Arginine side chain interactions and the role of arginine as a gating charge carrier in voltage sensitive ion channels

Gating charges in voltage-sensing domains (VSD) of voltage-sensitive ion channels and enzymes are carried on arginine side chains rather than lysine. This arginine preference may result from the unique hydration properties of the side chain guanidinium group which facilitates its movement through a hydrophobic plug that seals the center of the VSD, as suggested by molecular dynamics simulations. To test for side chain interactions implicit in this model we inspected interactions of the side chains of arginine and lysine with each of the 19 non-glycine amino acids in proteins in the protein data bank. The arginine guanidinium interacts with non-polar aromatic and aliphatic side chains above and below the guanidinium plane while hydrogen bonding with polar side chains is restricted to in-plane positions. In contrast, non-polar side chains interact largely with the aliphatic part of the lysine side chain. The hydration properties of arginine and lysine are strongly reflected in their respective interactions with non-polar and polar side chains as observed in protein structures and in molecular dynamics simulations, and likely underlie the preference for arginine as a mobile charge carrier in VSD

Ranolazine inhibition of hERG potassium channels: Drug–pore interactions and reduced potency against inactivation mutants

AbstractThe antianginal drug ranolazine, which combines inhibitory actions on rapid and sustained sodium currents with inhibition of the hERG/IKr potassium channel, shows promise as an antiarrhythmic agent. This study investigated the structural basis of hERG block by ranolazine, with lidocaine used as a low potency, structurally similar comparator. Recordings of hERG current (IhERG) were made from cell lines expressing wild-type (WT) or mutant hERG channels. Docking simulations were performed using homology models built on MthK and KvAP templates. In conventional voltage clamp, ranolazine inhibited IhERG with an IC50 of 8.03μM; peak IhERG during ventricular action potential clamp was inhibited ~62% at 10μM. The IC50 values for ranolazine inhibition of the S620T inactivation deficient and N588K attenuated inactivation mutants were respectively ~73-fold and ~15-fold that for WT IhERG. Mutations near the bottom of the selectivity filter (V625A, S624A, T623A) exhibited IC50s between ~8 and 19-fold that for WT IhERG, whilst the Y652A and F656A S6 mutations had IC50s ~22-fold and 53-fold WT controls. Low potency lidocaine was comparatively insensitive to both pore helix and S6 mutations, but was sensitive to direction of K+ flux and particularly to loss of inactivation, with an IC50 for S620T-hERG ~49-fold that for WT IhERG. Docking simulations indicated that the larger size of ranolazine gives it potential for a greater range of interactions with hERG pore side chains compared to lidocaine, in particular enabling interaction of its two aromatic groups with side chains of both Y652 and F656. The N588K mutation is responsible for the SQT1 variant of short QT syndrome and our data suggest that ranolazine is unlikely to be effective against IKr/hERG in SQT1 patients

Molecular basis of hERG potassium channel blockade by the class Ic antiarrhythmic flecainide

AbstractThe class Ic antiarrhythmic drug flecainide inhibits KCNH2-encoded “hERG” potassium channels at clinically relevant concentrations. The aim of this study was to elucidate the underlying molecular basis of this action. Patch clamp recordings of hERG current (IhERG) were made from hERG expressing cells at 37°C. Wild-type (WT) IhERG was inhibited with an IC50 of 1.49μM and this was not significantly altered by reversing the direction of K+ flux or raising external [K+]. The use of charged and uncharged flecainide analogues showed that the charged form of the drug accesses the channel from the cell interior to produce block. Promotion of WT IhERG inactivation slowed recovery from inhibition, whilst the N588K and S631A attenuated-inactivation mutants exhibited IC50 values 4–5 fold that of WT IhERG. The use of pore-helix/selectivity filter (T623A, S624A V625A) and S6 helix (G648A, Y652A, F656A) mutations showed <10-fold shifts in IC50 for all but V625A and F656A, which respectively exhibited IC50s 27-fold and 142-fold their WT controls. Docking simulations using a MthK-based homology model suggested an allosteric effect of V625A, since in low energy conformations flecainide lay too low in the pore to interact directly with that residue. On the other hand, the molecule could readily form π–π stacking interactions with aromatic residues and particularly with F656. We conclude that flecainide accesses the hERG channel from the cell interior on channel gating, binding low in the inner cavity, with the S6 F656 residue acting as a principal binding determinant

