S4 Charges Move Close to Residues in the Pore Domain during Activation in a K Channel

Voltage-gated ion channels respond to changes in the transmembrane voltage by opening or closing their ion conducting pore. The positively charged fourth transmembrane segment (S4) has been identified as the main voltage sensor, but the mechanisms of coupling between the voltage sensor and the gates are still unknown. Obtaining information about the location and the exact motion of S4 is an important step toward an understanding of these coupling mechanisms. In previous studies we have shown that the extracellular end of S4 is located close to segment 5 (S5). The purpose of the present study is to estimate the location of S4 charges in both resting and activated states. We measured the modification rates by differently charged methanethiosulfonate regents of two residues in the extracellular end of S5 in the Shaker K channel (418C and 419C). When S4 moves to its activated state, the modification rate by the negatively charged sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES−) increases significantly more than the modification rate by the positively charged [2-(trimethylammonium)ethyl] methanethiosulfonate, bromide (MTSET+). This indicates that the positive S4 charges are moving close to 418C and 419C in S5 during activation. Neutralization of the most external charge of S4 (R362), shows that R362 in its activated state electrostatically affects the environment at 418C by 19 mV. In contrast, R362 in its resting state has no effect on 418C. This suggests that, during activation of the channel, R362 moves from a position far away (>20 Å) to a position close (8 Å) to 418C. Despite its close approach to E418, a residue shown to be important in slow inactivation, R362 has no effect on slow inactivation or the recovery from slow inactivation. This refutes previous models for slow inactivation with an electrostatic S4-to-gate coupling. Instead, we propose a model with an allosteric mechanism for the S4-to-gate coupling

S4 Movement in a Mammalian HCN Channel

Hyperpolarization-activated, cyclic nucleotide–gated ion channels (HCN) mediate an inward cation current that contributes to spontaneous rhythmic firing activity in the heart and the brain. HCN channels share sequence homology with depolarization-activated Kv channels, including six transmembrane domains and a positively charged S4 segment. S4 has been shown to function as the voltage sensor and to undergo a voltage-dependent movement in the Shaker K+ channel (a Kv channel) and in the spHCN channel (an HCN channel from sea urchin). However, it is still unknown whether S4 undergoes a similar movement in mammalian HCN channels. In this study, we used cysteine accessibility to determine whether there is voltage-dependent S4 movement in a mammalian HCN1 channel. Six cysteine mutations (R247C, T249C, I251C, S253C, L254C, and S261C) were used to assess S4 movement of the heterologously expressed HCN1 channel in Xenopus oocytes. We found a state-dependent accessibility for four S4 residues: T249C and S253C from the extracellular solution, and L254C and S261C from the internal solution. We conclude that S4 moves in a voltage-dependent manner in HCN1 channels, similar to its movement in the spHCN channel. This S4 movement suggests that the role of S4 as a voltage sensor is conserved in HCN channels. In addition, to determine the reason for the different cAMP modulation and the different voltage range of activation in spHCN channels compared with HCN1 channels, we constructed a COOH-terminal–deleted spHCN. This channel appeared to be similar to a COOH-terminal–deleted HCN1 channel, suggesting that the main functional differences between spHCN and HCN1 channels are due to differences in their COOH termini or in the interaction between the COOH terminus and the rest of the channel protein in spHCN channels compared with HCN1 channels

Kinetic Relationship between the Voltage Sensor and the Activation Gate in spHCN Channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarizations that cause an inward movement of the positive charges in the fourth transmembrane domain (S4), which triggers channel opening. The mechanism of how the motion of S4 charges triggers channel opening is unknown. Here, we used voltage clamp fluorometry (VCF) to detect S4 conformational changes and to correlate these to the different activation steps in spHCN channels. We show that S4 undergoes two distinct conformational changes during voltage activation. Analysis of the fluorescence signals suggests that the N-terminal region of S4 undergoes conformational changes during a previously characterized mode shift in HCN channel voltage dependence, while a more C-terminal region undergoes an additional conformational change during gating charge movements. We fit our fluorescence and ionic current data to a previously proposed 10-state allosteric model for HCN channels. Our results are not compatible with a fast S4 motion and rate-limiting channel opening. Instead, our data and modeling suggest that spHCN channels open after only two S4s have moved and that S4 motion is rate limiting during voltage activation of spHCN channels

