Physics-based force fields for ligand-protein docking usually determine electrostatic energy with distance-dependent dielectric (DDD) functions, which do not fully account for the dielectric permittivity variance between approximately 2 in the protein core and approximately 80 in bulk water. Here we propose an atom-atom solvent exposure- and distance-dependent dielectric (SEDDD) function, which accounts for both electrostatic and dehydration energy components. Docking was performed using the ZMM program, the AMBER force field, and precomputed libraries of ligand conformers. At the seeding stage, hundreds of thousands of positions and orientations of conformers from the libraries were sampled within the rigid protein. At the refinement stage, the ten lowest-energy structures from the seeding stage were Monte Carlo-minimized with the flexible ligand and flexible protein. A search was considered a success if the root mean square deviation (RMSD) of the ligand atoms in the apparent global minimum from the x-ray structure was <2 A. Calculations on an examining set of 60 ligand-protein complexes with different DDD functions and solvent-exclusion energy revealed outliers in most of which the ligand-binding site was located at the protein surface. Using a training set of 16 ligand-protein complexes, which did not overlap with the examining set, we parameterized the SEDDD function to minimize the RMSD of the apparent global minima from the x-ray structures. Recalculation of the examining set with the SEDDD function demonstrated a 20% increase in the success rate versus the best-performing DDD function.

Ion permeation through voltage-gated sodium channels is modulated by various drugs and toxins. The atomistic mechanisms of action of many toxins are poorly understood. A steroidal alkaloid batrachotoxin (BTX) causes persistent channel activation by inhibiting inactivation and shifting the voltage dependence of activation to more negative potentials. Traditionally, BTX is considered to bind at the channel-lipid interface and allosterically modulate the ion permeation. However, amino acid residues critical for BTX action are found in the inner helices of all four repeats, suggesting that BTX binds in the pore. In the octapeptide segment IFGSFFTL in IIIS6 of a cockroach sodium channel BgNa V , besides Ser_3i15 and Leu_3i19, which correspond to known BTX-sensing residues of mammalian sodium channels, we found that Gly_3i14 and Phe_3i16 are critical for BTX action. Using these data along with published data as distance constraints, we docked BTX in the Kv1.2-based homology model of the open BgNa V channel. We arrived at a model in which BTX adopts a horseshoe conformation with the horseshoe plane normal to the pore axis. The BTX ammonium group is engaged in cation-interactions with Phe_3i16 and BTX moieties interact with known BTX-sensing residues in all four repeats. Oxygen atoms at the horseshoe inner surface constitute a transient binding site for permeating cations, whereas the bulky BTX molecule would resist the pore closure, thus causing persistent channel activation. Our study reinforces the concept that steroidal sodium channel agonists bind in the inner pore of sodium channels and elaborates the atomistic mechanism of BTX action.Voltage-gated sodium channels (Na V ) are responsible for the rapid rising phase of the action potential in nerve and muscle cells. The pore-forming ␣-subunit of Na V channels contains four repeats, and each repeat has six transmembrane helices (S1-S6). The S1-S4 helices form the voltage sensor domain.Positively charged S4 helices move outward in response to membrane depolarization. The S5 and S6 helices contribute to the pore-forming domain. The extracellular linkers connecting S5 and S6 helices form the four re-entrant P-loops, which contain the selectivity filter residues. In the absence of x-ray structures of Na V channels, their homology models, which are based on x-ray structures of potassium channels, are used to explain structure-activity relationships of various sodium channel ligands, including local anesthetics (1-4), steroidal activators (5-8), and pyrethroid insecticides (9, 10). Recent reinterpretation of data on substituted cysteine accessibility of the Ca V 2.1 channel (11) in view of a the channel homology model, which is based on the x-ray structure of the open voltage-gated potassium channel Kv1.2 (12), further supports a general similarity of the inner pore architecture in different voltage-gated cationic channels (13).Sodium channels are targets for numerous drugs and naturally occurring toxins. Batrachotoxin (BTX) 4 is a steroidal sodium-channel agonist, which...

