers of exercise-induced muscle damage (EIMD) and inflammation obtained after a simulated trail running race. Endurance trained males (n = 11) completed two experimental trials separated by 1 month in a randomized crossover design; one trial involved passive recovery (PAS), the other a specific whole body cryotherapy (WBC) for 96 h post-exercise (repeated each day). For each trial, subjects performed a 48 min running treadmill exercise followed by PAS or WBC. The Interleukin (IL) -1 (IL-1), IL-6, IL-10, tumor necrosis factor alpha (TNF-α), protein C-reactive (CRP) and white blood cells count were measured at rest, immediately post-exercise, and at 24, 48, 72, 96 h in post-exercise recovery. A significant time effect was observed to characterize an inflammatory state (Pre vs. Post) following the exercise bout in all conditions (p<0.05). Indeed, IL-1β (Post 1 h) and CRP (Post 24 h) levels decreased and IL-1ra (Post 1 h) increased following WBC when compared to PAS. In WBC condition (p<0.05), TNF-α, IL-10 and IL-6 remain unchanged compared to PAS condition. Overall, the results indicated that the WBC was effective in reducing the inflammatory process. These results may be explained by vasoconstriction at muscular level, and both the decrease in cytokines activity pro-inflammatory, and increase in cytokines anti-inflammatory