High yield purification of full-length functional hERG K+ channels produced in Saccharomyces cerevisiae.

The hERG potassium channel is essential for repolarization of the cardiac action potential. Due to this vital function, absence of unintended and potentially life-threatening interactions with hERG is required for approval of new drugs. The structure of hERG is therefore one of the most sought-after. To provide purified hERG for structural studies and new hERG biomimetic platforms for detection of undesirable interactions, we have developed a hERG expression platform generating unprecedented amounts of purified and functional hERG channels. Full-length hERG, with or without a C-terminally fused green fluorescent protein (GFP) His 8-tag was produced from a codon-optimized hERG cDNA in Saccharomyces cerevisiae. Both constructs complemented the high potassium requirement of a knock-out Saccharomyces cerevisiae strain, indicating correct tetramer assembly in vivo. Functionality was further demonstrated by Astemizole binding to membrane embedded hERG-GFP-His 8 with a stoichiometry corresponding to tetramer assembly. The 156 kDa hERG-GFP protein accumulated to a membrane density of 1.6%. Fluorescence size exclusion chromatography of hERG-GFP-His 8 solubilized in Fos-Choline-12 supplemented with cholesteryl-hemisuccinate and Astemizole resulted in a monodisperse elution profile demonstrating a high quality of the hERG channels. hERG-GFP-His 8 purified by Ni-affinity chromatography maintained the ability to bind Astemizole with the correct stoichiometry indicating that the native, tetrameric structure was preserved. To our knowledge this is the first reported high-yield production and purification of full length, tetrameric and functional hERG. This significant breakthrough will be paramount in obtaining hERG crystal structures, and in establishment of new high-throughput hERG drug safety screening assays.

Characterization of transport mechanisms and determinants critical for Na+-dependent Pi symport of the PiT family paralogs human PiT1 and PiT2.

The general phosphate need in mammalian cells is accommodated by members of the P(i) transport (PiT) family (SLC20), which use either Na(+) or H(+) to mediate inorganic phosphate (P(i)) symport. The mammalian PiT paralogs PiT1 and PiT2 are Na(+)-dependent P(i) (NaP(i)) transporters and are exploited by a group of retroviruses for cell entry. Human PiT1 and PiT2 were characterized by expression in Xenopus laevis oocytes with (32)P(i) as a traceable P(i) source. For PiT1, the Michaelis-Menten constant for P(i) was determined as 322.5 +/- 124.5 microM. PiT2 was analyzed for the first time and showed positive cooperativity in P(i) uptake with a half-maximal activity constant for P(i) of 163.5 +/- 39.8 microM. PiT1- and PiT2-mediated Na(+)-dependent P(i) uptake functions were not significantly affected by acidic and alkaline pH and displayed similar Na(+) dependency patterns. However, only PiT2 was capable of Na(+)-independent P(i) transport at acidic pH. Study of the impact of divalent cations Ca(2+) and Mg(2+) revealed that Ca(2+) was important, but not critical, for NaP(i) transport function of PiT proteins. To gain insight into the NaP(i) cotransport function, we analyzed PiT2 and a PiT2 P(i) transport knockout mutant using (22)Na(+) as a traceable Na(+) source. Na(+) was transported by PiT2 even without P(i) in the uptake medium and also when P(i) transport function was knocked out. This is the first time decoupling of P(i) from Na(+) transport has been demonstrated for a PiT family member. Moreover, the results imply that putative transmembrane amino acids E(55) and E(575) are responsible for linking P(i) import to Na(+) transport in PiT2.

Co-expression of mCysLT1 receptors and IK channels in Xenopus laevis oocytes elicits LTD4-stimulated IK current, independent of an increase in [Ca2+]i.

Addition of LTD4 (10 nM) to Xenopus laevis oocytes expressing the mCysLT1 receptor together with hBK or hIK channels resulted in the activation of both channels secondary to an LTD4-induced increase in [Ca2+]i. In addition, the hIK channel is activated by low concentrations of LTD4 (<0.1 nM), which did not result in any increase in [Ca2+]i. Even though activation of hIK by low concentrations of LTD4 was independent of an increase in [Ca2+]i, a certain "permissive" level of [Ca2+]i was required for its activation, since buffering of intracellular Ca2+ by EGTA completely abolished the response to LTD4. Neither hTBAK1 nor hTASK2 was activated following stimulations with LTD4 (0.1 and 100 nM).

KCNE4 is an inhibitory subunit to Kv1.1 and Kv1.3 potassium channels.

Kv1 potassium channels are widely distributed in mammalian tissues and are involved in a variety of functions from controlling the firing rate of neurons to maturation of T-lymphocytes. Here we show that the newly described KCNE4 beta-subunit has a drastic inhibitory effect on currents generated by Kv1.1 and Kv1.3 potassium channels. The inhibition is found on channels expressed heterologously in both Xenopus oocytes and mammalian HEK293 cells. mKCNE4 does not inhibit Kv1.2, Kv1.4, Kv1.5, or Kv4.3 homomeric complexes, but it does significantly reduce current through Kv1.1/Kv1.2 and Kv1.2/Kv1.3 heteromeric complexes. Confocal microscopy and Western blotting reveal that Kv1.1 is present at the cell surface together with KCNE4. Real-time RT-PCR shows a relatively high presence of mKCNE4 mRNA in several organs, including uterus, kidney, lung, intestine, and in embryo, whereas a much lower mRNA level is detected in the heart and in five different parts of the brain. Having the broad distribution of Kv1 channels in mind, the demonstrated inhibitory property of KCNE4-subunits could locally and/or transiently have a dramatic influence on cellular excitability and on setting resting membrane potentials.