Molecular Movement of the Voltage Sensor in a K Channel

The X-ray crystallographic structure of KvAP, a voltage-gated bacterial K channel, was recently published. However, the position and the molecular movement of the voltage sensor, S4, are still controversial. For example, in the crystallographic structure, S4 is located far away (>30 Å) from the pore domain, whereas electrostatic experiments have suggested that S4 is located close (<8 Å) to the pore domain in open channels. To test the proposed location and motion of S4 relative to the pore domain, we induced disulphide bonds between pairs of introduced cysteines: one in S4 and one in the pore domain. Several residues in S4 formed a state-dependent disulphide bond with a residue in the pore domain. Our data suggest that S4 is located close to the pore domain in a neighboring subunit. Our data also place constraints on possible models for S4 movement and are not compatible with a recently proposed KvAP model

Adaptive Governance and Resilience Capacity of Farms: The Fit Between Farmers’ Decisions and Agricultural Policies

Greater resilience is needed for farms to deal with shocks and disturbances originating from economic, environmental, social and institutional challenges, with resilience achieved by adequate adaptive governance. This study focuses on the resilience capacity of farms in the context of multi-level adaptive governance. We define adaptive governance as adjustments in decision-making processes at farm level and policy level, through changes in management practices and policies in response to identified challenges and the delivery of desired functions (e.g. private and public goods) to be attained. The aim of the study is twofold. First, we investigate how adaptive governance processes at farm level and policy level influence the resilience capacity of farms in terms of robustness, adaptability and transformability. Second, we investigate the “fit” between the adaptive governance processes at farm level and policy level to enable resilience. We study primary egg and broiler production in Sweden taking into consideration economic, social and environmental challenges. We use semi-structured interviews with 17 farmers to explain the adaptive processes at farm level and an analysis of policy documents from the Common Agricultural Policy program 2014–2020, to explain the intervention actions taken by the Common Agricultural Policy. Results show that neither the farm level nor policy level adaptive processes on their own have the capacity to fully enable farms to be robust, adaptable and transformable. While farm level adaptive processes are mainly directed toward securing the robustness and adaptability of farms, policy level interventions are targeted at enabling adaptability. The farm- and the policy level adaptive processes do not “fit” for attaining robustness and transformability

High Density Circumstellar Interaction in the Luminous Type IIn SN 2010jl: The first 1100 days

HST and ground based observations of the Type IIn SN 2010jl are analyzed, including photometry, spectroscopy in the ultraviolet, optical and NIR bands, 26-1128 days after first detection. At maximum the bolometric luminosity was $\sim 3\times10^{43}$ erg/s and even at 850 days exceeds $10^{42}$ erg/s. A NIR excess, dominating after 400 days, probably originates in dust in the circumstellar medium (CSM). The total radiated energy is $> 6.5\times10^{50}$ ergs, excluding the dust component. The spectral lines can be separated into one broad component due to electron scattering, and one narrow with expansion velocity $\sim 100$ km/s from the CSM. The broad component is initially symmetric around zero velocity but becomes blueshifted after $\sim 50$ days, while remaining symmetric about a shifted centroid velocity. Dust absorption in the ejecta is unlikely to explain the line shifts, and we attribute the shift instead to acceleration by the SN radiation. From the optical lines and the X-ray and dust properties, there is strong evidence for large scale asymmetries in the CSM. The ultraviolet lines indicate CNO processing in the progenitor, while the optical shows a number of narrow coronal lines excited by the X-rays. The bolometric light curve is consistent with a radiative shock in an $r^{-2}$ CSM with a mass loss rate of $\sim 0.1$ M_sun/yr. The total mass lost is $> 3$ M_sun. These properties are consistent with the SN expanding into a CSM characteristic of an LBV progenitor with a bipolar geometry. The apparent absence of nuclear processing is attributed to a CSM still opaque to electron scattering.Comment: ApJ in press. Updated and changed after referees comment