Voltage-gated sodium and calcium channels play key roles in the physiology of excitable cells. The alpha-1 subunit of these channels folds from a polypeptide chain of four homologous repeats. In each repeat, the cytoplasmic halves of the pore-lining helices contain exceptionally conserved asparagines. Such conservation implies important roles, which are unknown. Mutations of the asparagines affect activation and inactivation gating as well as the action of pore-targeting ligands, including local anesthetics and steroidal agonists batrachotoxin and veratridine. In the absence of the open-channel structures, underlying mechanisms are unclear. Here, we modeled the pore module of Cav1.2 and Nav1.4 channels and their mutants in the open and closed states using the X-ray structures of potassium and sodium channels as templates. The energy of each model was Monte Carlo-minimized. The asparagines do not face the pore in the modeled states. In the open-channel models, the asparagine residue in a given repeat forms an inter-repeat H-bond with a polar residue, which is typically nine positions downstream from the conserved asparagine in the preceding repeat. The H-bonds, which are strengthened by surrounding hydrophobic residues, would stabilize the open channel and shape the open-pore geometry. According to our calculation, the latter is much more sensitive to mutations of the asparagines than the closed-pore geometry. Rearrangement of inter-repeat contacts may explain effects of these mutations on the voltage dependence of activation and inactivation and action of pore-targeting ligands.

Batrachotoxin (BTX), a steroidal alkaloid, and pyrethroid insecticides bind to distinct but allosterically coupled receptor sites on voltage-gated sodium channels and cause persistent channel activation. BTX presumably binds in the inner pore, whereas pyrethroids are predicted to bind at the lipid-exposed cavity formed by the short intracellular linker-helix IIS4-S5 and transmembrane helices IIS5 and IIIS6. The alkylamide insecticide (2E,4E)-N-(1,2-dimethylpropyl)-6-(5-bromo-2-naphthalenyl)-2,4-hexadienamide (BTG 502) reduces sodium currents and antagonizes the action of BTX on cockroach sodium channels, suggesting that it also binds inside the pore. However, a pyrethroid-sensing residue, Phe 3i17 in IIIS6, which does not face the pore, is essential for the activity of BTG 502 but not for BTX. In this study, we found that three additional deltamethrin-sensing residues in IIIS6, Ile 3i12 , Gly 3i14 , and Phe 3i16 (the latter two are also BTX-sensing), and three BTX-sensing residues, Ser 3i15 and Leu 3i19 in IIIS6 and Phe 4i15 in IVS6, are all critical for BTG 502 action on cockroach sodium channels. Using these data as constraints, we constructed a BTG 502 binding model in which BTG 502 wraps around IIIS6, probably making direct contacts with all of the above residues on the opposite faces of the IIIS6 helix, except for the putative gating hinge Gly 3i14 . BTG 502 and its inactive analog DAP 1855 antagonize the action of deltamethrin. The antagonism was eliminated by mutations of Ser 3i15 , Phe 3i17 , Leu 3i19 , and Phe 4i15 but not by mutations of Ile 3i12 , Gly 3i14 , and Phe 3i16 . Our analysis revealed a unique mode of action of BTG 502, its receptor site overlapping with those of both BTX and deltamethrin.

interacts with CaV1.2 in a similar domain as calmodulin (CaM). The purpose of this study is test the hypothesis that CaM and CCt compete for functional interaction with CaV1.2. ICa,L and barium current (IBa,L) was recorded from HEK 293 cells transfected with CaV1.2 þ CaVbeta2a. This background was compared to cells additionally transfected with CaM and/or CCt. The CaV1.2 expressed was deleted at position 1733 (numbering based on rabbit sequence), and CCt corresponded to amino acids 1821-2171. ICa,L and IBa,L was recorded in each cell, and we compared the increase of current, the shift of activation midpoint, and current kinetics of ICa,L versus IBa,L within a given cell. Maximal conductance ratio Ca/Ba is ~0.4 for CaV1.2þCaVbeta2a expression. Addition of CaM co-expression does not alter Ca /Ba conductance. CCt co-expression significantly increases the relative Ca /Ba ratio 2-fold, and this effect is reversed by CCtþCaM co-expression. Examination of the peak I(V) curves suggests that midpoint of activation was not affected, and ICa,L density is not different in for all transfection conditions. We conclude that CCt attenuation of conductance occurs only with Ba, and is consistent with a Ca alleviation of CCt block. Thus, CaM and Ca functionally compete to limit CCt auto-inhibition of CaV1.2 current.