Inhibition of the hERG Potassium Channel by a Methanesulphonate-Free E-4031 Analogue

hERG (human Ether-à-go-go Related Gene)-encoded potassium channels underlie the cardiac rapid delayed rectifier (IKr) potassium current, which is a major target for antiarrhythmic agents and diverse non-cardiac drugs linked to the drug-induced form of long QT syndrome. E-4031 is a high potency hERG channel inhibitor from the methanesulphonanilide drug family. This study utilized a methanesulphonate-lacking E-4031 analogue, “E-4031-17”, to evaluate the role of the methanesulphonamide group in E-4031 inhibition of hERG. Whole-cell patch-clamp measurements of the hERG current (IhERG) were made at physiological temperature from HEK 293 cells expressing wild-type (WT) and mutant hERG constructs. For E-4031, WT IhERG was inhibited by a half-maximal inhibitory concentration (IC50) of 15.8 nM, whilst the comparable value for E-4031-17 was 40.3 nM. Both compounds exhibited voltage- and time-dependent inhibition, but they differed in their response to successive applications of a long (10 s) depolarisation protocol, consistent with greater dissociation of E-4031-17 than the parent compound between applied commands. Voltage-dependent inactivation was left-ward voltage shifted for E-4031 but not for E-4031-17; however, inhibition by both compounds was strongly reduced by attenuated-inactivation mutations. Mutations of S6 and S5 aromatic residues (F656V, Y652A, F557L) greatly attenuated actions of both drugs. The S624A mutation also reduced IhERG inhibition by both molecules. Overall, these results demonstrate that the lack of a methanesulphonate in E-4031-17 is not an impediment to high potency inhibition of IhERG

The deep distributions of helium isotopes, radiocarbon, and noble gases along the U.S. GEOTRACES East Pacific Zonal Transect (GP16)

Author Posting. © The Author(s), 2017. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Marine Chemistry 201 (2018): 167-182, doi:10.1016/j.marchem.2017.03.009.We report the deep distributions of noble gases, helium isotopes, and radiocarbon measured during the U.S. GEOTRACES GP16 East Pacific Zonal Transect between 152 and 77°W at 12- 15°S in the South Pacific. The dominant feature is an intense tongue of hydrothermal effluent that extends more than 4,000 km westward from the East Pacific Rise (EPR) at ~2500m depth. The patterns reveal significant “downstream” variations in water mass structure, advection, and mixing that belie the simple perception of a continuous plume extending westward from the EPR. For example, one feature observed at 120°W, 14°S has tracer signatures that are consistent with a water mass originating from an area as much as 2,000 km south of this section, suggesting a quasi-permanent northward flow on the western flank of the EPR. Helium isotope variations in the plume show a uniquely high 3He/4He source in the tongue compared with typical mid-ocean ridge basalts (MORB), consistent with the anomalously high ratios observed in MORB glasses from the EPR segment just south of this transect. The water column data also reveal that the background 3He/4He east of the EPR is significantly lower than values characteristic of MORB, suggesting an additional, more geographically distributed radiogenic 4He flux of order 107 mol/y into the deep Pacific. In the western end of the section, incoming bottom waters have relatively less hydrothermal hydrothermal helium, more radiocarbon, and more oxygen, as well as negative saturation anomalies for the heavy noble gases (Ar, Kr, and Xe). During the basin-scale upwelling of this water, diapycnal mixing serves to erase these negative anomalies. The relative magnitudes of the increases for the heavy noble gases (Ar, Kr, and Xe) are quantitatively consistent with this process. This leads us to estimate the relatively smaller effects on He and Ne saturations, which range from near zero to 0.2% and 0.3% respectively. With this information, we are able to refine our estimates of the magnitude of 3He and 4He excesses and the absolute 3He/4He ratio of non-atmospheric helium introduced into deep Pacific waters.The work was funded under National Science Foundation grant number OCE-1232991 for WJJ and OCE-1130870 for CRG