Structure of the CLC-1 chloride channel from Homo sapiens.

CLC channels mediate passive Cl- conduction, while CLC transporters mediate active Cl- transport coupled to H+ transport in the opposite direction. The distinction between CLC-0/1/2 channels and CLC transporters seems undetectable by amino acid sequence. To understand why they are different functionally we determined the structure of the human CLC-1 channel. Its 'glutamate gate' residue, known to mediate proton transfer in CLC transporters, adopts a location in the structure that appears to preclude it from its transport function. Furthermore, smaller side chains produce a wider pore near the intracellular surface, potentially reducing a kinetic barrier for Cl- conduction. When the corresponding residues are mutated in a transporter, it is converted to a channel. Finally, Cl- at key sites in the pore appear to interact with reduced affinity compared to transporters. Thus, subtle differences in glutamate gate conformation, internal pore diameter and Cl- affinity distinguish CLC channels and transporters

The mechanosensitive ion channel TRAAK is localized to the mammalian node of Ranvier.

TRAAK is a membrane tension-activated K+ channel that has been associated through behavioral studies to mechanical nociception. We used specific monoclonal antibodies in mice to show that TRAAK is localized exclusively to nodes of Ranvier, the action potential propagating elements of myelinated nerve fibers. Approximately 80 percent of myelinated nerve fibers throughout the central and peripheral nervous system contain TRAAK in what is likely an all-nodes or no-nodes per axon fashion. TRAAK is not observed at the axon initial segment where action potentials are first generated. We used polyclonal antibodies, the TRAAK inhibitor RU2 and node clamp amplifiers to demonstrate the presence and functional properties of TRAAK in rat nerve fibers. TRAAK contributes to the leak K+ current in mammalian nerve fiber conduction by hyperpolarizing the resting membrane potential, thereby increasing Na+ channel availability for action potential propagation. We speculate on why nodes of Ranvier contain a mechanosensitive K+ channel

The Molecular Basis of Texture in Mashed Potato

Mash production often involves thermal pre-treatments (pre-cooks) that are designed to increase the physical strength and restrict cell separation of cooked potatoes prior to mashing. This improves the mash quality. During pre-cooking, the control of starch swelling pressures during cooking and/or the activation of pectin methylesterase (PME) prior to cooking can occur. The aim of this thesis was to examine changes in texture of cooked potatoes caused by these pre-cook treatments prior to mashing. Cooking was carried out in laboratory conditions. During steam cooking (one-stage cooking) the potatoes quickly reached 60-6

Two Separate Interfaces between the Voltage Sensor and Pore Are Required for the Function of Voltage-Dependent K+ Channels

Voltage-dependent K+ (Kv) channels gate open in response to the membrane voltage. To further our understanding of how cell membrane voltage regulates the opening of a Kv channel, we have studied the protein interfaces that attach the voltage-sensor domains to the pore. In the crystal structure, three physical interfaces exist. Only two of these consist of amino acids that are co-evolved across the interface between voltage sensor and pore according to statistical coupling analysis of 360 Kv channel sequences. A first co-evolved interface is formed by the S4-S5 linkers (one from each of four voltage sensors), which form a cuff surrounding the S6-lined pore opening at the intracellular surface. The crystal structure and published mutational studies support the hypothesis that the S4-S5 linkers convert voltage-sensor motions directly into gate opening and closing. A second co-evolved interface forms a small contact surface between S1 of the voltage sensor and the pore helix near the extracellular surface. We demonstrate through mutagenesis that this interface is necessary for the function and/or structure of two different Kv channels. This second interface is well positioned to act as a second anchor point between the voltage sensor and the pore, thus allowing efficient transmission of conformational changes to the pore's gate

A Gating Charge Transfer Center in Voltage Sensors

Voltage sensors regulate the conformations of voltage-dependent ion channels and enzymes. Their nearly switchlike response as a function of membrane voltage comes from the movement of positively charged amino acids, arginine or lysine, across the membrane field. We used mutations with natural and unnatural amino acids, electrophysiological recordings, and x-ray crystallography to identify a charge transfer center in voltage sensors that facilitates this movement. This center consists of a rigid cyclic "cap" and two negatively charged amino acids to interact with a positive charge. Specific mutations induce a preference for lysine relative to arginine. By placing lysine at specific locations, the voltage sensor can be stabilized in different conformations, which enables a dissection of voltage sensor movements and their relation to ion channel opening

A Gating Charge Transfer Center in Voltage Sensors

Voltage sensors regulate the conformations of voltage-dependent ion channels and enzymes. Their nearly switchlike response as a function of membrane voltage comes from the movement of positively charged amino acids, arginine or lysine, across the membrane field. We used mutations with natural and unnatural amino acids, electrophysiological recordings, and x-ray crystallography to identify a charge transfer center in voltage sensors that facilitates this movement. This center consists of a rigid cyclic "cap" and two negatively charged amino acids to interact with a positive charge. Specific mutations induce a preference for lysine relative to arginine. By placing lysine at specific locations, the voltage sensor can be stabilized in different conformations, which enables a dissection of voltage sensor movements and their relation to ion channel opening

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival