Transmembrane Extension and Oligomerization of the CLIC1 Chloride Intracellular Channel Protein upon Membrane Interaction

Chloride intracellular channel proteins (CLICs) differ from most ion channels as they can exist in both soluble and integral membrane forms. The CLICs are expressed as soluble proteins but can reversibly autoinsert into the membrane to form active ion channels. For CLIC1, the interaction with the lipid bilayer is enhanced under oxidative conditions. At present, little evidence is available characterizing the structure of the putative oligomeric CLIC integral membrane form. Previously, fluorescence resonance energy transfer (FRET) was used to monitor and model the conformational transition within CLIC1 as it interacts with the membrane bilayer. These results revealed a large-scale unfolding between the C- and N-domains of CLIC1 as it interacts with the membrane. In the present study, FRET was used to probe lipid-induced structural changes arising in the vicinity of the putative transmembrane region of CLIC1 (residues 24–46) under oxidative conditions. Intramolecular FRET distances are consistent with the model in which the N-terminal domain inserts into the bilayer as an extended α-helix. Further, intermolecular FRET was performed between fluorescently labeled CLIC1 monomers within membranes. The intermolecular FRET shows that CLIC1 forms oligomers upon oxidation in the presence of the membranes. Fitting the data to symmetric oligomer models of the CLIC1 transmembrane form indicates that the structure is large and most consistent with a model comprising approximately six to eight subunits

Effects of Calcium Binding and the Hypertrophic Cardiomyopathy A8V Mutation on the Dynamic Equilibrium between Closed and Open Conformations of the Regulatory N‑Domain of Isolated Cardiac Troponin C

Troponin C (TnC) is the calcium-binding subunit of the troponin complex responsible for initiating striated muscle contraction in response to calcium influx. In the skeletal TnC isoform, calcium binding induces a structural change in the regulatory N-domain of TnC that involves a transition from a closed to open structural state and accompanying exposure of a large hydrophobic patch for troponin I (TnI) to subsequently bind. However, little is understood about how calcium primes the N-domain of the cardiac isoform (cTnC) for interaction with the TnI subunit as the open conformation of the regulatory domain of cTnC has been observed only in the presence of bound TnI. Here we use paramagnetic relaxation enhancement (PRE) to characterize the closed to open transition of isolated cTnC in solution, a process that cannot be observed by traditional nuclear magnetic resonance methods. Our PRE data from four spin-labeled monocysteine constructs of isolated cTnC reveal that calcium binding triggers movement of the N-domain helices toward an open state. Fitting of the PRE data to a closed to open transition model reveals the presence of a small population of cTnC molecules in the absence of calcium that possess an open conformation, the level of which increases substantially upon Ca²⁺ binding. These data support a model in which calcium binding creates a dynamic equilibrium between the closed and open structural states to prime cTnC for interaction with its target peptide. We also used PRE data to assess the structural effects of a familial hypertrophic cardiomyopathy point mutation located within the N-domain of cTnC (A8V). The PRE data show that the Ca²⁺ switch mechanism is perturbed by the A8V mutation, resulting in a more open N-domain conformation in both the apo and holo states

Cell-attached recordings of HEK cells transiently transfected with WT CLIC1 and R29A CLIC1 protein.

<p>Single-channel current traces are shown for WT (A) and R29A (B) transfected cells. The voltage steps are from -5 to + 25 mV (10 mV step increment). Single-channel current/voltage plots are shown in (C) for WT (□) and R29A (○). Average single-channel conductance of 12.3 ± 0.1 pS for WT and 13.1 ± 0.3 pS for R29A and an extrapolated reversal potential of -63 ± 0.4 mV and -61 ± 0.6 mV, respectively. The open probability obtained at each membrane potential is shown in (D) for WT (□) and R29A (○).</p

Cell-attached recordings of HEK cells transiently transfected with WT and K37A CLIC1 protein.

<p>Single-channel current traces are shown for WT (A) and K37A (B) CLIC1 transfected cells. The membrane voltage was clamped at different values (indicated on the left of each trace) in 10 mV step increments. Single-channel current/voltage plots are shown in (C) for WT (□) and K37A (○). Average single-channel conductance of 12.1 ± 0.6 pS for WT and 17.4 ± 0.8 pS for K37A with an extrapolated reversal potential of -58 ± 0.7 mV and -56 ± 1.2 mV, respectively, was calculated. The open probability obtained at each membrane potential is shown in (D) for WT (□) and K37A (○).</p

CLIC1 ion channel activity from Tip-Dip bilayer experiments.

