Nicoletta Savalli

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.

Voltage-dependent conformational changes in human Ca2+- and voltage-activated K+ channel, revealed by voltage-clamp fluorometry

Large conductance voltage- and Ca2+-activated K+ (BKCa) channels regulate important physiological processes such as neurotransmitter release and vascular tone. BKCa channels possess a voltage sensor mainly represented by the S4 transmembrane domain. Changes in membrane potential displace the voltage sensor, producing a conformational change that leads to channel opening. By site-directed fluorescent labeling of residues in the S3–S4 region and by using voltage clamp fluorometry, we have resolved the conformational changes the channel undergoes during activation. The voltage dependence of these conformational changes (detected as changes in fluorescence emission, fluorescence vs. voltage curves) always preceded the channel activation curves, as expected for protein rearrangements associated to the movement of the voltage sensor. Extremely slow conformational changes were revealed by fluorescent labeling of position 202, elicited by a mutual interaction of the fluorophore with the adjacent tryptophan 203.

Operation of the voltage sensor of a human voltage- and Ca2+-activated K+ channel

Voltage sensor domains (VSDs) are structurally and functionally conserved protein modules that consist of four transmembrane segments (S1–S4) and confer voltage sensitivity to many ion channels. Depolarization is sensed by VSD-charged residues residing in the membrane field, inducing VSD activation that facilitates channel gating. S4 is typically thought to be the principal functional component of the VSD because it carries, in most channels, a large portion of the VSD gating charge. The VSDs of large-conductance, voltage- and Ca2+-activated K+ channels are peculiar in that more gating charge is carried by transmembrane segments other than S4. Considering its “decentralized” distribution of voltage-sensing residues, we probed the BKCa VSD for evidence of cooperativity between charge-carrying segments S2 and S4. We achieved this by optically tracking their activation by using voltage clamp fluorometry, in channels with intact voltage sensors and charge-neutralized mutants. The results from these experiments indicate that S2 and S4 possess distinct voltage dependence, but functionally interact, such that the effective valence of one segment is affected by charge neutralization in the other. Statistical-mechanical modeling of the experimental findings using allosteric interactions demonstrates two mechanisms (mechanical coupling and dynamic focusing of the membrane electric field) that are compatible with the observed cross-segment effects of charge neutralization.

Modes of Operation of the BKCa Channel β2 Subunit

The β2 subunit of the large conductance Ca2+- and voltage-activated K+ channel (BKCa) modulates a number of channel functions, such as the apparent Ca2+/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the β2 subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BKCa channels (hSlo), we have investigated the mechanisms of operation of the β2 subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with β2 subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the β2 subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BKCa channels coexpressed with the β2 subunit. The experimental observations are consistent with the view that the β2 subunit of BKCa channels facilitates channel activation by changing the voltage sensor equilibrium and that the β2-induced inactivation process does not follow a typical N-type mechanism.

Functional heterogeneity of the four voltage sensors of a human L-type calcium channel

Significance Intracellular Ca2+ concentration is a chemical message upstream of critical physiological processes: synaptic and endocrine signaling, muscle contraction, etc. In excitable cells, calcium influx is controlled by voltage-gated calcium (CaV) channels. The CaV protein possesses four homologous but nonidentical voltage-sensing domains (VSDs), each potentially acting as a gatekeeper for the calcium signal. However, the contribution of each VSD to calcium entry is unclear. By optically tracking the voltage-dependent movements of each human CaV1.2 VSD, we revealed the conformational rearrangements that precede and govern excitation-evoked calcium signaling: VSDs II and III primarily control CaV1.2 conduction. Global structure-based kinetic modeling also revealed the participation of VSD I, implicating principles of protein allostery in the CaV VSD–pore coupling mechanism. Excitation-evoked Ca2+ influx is the fastest and most ubiquitous chemical trigger for cellular processes, including neurotransmitter release, muscle contraction, and gene expression. The voltage dependence and timing of Ca2+ entry are thought to be functions of voltage-gated calcium (CaV) channels composed of a central pore regulated by four nonidentical voltage-sensing domains (VSDs I–IV). Currently, the individual voltage dependence and the contribution to pore opening of each VSD remain largely unknown. Using an optical approach (voltage-clamp fluorometry) to track the movement of the individual voltage sensors, we discovered that the four VSDs of CaV1.2 channels undergo voltage-evoked conformational rearrangements, each exhibiting distinct voltage- and time-dependent properties over a wide range of potentials and kinetics. The voltage dependence and fast kinetic components in the activation of VSDs II and III were compatible with the ionic current properties, suggesting that these voltage sensors are involved in CaV1.2 activation. This view is supported by an obligatory model, in which activation of VSDs II and III is necessary to open the pore. When these data were interpreted in view of an allosteric model, where pore opening is intrinsically independent but biased by VSD activation, VSDs II and III were each found to supply ∼50 meV (∼2 kT), amounting to ∼85% of the total energy, toward stabilizing the open state, with a smaller contribution from VSD I (∼16 meV). VSD IV did not appear to participate in channel opening.

