Delayed sample arrival at the laboratory does not lead to more false negatives in the Danish population screening for colorectal cancer.

In Denmark, biennial population screening for colorectal cancer was introduced in 2014 for all aged 50-74 years. Five laboratories representative for the regional division of Denmark perform the immunochemical testing of faecal occult blood in the screening samples (iFOBT, OC-Sensor (Eiken Chemical, cut-off 100 µg/L)). In July 2016, a new agreement on the public post-delivery entailed an increased lag time (five days) from the screening participant drops the screening sample into a mail-box until sample arrival at the laboratories. Previous work had reported that a lag time above five days led to more false negative iFOBT tests. We investigated if this was true also under Danish conditions. We performed two stability tests; one with sample storage at 30 °C for 14 days (N = 60), and another with sample storage at room temperature for 13 days (N = 10). We extracted data from our laboratory information system (LABKA) on all iFOBT tests performed in the entire Central Denmark Region (N = 104,328 patients) during the last six months for each calendar year 2014-16. For each year, we computed the distribution of iFOBT tests below and above cut-off. Our stability tests showed no positive samples switching to false negative after storage; however, some negative samples turned false positive, especially at 30 °C. The data showed no change in the distribution of iFOBT tests below and above cut-off after July 2016. We found no evidence that an enhanced lag time increased the number of false negative iFOBT tests in the Danish screening program for colorectal cancer.

Isotope labeling strategies for analysis of an ion channel cytoplasmic domain by NMR spectroscopy.

As large, multimeric, integral membrane proteins, ion channels pose technical challenges to analysis by NMR spectroscopy. Here we present a strategy to overcome some of these technical hurdles, using a representative ion channel modulatory domain, the regulator of K(+) conductance (RCK) domain from a K(+) channel cloned from Thermoplasma volcanium. By introducing a mutation to limit the stoichiometry of the octameric RCK domain "gating ring" complex to its dimeric building block, NMR spectral resolution can be greatly improved. Here we present protocols for efficient production of highly deuterated, uniformly (15)N-labeled protein, as well as protein containing (15)N-labeling to specific amino acid types. These labeling strategies can be applied to improve spectral resolution and facilitate sequential resonance assignments.

Structure and function of multiple Ca2+-binding sites in a K+ channel regulator of K+ conductance (RCK) domain.

Regulator of K(+) conductance (RCK) domains control the activity of a variety of K(+) transporters and channels, including the human large conductance Ca(2+)-activated K(+) channel that is important for blood pressure regulation and control of neuronal firing, and MthK, a prokaryotic Ca(2+)-gated K(+) channel that has yielded structural insight toward mechanisms of RCK domain-controlled channel gating. In MthK, a gating ring of eight RCK domains regulates channel activation by Ca(2+). Here, using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca(2+)-binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca(2+)-binding sites, resulting in a stoichiometry of 24 Ca(2+) ions per channel, is consistent with the steep relation between [Ca(2+)] and MthK channel activity. Comparison of Ca(2+)-bound and unliganded RCK domains suggests a physical mechanism for Ca(2+)-dependent conformational changes that underlie gating in this class of channels.

Allosteric mechanism of Ca2+ activation and H+-inhibited gating of the MthK K+ channel.

MthK is a Ca(2+)-gated K(+) channel whose activity is inhibited by cytoplasmic H(+). To determine possible mechanisms underlying the channel's proton sensitivity and the relation between H(+) inhibition and Ca(2+)-dependent gating, we recorded current through MthK channels incorporated into planar lipid bilayers. Each bilayer recording was obtained at up to six different [Ca(2+)] (ranging from nominally 0 to 30 mM) at a given [H(+)], in which the solutions bathing the cytoplasmic side of the channels were changed via a perfusion system to ensure complete solution exchanges. We observed a steep relation between [Ca(2+)] and open probability (Po), with a mean Hill coefficient (n(H)) of 9.9 +/- 0.9. Neither the maximal Po (0.93 +/- 0.005) nor n(H) changed significantly as a function of [H(+)] over pH ranging from 6.5 to 9.0. In addition, MthK channel activation in the nominal absence of Ca(2+) was not H(+) sensitive over pH ranging from 7.3 to 9.0. However, increasing [H(+)] raised the EC(50) for Ca(2+) activation by approximately 4.7-fold per tenfold increase in [H(+)], displaying a linear relation between log(EC(50)) and log([H(+)]) (i.e., pH) over pH ranging from 6.5 to 9.0. Collectively, these results suggest that H(+) binding does not directly modulate either the channel's closed-open equilibrium or the allosteric coupling between Ca(2+) binding and channel opening. We can account for the Ca(2+) activation and proton sensitivity of MthK gating quantitatively by assuming that Ca(2+) allosterically activates MthK, whereas H(+) opposes activation by destabilizing the binding of Ca(2+).

