Anna Stary

Toward a Consensus Model of the hERG Potassium Channel

Malfunction of hERG potassium channels, due to inherited mutations or inhibition by drugs, can cause long QT syndrome, which can lead to life‐threatening arrhythmias. A three‐dimensional structure of hERG is a prerequisite to understand the molecular basis of hERG malfunction. To achieve a consensus model, we carried out an extensive analysis of hERG models based on various alignments of helix S5. We analyzed seven models using a combination of conventional geometry/packing/normality validation methods as well as molecular dynamics simulations and molecular docking. A synthetic test set with the X‐ray crystal structure of Kv1.2 with artificially shifted S5 sequences modeled into the structure served as a reference case. We docked the known hERG inhibitors (+)‐cisapride, (S)‐terfenadine, and MK‐499 into the hERG models and simulation snapshots. None of the single analyses unambiguously identified a preferred model, but the combination of all three revealed that there is only one model that fulfils all quality criteria. This model is confirmed by a recent mutation scanning experiment (P. Ju, G. Pages, R. P. Riek, P. C. Chen, A. M. Torres, P. S. Bansal, S. Kuyucak, P. W. Kuchel, J. I. Vandenberg, J. Biol. Chem. 2009, 284, 1000–1008). 1 We expect the modeled structure to be useful as a basis both for computational studies of channel function and kinetics as well as the design of experiments.

The anti-protozoal drug pentamidine blocks KIR2.x-mediated inward rectifier current by entering the cytoplasmic pore region of the channel

Background and purpose:  Pentamidine is a drug used in treatment of protozoal infections. Pentamidine treatment may cause sudden cardiac death by provoking cardiac arrhythmias associated with QTc prolongation and U‐wave alterations. This proarrhythmic effect was linked to inhibition of hERG trafficking, but not to acute block of ion channels contributing to the action potential. Because the U‐wave has been linked to the cardiac inward rectifier current (IK1), we examined the action and mechanism of pentamidine‐mediated IK1 block.

Probing the Architecture of an L-type Calcium Channel with a Charged Phenylalkylamine EVIDENCE FOR A WIDELY OPEN PORE AND DRUG TRAPPING

Voltage-gated calcium channels are in a closed conformation at rest and open temporarily when the membrane is depolarized. To gain insight into the molecular architecture of Cav1.2, we probed the closed and open conformations with the charged phenylalkylamine (-)devapamil ((-)qD888). To elucidate the access pathway of (-)D888 to its binding pocket from the intracellular side, we used mutations replacing a highly conserved Ile-781 by threonine/proline in the pore-lining segment IIS6 of Cav1.2 (1). The shifted channel gating of these mutants (by 30–40 mV in the hyperpolarizing direction) enabled us to evoke currents with identical kinetics at different potentials and thus investigate the effect of the membrane potentials on the drug access per se. We show here that under these conditions the development of channel block by (-)qD888 is not affected by the transmembrane voltage. Recovery from block at rest was, however, accelerated at more hyperpolarized voltages. These findings support the conclusion that Cav1.2 must be opening widely to enable free access of the charged (-)D888 molecule to its binding site, whereas drug dissociation from the closed channel conformation is restricted by bulky channel gates. The functional data indicating a location of a trapped (-)D888 molecule close to the central pore region are supported by a homology model illustrating that the closed Cav1.2 is able to accommodate a large cation such as (-)D888.

Structural model of the Ca(V)1.2 pore.

Understanding the structure and functional mechanisms of voltage-gated calcium channels remains a major task in membrane biophysics. In the absence of three dimensional structures, homology modelling techniques are the method of choice, to address questions concerning the structure of these channels. We have developed models of the open Cav1.2 pore, based on the crystal structure of the mammalian voltage-gated potassium channel Kv1.2 and a model of the bacterial sodium channel NaChBac. Our models are developed to be consistent with experimental data and modelling criteria. The models highlight major differences between voltage-gated potassium and calcium channels, in the P segments, as well as the inner pore helices. Molecular dynamics simulations support the hypothesis of a clockwise domain arrangement and experimental observations of asymmetric calcium channel behaviour. In the accompanying paper these models were used to study structural effects of a channelopathy mutation.

Coupled and independent contributions of residues in IS6 and IIS6 to activation gating of CaV1.2.

Voltage dependence and kinetics of CaV1.2 activation are affected by structural changes in pore-lining S6 segments of the α1-subunit. Significant effects are induced by either proline or threonine substitutions in the lower third of segment IIS6 (“bundle crossing region”), where S6 segments are likely to seal the channel in the closed conformation (Hohaus, A., Beyl, S., Kudrnac, M., Berjukow, S., Timin, E. N., Marksteiner, R., Maw, M. A., and Hering, S. (2005) J. Biol. Chem. 280, 38471–38477). Here we report that S435P in IS6 results in a large shift of the activation curve (-25.9 ± 1.2 mV) and slower current kinetics. Threonine substitutions at positions Leu-429 and Leu-434 induced a similar kinetic phenotype with shifted activation curves (L429T by -6.6 ± 1.2 and L434T by -12.1 ± 1.7 mV). Inactivation curves of all mutants were shifted to comparable extents as the activation curves. Interdependence of IS6 and IIS6 mutations was analyzed by means of mutant cycle analysis. Double mutations in segments IS6 and IIS6 induce either additive (L429T/I781T, -34.1 ± 1.4 mV; L434T/I781T, -40.4 ± 1.3 mV; L429T/L779T, -12.6 ± 1.3 mV; and L434T/L779T, -22.4 ± 1.3 mV) or nonadditive shifts of the activation curves along the voltage axis (S435P/I781T, -33.8 ± 1.4 mV). Mutant cycle analysis revealed energetic coupling between residues Ser-435 and Ile-781, whereas other paired mutations in segments IS6 and IIS6 had independent effects on activation gating.

Molecular dynamics and mutational analysis of a channelopathy mutation in the IIS6 helix of Ca V 1.2

A channelopathy mutation in segment IIS6 of CaV1.4 (I745T) has been shown to cause severe visual impairment by shifting the activation and inactivation curves to more hyperpolarised voltages and slowing activation and inactivation kinetics. A similar gating phenotype is caused by the corresponding mutation, I781T, in CaV1.2 (midpoint of activation curve (V0.5) shifted to -37.7 ± 1.2 mV). We show here that wild type gating can partially be restored by a helix stabilising rescue mutation N785A. V0.5 of I781T/N785A (V0.5 = -21.5 ± 0.6 mV) was shifted back towards wild type (V0.5 = -9.9±1.1 mV). Homology models developed in our group (see accompanying article for details) were used to perform MD-simulations on wild-type and mutant channels. Systematic changes in segment IIIS6 (M1187 - F1194) and in helix IIS6 (N785-L786) were observed. The simulated structural changes in S6 segments of I781T/N785A were less pronounced than in I781T. A delicate balance between helix flexibility and stability enabling the formation of hydrophobic seals at the inner channel mouth appears to be important for wild type CaV1.2 gating. Our study illustrates that effects of mutations in the lower part of IIS6 may not be localized to the residue or even segment being mutated, but may affect conformations of interacting segments.