<p>Current recordings from -80 to +80 mV, 20 mV interval, are shown in (A) for wild type CLIC1 (left), K37A (center) and R29A (right) CLIC1 protein. The upper panel of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074523#pone-0074523-g001" target="_blank">Figure 1B</a> depicts the single-channel current-voltage data for WT (□) and K37A (○) CLIC1 proteins. The average single-channel conductance differs between WT and K37A, calculated as 30.1 ± 0.2 and 42.4 ± 0.2 pS, respectively (n = 5, p < 0.001). In contrast, the channel open probability of the K37A mutation is very similar to the WT (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074523#pone-0074523-g001" target="_blank">Figure 1B</a>, lower panel). The current/voltage relationships for both WT (□) and R29A CLIC1 (○) are shown in (C). The single-channel conductance (upper panel), is 30.1 ± 0.2 and 29 ± 0.2 pS for WT and R29A, respectively. The average open probability for WT (□) and R29A mutated protein (○) is shown in the lower panel.</p

Open (τ_open) and close (τ_close) time constants of the WT and K37A CLIC1 ion channel.

<p>In panels (A) and (B), membrane potentials were held at +35 mV while panels (C) and (D) concern cell membrane potentials held at -5 mV for WT (A, C) and K37A (B, D) CLIC1 transfected HEK cells. (A) to (D) shows the open (left) and close (right) time distributions for each condition. Four seconds of single channel recordings appear as inserts in the corresponding panels for each condition. The open and close time distribution histograms were fitted by a double exponential decay function and plotted on a semi-logarithmic scale. Panels (E) and (F) depict open and close time distributions as a function of membrane potential for WT (□) and K37A (○) transfected HEK cells.</p

Whole-cell current in R29A transfected HEK cells.

<p>Family of whole-currents for WT (A) and R29A (B) transfected HEK cells (top panels). Voltage steps lasting 800 ms from holding potential of -30 mV to membrane potential of -80 to +100 mV (20 mV step increment). The middle panels depict the whole-cell currents after perfusion of 50 µM of IAA94. The bottom panels represent the IAA94-sensitive currents obtained by subtraction of the middle panel current from the upper panel current. Note that the IAA94-sensitive currents are plotted on a different scale with the scale bars on the right hand side of the figure. (C) an example of current/voltage relationship of the IAA94-sensitive current from a WT (□) and a R29A (○) transfected HEK cell. (D) Averaged G/V plots from IAA94-sensitive current of WT (□) and R29A mutant (○), from 5 independent experiments.</p

Endogenous CLIC1 is not expressed on the plasma membrane of untransfected HEK cells.

<p>(A) Family of currents for an untransfected HEK293 cell at varying applied voltages from -60 to +60 mV with 20 mV increment. Top panel: whole cell current in resting conditions; middle panel: after IAA94 perfusion; lower panel: IAA94-sensitive (CLIC1-mediated) current obtained from subtraction. (B) i/V curve of an IAA94-sensitive current in WT CLIC1 transfected HEK293 cell (□), and in an untransfected HEK293 cell (○). (C) Plot of the average of the IAA94-sensitive current as a percentage of the control current of untransfected HEK cells (n=5). Data are shown as mean ± SEM. CLIC1-mediated current is completely absent in untransfected HEK cells.</p

Proteins differentially expressed in the intra-cellular proteomes of controls MSCs and MSCs labeled with nanodiamonds.

<p>Control adipose-derived mesenchymal stem cells (MSCs) or MSCs labeled with nanodiamonds were held in culture for 72 hours in Standard Media. The MSCs were subsequently collected, processed and the tryptic peptides were labeled with an isobaric tag for iTRAQ analysis using a Qstar Elite. The experiment was repeated in triplicate. A total of 3059 proteins were identified. The fold-change values of the proteins which changed by >1.2 and <0.8 and had a p-value<0.05 in one or more experiments have been included. The fold-change values that did not meet these criteria in each experiment are depicted in brackets. For each of these proteins, the UniProt identification number, protein name, percentage of protein coverage (and number of unique peptides contributing to the sequence coverage), and fold change between the proteins in the MSCs labeled with nanodiamonds versus control MSCs, for all replicates, are included.</p

Proteins differentially expressed in the intra-cellular proteomes of controls MSCs and MSCs labeled with M-SPIO particles.

<p>Control MSCs or MSCs labeled with M-SPIO particles were held in culture for 72 hours in Standard Media. The MSCs were subsequently collected, processed and the tryptic peptides were labeled with an isobaric tag for iTRAQ analysis using a Q-Star Elite. The experiment was repeated in triplicate. A total of 3059 proteins were identified. The fold-change values of the proteins which changed by >1.2 and <0.8 and had a p-value<0.05 in one or more experiments have been included. The fold-change values that did not meet these criteria in each experiment are depicted in brackets. A blank space indicates that a relative fold-change was not obtained for the specified protein in that replicate. For each of these proteins, the UniProt identification number, protein name, percentage of protein coverage (and number of unique peptides contributing to the sequence coverage), and fold change between the proteins in the MSCs labeled with M-SPIO particles versus control MSCs, for all replicates, are included.</p