Bimane fluorescence scanning suggests secondary structure near the S3-S4 linker of BK channels.

Gating of large conductance Ca(2+)-activated K(+) channels (BK or maxi-K channels) is controlled by a Ca(2+)-sensor, formed by the channel cytoplasmic C-terminal domain, and a voltage sensor, formed by its S0-S4 transmembrane helices. Here we analyze structural properties of a portion of the BK channel voltage sensing domain, the S3-S4 linker, using fluorescence lifetime spectroscopy. Single residues in the S3-S4 linker region were substituted with cysteine, and the cysteine-substituted mutants were expressed in CHO cells and covalently labeled with the sulfhydryl-reactive fluorophore monobromo-trimethylammonio-bimane (qBBr). qBBr fluorescence is quenched by tryptophan and, to a lesser extent, tyrosine side chains. We found that qBBr fluorescence in several of the labeled cysteine-substituted channels shows position-specific quenching, as indicated by increase of the brief lifetime component of the qBBr fluorescence decay. Quenching was reduced with the mutation W203F (in the S4 segment), suggesting that Trp-203 acts as a quenching group. Our results suggest a working hypothesis for the secondary structure of the BK channel S3-S4 region, and places residues Leu-204, Gly-205, and Leu-206 within the extracellular end of the S4 helix.

Escherichia coli RNase P RNA: substrate ribose modifications at G+1, but not nucleotide -1/+73 base pairing, affect the transition state for cleavage chemistry.

The temperature dependence of processing of precursor tRNA(Gly) (ptRNA(Gly)) variants carrying a single 2'-OCH(3) or locked nucleic acid (LNA) modification at G+1 by Escherichia coli endoribonuclease P RNA was studied at rate-limiting chemistry. We show, for the first time, that these ribose modifications at nucleotide +1 increase the activation energy and alter the activation parameters for the transition state of hydrolysis at the canonical (c(0)) cleavage site (between nucleotides -1 and +1). The modified substrates, particularly the one with LNA at G+1, caused an increase in the activation enthalpy Delta H(double dagger), which was partly compensated for by a simultaneous increase in the activation entropy DeltaS(double dagger). NMR imino proton spectra of model acceptor stems derived from the same ptRNA variants unveiled that a riboT or U at -1 forms two hydrogen bonds with U+73, thus extending the acceptor stem by 1 bp. The non-canonical base pair is substantially stabilized by LNA substitution at nucleotides -1 or +1. To address if the activation energy increase owing to LNA at G+1 stems from dissociation of the U(-1)-U(+73) base pair as a prerequisite for interaction of U(+73) with U294 in endoribonuclease P RNA, we tested a ptRNA(Gly) variant that is capable of forming an extra C(-1)-G(+73) Watson-Crick base pair. However, compared with a control ptRNA (C at -1, U at +73), no significant change in activation parameters was observed for this ptRNA. Thus, our results argue against the possibility that breaking of an additional base pair at the end of the acceptor stem may present an energetic barrier for reaching the transition state of the chemical step for cleavage at the canonical (c(0)) phosphodiester.

Molecular architecture and divalent cation activation of TvoK, a prokaryotic potassium channel.