The Contribution of RCK Domains to Human BK Channel Allosteric Activation

Background: In BK channels, Ca2+ and voltage sensors are allosterically connected to the pore. Results: We optically resolved voltage sensor rearrangements, initiated by Ca2+ binding to the intracellular domains RCK1 and RCK2. Impairing the RCK2 abolished this allosteric effect. Conclusion: The RCK2 Ca2+ sensor is required for the allosteric facilitation of voltage sensor activation. Significance: RCK1 and RCK2 Ca2+ sensors are not functionally homologous. Large conductance voltage- and Ca2+-activated K+ (BK) channels are potent regulators of cellular processes including neuronal firing, synaptic transmission, cochlear hair cell tuning, insulin release, and smooth muscle tone. Their unique activation pathway relies on structurally distinct regulatory domains including one transmembrane voltage-sensing domain (VSD) and two intracellular high affinity Ca2+-sensing sites per subunit (located in the RCK1 and RCK2 domains). Four pairs of RCK1 and RCK2 domains form a Ca2+-sensing apparatus known as the “gating ring.” The allosteric interplay between voltage- and Ca2+-sensing apparati is a fundamental mechanism of BK channel function. Using voltage-clamp fluorometry and UV photolysis of intracellular caged Ca2+, we optically resolved VSD activation prompted by Ca2+ binding to the gating ring. The sudden increase of intracellular Ca2+ concentration ([Ca2+]i) induced a hyperpolarizing shift in the voltage dependence of both channel opening and VSD activation, reported by a fluorophore labeling position 202, located in the upper side of the S4 transmembrane segment. The neutralization of the Ca2+ sensor located in the RCK2 domain abolished the effect of [Ca2+]i increase on the VSD rearrangements. On the other hand, the mutation of RCK1 residues involved in Ca2+ sensing did not prevent the effect of Ca2+ release on the VSD, revealing a functionally distinct interaction between RCK1 and RCK2 and the VSD. A statistical-mechanical model quantifies the complex thermodynamics interplay between Ca2+ association in two distinct sites, voltage sensor activation, and BK channel opening.

Functional Role of CLIC1 Ion Channel in Glioblastoma-Derived Stem/Progenitor Cells

Background Chloride channels are physiologically involved in cell division and motility. Chloride intracellular channel 1 (CLIC1) is overexpressed in a variety of human solid tumors compared with normal tissues, suggesting a potential involvement of CLIC1 in the regulation of tumorigenesis. This led us to investigate the role of CLIC1 in gliomagenesis. Methods We used the neurosphere system to isolate stem/progenitor cells from human glioblastomas (GBMs). CLIC1 targeting in GBM neurospheres was achieved by both lentiviral-mediated short-hairpin RNA transduction and CLIC1 antibody treatment, and its effect on stem-like properties was analyzed in vitro by proliferation and clonogenic assays and in vivo by orthotopic injection in immunocompromised mice. Channel activity was studied by perforated patch clamp technique. Differences in expression were analyzed by analysis of variance with Tamhane’s multiple comparison test. Kaplan–Meier analyses and log-rank test were used to assess survival. All statistical tests were two-sided. Results CLIC1 was statistically significantly overexpressed in GBMs compared with normal brain tissues (P < .001) with a better survival of patients with CLIC1 low-expressing tumors (CLIC1low vs CLIC1high survival: χ2 = 74.35; degrees of freedom = 1; log-rank P < .001). CLIC1 was variably expressed in patient-derived GBM neurospheres and was found enriched in the stem/progenitor compartment. CLIC1 silencing reduced proliferative (P < .01), clonogenic (P < .01), and tumorigenic capacity (P < .05) of stem/progenitor cells. The reduction of CLIC1 chloride currents with a specific CLIC1 antibody mirrored the biological effects of CLIC1 silencing in GBM patient–derived neurospheres. Conclusions Reduced gliomagenesis after CLIC1 targeting in tumoral stem/progenitor cells and the finding that CLIC1 expression is inversely associated with patient survival suggest CLIC1 as a potential target and prognostic biomarker.

The α2δ-1 subunit remodels CaV1.2 voltage sensors and allows Ca2+ influx at physiological membrane potentials.

Voltage-sensing domains (VSDs) in voltage-gated calcium channels sense the potential difference across membranes and interact with the pore to open it. Savalli et al. find that the accessory subunit α2δ-1 increases the sensitivity of VSDs I–III and also their efficiency of coupling to the pore.

Bcl-2 down modulation in WEHI-3B/CTRES cells resistant to Cholera Toxin (CT)-induced apoptosis

The very different effects of Cholera Toxin (CT) on cell growth and proliferation may depend on the type of ganglioside receptors in cell membranes and different signal transduction mechanisms triggered, but other functions related to the drug resistance mechanisms can not be excluded. The effect of CT treatment on the “in vitro” clonogenicity, the Population Doubling Time (PDT), apoptosis, PKA activation and Bax and Bcl-2 expression was evaluated in WEHI-3B cell line and its CT-resistant subclone (WEHI-3B/CTRES). In WEHI-3B parental cells the dramatic accumulation of cAMP induced by CT correlated well with PKA activation, increased PDT value, inhibition of clonogenicity and apoptosis. H-89 treatment inhibited PKA activation by CT but did not protect the cells from apoptosis and growth inhibition. In WEHI-3B/CTRES no significant CT-dependent accumulation of cAMP occurred with any increase of PKA activity and PDT. In CT resistant cells (WEHI-3B/CTRES), Bcl-2 expression was down regulated by both CT or drug treatment (eg., ciprofloxacin, CPX) although these cells were protected from CT-dependent apoptosis but not from drug-induced apoptosis. Differently from other cell models described, down regulation of Bcl-2 is proved to be independent on cAMP accumulation and PKA activation. Our observations support the implication of cAMP dependent kinase (PKA) in the inhibition of WEHI-3B cells growth and suggest that, in WEHI-3B/CTRES, Bcl-2 expression could be modulated by CT in the absence of cAMP accumulation. Also in consideration of many contradictory data reported in literature, our cell models (of one sensitive parental cell strain and two clones with different uncrossed specific resistance to CT and CPX) provides a new and interesting tool for better investigating the relationship between the CT signal transduction mechanisms and Bcl-2 expression and function.