The human ether-a-go-go related gene (hERG) encodes the voltage-gated K(+) channel that underlies the rapidly activating delayed-rectifier current in cardiac myocytes. hERG is synthesized in the endoplasmic reticulum (ER) as an "immature" N-linked glycoprotein and is terminally glycosylated in the Golgi apparatus. Most hERG missense mutations linked to long QT syndrome type 2 (LQT2) reduce the terminal glycosylation and functional expression. We tested the hypothesis that a distinct pre-Golgi compartment negatively regulates the trafficking of some LQT2 mutations to the Golgi apparatus. We found that treating cells in nocodazole, a microtubule depolymerizing agent, altered the subcellular localization, functional expression, and glycosylation of the LQT2 mutation G601S-hERG differently from wild-type hERG (WT-hERG). G601S-hERG quickly redistributed to peripheral compartments that partially colocalized with KDEL (Lys-Asp-Glu-Leu) chaperones but not calnexin, Sec31, or the ER golgi intermediate compartment (ERGIC). Treating cells in E-4031, a drug that increases the functional expression of G601S-hERG, prevented the accumulation of G601S-hERG to the peripheral compartments and increased G601S-hERG colocalization with the ERGIC. Coexpressing the temperature-sensitive mutant G protein from vesicular stomatitis virus, a mutant N-linked glycoprotein that is retained in the ER, showed it was not restricted to the same peripheral compartments as G601S-hERG at nonpermissive temperatures. We conclude that the trafficking of G601S-hERG is negatively regulated by a microtubule-dependent compartment within the ER. Identifying mechanisms that prevent the sorting or promote the release of LQT2 channels from this compartment may represent a novel therapeutic strategy for LQT2.

KCNH2 encodes the Kv11.1 channel, which conducts the rapidly activating delayed rectifier K+ current (IKr) in the heart. KCNH2 mutations cause type 2 long QT syndrome (LQT2), which increases the risk for life-threatening ventricular arrhythmias. LQT2 mutations are predicted to prolong the cardiac action potential (AP) by reducing IKr during repolarization. Kv11.1 contains several conserved basic amino acids in the fourth transmembrane segment (S4) of the voltage sensor that are important for normal channel trafficking and gating. This study sought to determine the mechanism(s) by which LQT2 mutations at conserved arginine residues in S4 (R531Q, R531W or R534L) alter Kv11.1 function. Western blot analyses of HEK293 cells transiently expressing R531Q, R531W or R534L suggested that only R534L inhibited Kv11.1 trafficking. Voltage-clamping experiments showed that R531Q or R531W dramatically altered Kv11.1 current (IKv11.1) activation, inactivation, recovery from inactivation and deactivation. Coexpression of wild type (to mimic the patients’ genotypes) mostly corrected the changes in IKv11.1 activation and inactivation, but deactivation kinetics were still faster. Computational simulations using a human ventricular AP model showed that accelerating deactivation rates was sufficient to prolong the AP, but these effects were minimal compared to simply reducing IKr. These are the first data to demonstrate that coexpressing wild type can correct activation and inactivation dysfunction caused by mutations at a critical voltage-sensing residue in Kv11.1. We conclude that some Kv11.1 mutations might accelerate deactivation to cause LQT2 but that the ventricular AP duration is much more sensitive to mutations that decrease IKr. This likely explains why most LQT2 mutations are nonsense or trafficking-deficient.

A quantum calculation (DFT) has been carried out on the crown ether (CE) 14-C-4, together with up to 14 methanol molecules, or 27 water molecules, plus one ion. The free energy of complex formation is known experimentally for both Na þ and K þ ions, allowing comparison for methanol; neither ion can be complexed from bulk water. We calculate that the ions could be complexed from a more limited water solvent shell. In order to avoid leaving an unrealistic CE-vapor interface, 8 water molecules or 4 methanol molecules were placed on the side of the CE opposite the ion. Thus ''bulk solvation'' was 10 methanol or 19 water molecules. In addition to optimizing the geometry at B3LYP/6-311þþG** level, we did frequency calculations to obtain the thermodynamic quantities. The errors average approximately 2 kcal/mole. The methanol complex formation values are, for K þ , À2.40kcal/mole (calculated), À1.80 kcal/ mole (experimental); for Na þ , À5.4 kcal/mole(calculated), À2.2 or-3.0 kcal/ mole (experimental, from 2 laboratories). For water, there is only the qualitative observation that complexes do not form; however, in the calculation, removing 7 water molecules results in only one shell of solvation. The free energy of complex formation is then negative, so that a complex could form. Since the system has over 100 atoms, it is large enough to model the interaction of a protein with an ion at the surface of the protein (see abstract of Kariev and Green: ''Quantum calculations on the KcsA channel 1/4rdquo;), suggesting that quantum calculation is useful in a case where polarizability and charge transfer, neither present in standard molecular dynamics, are important. (Acknowledgement for calculations: W.R. Wiley supercomputer facility of the EMSL at PNNL).

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M-channels are slowly activated, non-inactivating, voltage-dependent potassium channels. Heteromeric assembly of subunits, encoded by two members of the KCNQ gene family KCNQ2 and KCNQ3, recapitulate the functional properties of the M-current. KCNQ2 and KCNQ3 are co-expressed on the cell body and dendrites of hippocampal and cortical neurons. Importantly,