RCK (regulator of conductance of potassium) domains form a family of ligand-binding domains found in many prokaryotic K+ channels and transport proteins. Although many RCK domains contain an apparent nucleotide binding motif, some are known instead to bind Ca2+, which can then facilitate channel opening. Here we report on the molecular architecture and ligand activation properties of an RCK-containing potassium channel cloned from the prokaryote Thermoplasma volcanium. This channel, called TvoK, is of an apparent molecular mass and subunit composition that is consistent with the hetero-octameric configuration hypothesized for the related MthK (Methanobacterium thermoautotrophicum potassium) channel, in which four channel-tethered RCK domains coassemble with four soluble (untethered) RCK domains. The expression of soluble TvoK RCK subunits arises from an unconventional UUG start codon within the TvoK gene; silent mutagenesis of this alternative start codon abolishes expression of the soluble form of the TvoK RCK domain. Using single channel recording of purified, reconstituted TvoK, we found that the channel is activated by Ca2+ as well as Mg2+, Mn2+, and Ni2+. This non-selective divalent activation is in contrast with the activation properties of MthK, which is selectively activated by Ca2+. Transplantation of the TvoK RCK domain into MthK generates a channel that can be activated by Mg2+, illustrating that the Mg2+ binding site is likely contained within the RCK domain. We present a working hypothesis for TvoK gating in which the binding of either Ca2+ or Mg2+ can contribute approximately 5 kcal/mol toward stabilization of the open conformation of the channel.

Glutamic acid-rich proteins of rod photoreceptors are natively unfolded.

The outer segment of vertebrate photoreceptors is a specialized compartment that hosts all the signaling components required for visual transduction. Specific to rod photoreceptors is an unusual set of three glutamic acid-rich proteins (GARPs) as follows: two soluble forms, GARP1 and GARP2, and the N-terminal cytoplasmic domain (GARP' part) of the B1 subunit of the cyclic GMP-gated channel. GARPs have been shown to interact with proteins at the rim of the disc membrane. Here we characterized native GARP1 and GARP2 purified from bovine rod photoreceptors. Amino acid sequence analysis of GARPs revealed structural features typical of "natively unfolded" proteins. By using biophysical techniques, including size-exclusion chromatography, dynamic light scattering, NMR spectroscopy, and circular dichroism, we showed that GARPs indeed exhibit a large degree of intrinsic disorder. Analytical ultracentrifugation and chemical cross-linking showed that GARPs exist in a monomer/multimer equilibrium. The results suggested that the function of GARP proteins is linked to their structural disorder. They may provide flexible spacers or linkers tethering the cyclic GMP-gated channel in the plasma membrane to peripherin at the disc rim to produce a stack of rings of these protein complexes along the long axis of the outer segment. GARP proteins could then provide the environment needed for protein interactions in the rim region of discs.

Regulation of the expression of mouse TAP-associated glycoprotein (tapasin) by cytokines.

The expression of antigen presenting MHC class I molecules can be enhanced through cytokines, e.g. upon infection with bacteria or viruses, either directly by enhancing class I gene transcription or by increasing the amounts of accessory proteins of the loading complex. Tapasin plays a significant role in the peptide loading of class I molecules. Here, we describe recognition motifs of cytokine inducible transcription factors in the promoter region of the mouse tapasin gene, most of them clustered within the 140 base pairs upstream of the start codon. Tapasin mRNA was strongly induced in vivo after infection with the facultatively intracellular bacterium Listeria monocytogenes in an IFN-gamma-dependent fashion. Accordingly, both tapasin mRNA and protein were strongly induced in a time and dose dependent manner in embryonic fibroblasts treated with the cytokines IFN-gamma and IFN-beta, and weakly induced after treatment with TNF-alpha. Co-stimulation of tapasin by TNF-alpha and IFN-gamma resulted in a weak synergistic effect. Using fibroblasts either lacking IRF-1 or inhibited in protein synthesis we show that secondary transcription factors are necessary for a maximal stimulation of tapasin expression upon IFN-gamma stimulation. The sequential induction of TAP1, LMP2, and tapasin before the stimulated expression of class I heavy chain is discussed.