Voltage sensor domains (VSDs) regulate ion channels and enzymes by transporting electrically charged residues across a hydrophobic VSD constriction called the gating pore or hydrophobic plug. How the gating pore controls the gating charge movement presently remains debated. Here, using saturation mutagenesis and detailed analysis of gating currents from gating pore mutations in the Shaker Kv channel, we identified statistically highly significant correlations between VSD function and physicochemical properties of gating pore residues. A necessary small residue at position S240 in S1 creates a "steric gap" that enables an intracellular access pathway for the transport of the S4 Arg residues. In addition, the stabilization of the depolarized VSD conformation, a hallmark for most Kv channels, requires large side chains at positions F290 in S2 and F244 in S1 acting as "molecular clamps," and a hydrophobic side chain at position I237 in S1 acting as a local intracellular hydrophobic barrier. Finally, both size and hydrophobicity of I287 are important to control the main VSD energy barrier underlying transitions between resting and active states. Taken together, our study emphasizes the contribution of several gating pore residues to catalyze the gating charge transfer. This work paves the way toward understanding physicochemical principles underlying conformational dynamics in voltage sensors.energy landscape | kinetic model | global fit

The prokaryotic KcsA channel is gated at the helical bundle crossing by intracellular protons and inactivates at the extracellular selectivity filter. The C-terminal transmembrane helix has to undergo a conformational change for potassium ions to access the central cavity. Whereas a partial opening of the tetrameric channel is suggested to be responsible for subconductance levels of ion channels, including KcsA, a cooperative opening of the 4 subunits is postulated as the final opening step. In this study, we used single-channel fluorescence spectroscopy of KcsA to directly observe the movement of each subunit and the temporal correlation between subunits. Purified KcsA channels labeled at the C terminus near the bundle crossing have been inserted into supported lipid bilayer, and the fluorescence traces analyzed by means of a cooperative or independent Markov model. The analysis revealed that the 4 subunits do not move fully independently but instead showed a certain degree of cooperativity. However, the 4 subunits do not simply open in 1 concerted step.gating ͉ ion channel ͉ single-molecule

Distance determination from an echo intensity modulation obtained by pulsed double electron-electron resonance (DEER) experiment is a mathematically ill-posed problem. Tikhonov regularization yields distance distributions that can be difficult to interpret, especially in system with multiple discrete distance distributions. Here, we show that by using geometric fit constraints in symmetric homo-oligomeric protein systems, we were able to increase the accuracy of a model-based fit solution based on a sum of Gaussian distributions. Our approach was validated on two different ion channels of known oligomeric states, KcsA (a tetramer) and CorA (a pentamer). Statistical analysis of the resulting fits was integrated within our method to help the experimenter evaluate the significance of a symmetry-constrained vs. standard model distribution fit and to examine multi-distance confidence regions. This approach was used to quantitatively evaluate the role of the C-terminal domain (CTD) on the flexibility and conformation of the activation gate of the K+ channel KcsA. Our analysis reveals a significant increase in the dynamics of the inner bundle gate upon opening. Also, it explicitly demonstrates the degree to which the CTD restricts the motion of the lower gate at rest and during activation gating.

SUMMARY Proteins may undergo multiple conformational changes required for their function. One strategy used to estimate target site positions in unknown structural conformations involves single-pair resonance energy transfer (RET) distance measurements. However, interpretation of inter-residue distances is difficult when applied to three-dimensional structural rearrangements, especially in homomeric systems. We developed a novel method using inverse trilateration/triangulation to map target sites within a homomeric protein in all defined states with simultaneous functional recordings. The procedure accounts for probe diffusion to accurately determine the three-dimensional position and confidence region of lanthanide LRET donors attached to a target site (one/subunit), relative to a single fluorescent acceptor placed in a static site. As a first application, the method is used to determine the position of a functional voltage-gated potassium channel’s voltage sensor. Our results verify the crystal structure relaxed conformation and report on the resting and active conformations for which crystal structures are not available.

Large-conductance Ca 2+ -and voltage-activated K + (BK) channels are involved in a large variety of physiological processes. Regulatory β-subunits are one of the mechanisms responsible for creating BK channel diversity fundamental to the adequate function of many tissues. However, little is known about the structure of its voltage sensor domain. Here, we present the external architectural details of BK channels using lanthanide-based resonance energy transfer (LRET). We used a genetically encoded lanthanide-binding tag (LBT) to bind terbium as a LRET donor and a fluorophore-labeled iberiotoxin as the LRET acceptor for measurements of distances within the BK channel structure in a living cell. By introducing LBTs in the extracellular region of the α-or β1-subunit, we determined (i) a basic extracellular map of the BK channel, (ii) β1-subunit-induced rearrangements of the voltage sensor in α-subunits, and (iii) the relative position of the β1-subunit within the α/β1-subunit complex.lanthanide resonance energy transfer | BK channels | β1-subunit I mportant physiological processes involve Ca 2+ entry into cells mediated by voltage-dependent Ca 2+ channels. This divalent cation influx is essential for life because it permits, for example, the adequate functioning of smooth muscle or neurosecretion to occur. Some mechanism must be put into action, however, to control Ca 2+ influx, either to dampen or to stop the physiological effects of the cytoplasmic increase in Ca 2+. In many cases, this dampening mechanism is accomplished by one of the most broadly expressed channels in mammals: the large-conductance Ca 2+ -and voltage-activated K + (BK) channel (1-3). Because there is a single gene coding for the BK channel (Slowpoke KNCMA1), channel diversity must be a consequence of alternative splicing and/or interaction with regulatory subunits. In fact, both mechanisms account for BK channel diversity, but the most dramatic changes in BK channel properties are brought about through the interaction with regulatory subunits, membrane-integral proteins, denominated BK β-subunits (β1-β4) (4-7) and the recently discovered γ-subunits (γ1-γ4) (8, 9).Structurally, the BK channel is a homotetramer of its poreforming α-subunit and is a member of the voltage-dependent potassium (Kv) channel family. Distinct from Kv channels, however, BK channel subunits are composed of seven transmembrane domains S0-S6 (10, 11). Little is known about the detailed structure of the membrane-spanning portion of the BK channel, or of the α/β1-subunit complex. Here, we used a variant of Förster resonance energy transfer (FRET), called lanthanide-based resonance energy transfer (LRET), to determine the positions of the N terminus (NT) and S0, S1, and S2 transmembrane segments of the α-subunit of the BK channel, as well as the position of the β1-subunit in the α/β1-subunit complex. LRET uses luminescent lanthanides (e.g., Tb 3+ ) as donor instead of conventional fluorophores. This technique has been successfully used to measure intramolecular distances and to...

current. The magnitude of the gating charge in Kv1.2 potassium channel is calculated from more than 1microsecond of all-atom molecular dynamics simulation. Free energy calculations are performed to determine the individual contribution of several (nine) charged residues of the VSD to the gating charge. The total gating charge obtained for the refined models of the channel is~10.5e, indicating that the refined model of the closed resting state most likely represents an intermediate conformation that precedes closing of the channel. Through steered molecular dynamics (SMD) simulations we identify a closed conformation of the channel, corresponding to a gating charge of 12.7e, in accord with experimental values obtained for the Shaker potassium channel. 2694-PosPathway Calculation of the Conformational Transition of the Voltage Sensor Domain in the Kv1.2 Channel

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