The gating behaviour and pharmacological sensitivity of HERG are remarkably different from the corresponding properties of M‐eag, a structurally similar member of the Eag family of potassium channels. In contrast to HERG, M‐eag exhibits no apparent inactivation and little rectification, and is insensitive to the class III antiarrhythmic drug E‐4031. We generated chimeric channels of HERG and M‐eag sequences and made point mutations to identify the region necessary for rapid inactivation in HERG. This region includes the P region and half of the S6 putative transmembrane domain, including sites not previously associated with inactivation and rectification in HERG. Transfer of a small segment of the HERG polypeptide to M‐eag, consisting largely of the P region and part of the S6 transmembrane domain, is sufficient to confer rapid inactivation and E‐4031 sensitivity to M‐eag. This region differs from the corresponding region in M‐eag by only fifteen residues. Previous hypotheses that rapid inactivation of HERG channels occurs by a C‐type inactivation mechanism are supported by the parallel effects on rates of HERG inactivation and Shaker C‐type inactivation by a series of mutations at two equivalent sites in the polypeptide sequences. In addition to sites homologous to those previously described for C‐type inactivation in Shaker, inactivation in HERG involves a residue in the upstream P region not previously associated with C‐type inactivation. Although this site is equivalent to one implicated in P‐type inactivation in Kv2.1 channels, our data are most consistent with a single, C‐type inactivation mechanism.
The nuclear accessory protein in porcine intestinal nuclear extracts that activates the binding of the vitamin D receptor to its vitamin D response elements has been highly purified. It contains a protein that binds 9-cis- [H] retinoic acid, was detected on immunoblots with an anti-retinoid X receptor (RXR) peptide antibody, and supports the binding of retinoic acid receptor y to the retinoic acid receptor ,B gene response element. Most important, the two specific complexes formed by porcine nuclear extract with the vitamin D response elements from either the osteocalcin gene or the rat 24-hydroxylase gene are shifted to a larger complex by both an anti-vitamin D receptor antibody and an anti-RXR antibody, leaving no doubt that in vivo the nuclear accessory factor for the vitamin D receptor in the intestine is an RXR protein.Vitamin D regulates calcium homeostasis by the mobilization of calcium from bone, reabsorption of calcium in the kidney, and absorption of calcium from the intestine. The physiologically active metabolite of vitamin D 1,25-dihydroxyvitamin D3 [1,2D3] is known to exert these functions by binding to a transcription factor, the vitamin D receptor (VDR) (1). The VDR is a member of the steroid/thyroid receptor superfamily, which also includes nonsteroid binding receptors such as the retinoic acid receptors (RARs) and retinoid X receptors (RXRs) (2). VDR and RAR are members of a subset of this family that can bind to response elements as heterodimers and recognize response elements consisting of direct repeats (3).Several reports have demonstrated that RXRs can function in vitro as accessory factor(s) (e.g., heterodimerize) with VDR and other receptors. Heterodimerization of RXRs with VDRs, thyroid receptors, and RARs increases the affinity of these receptors for their response elements. When RXR cDNAs are cotransfected with these receptors, the transcriptional response to the respective ligand is increased (4-9). These studies provide evidence for a possible role of RXRs as a link between several ligand-induced pathways.A mammalian-derived nuclear accessory factor (NAF) required for tight binding of VDR to vitamin D response elements (DREs) has been described (10-12). The determination of the molecular weight of NAF from pig intestinal nuclear extract (PINE), a major vitamin D target tissue, revealed a molecular mass of 59-64 kDa (12). MacDonald et al. (13) MATERIALS AND METHODS Chemicals. 1,25-(OH)2D3 was purchased from Tetrionics (Madison, WI). 9-cis-Retinoic acid (RA) was synthesized by silver oxide oxidation of 9-cisretinaldehyde (Aldrich). Radioactive 9-cis-RA was synthesized from all-trans-[11,12-3H]RA (48.7 Ci/mmol; 1 Ci = 37 GBq; DuPont/NEN) by photochemical isomerization (14) and was purified by HPLC using a C18 Zorbax-ODS column as described (14). The purity of 9-cis- [11,12-3H]
The baculovirus genetic expression system has been used to produce murine retinoic acid receptor (RAR) type Y in Spodopterafrugiperda insect cells and Manduwa sexta insect larvae. A hydroxyapatite binding assay revealed production levels of 300 pmol of unoccupied receptor per mg of protein in insect cells, whereas levels from infected insect larvae were found to average 100 pmol of RARy per mg of protein.The cytosolic preparation from infected insect cells exhibited an equilibrium dissociation constant of 2.1 nM as determined by a retinoic acid saturation analysis plotted by the method of This distinction between RARy and the vitamin D receptor suggests a difference in the molecular requirements by these two receptors for specific binding of their respective DNA response elements.
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