SummaryWe describe the development and characterization of a mouse and human epithelial cell monolayer platform of the small and large intestines, with a broad range of potential applications including the discovery and development of minimally systemic drug candidates. Culture conditions for each intestinal segment were optimized by correlating monolayer global gene expression with the corresponding tissue segment. The monolayers polarized, formed tight junctions, and contained a diversity of intestinal epithelial cell lineages. Ion transport phenotypes of monolayers from the proximal and distal colon and small intestine matched the known and unique physiology of these intestinal segments. The cultures secreted serotonin, GLP-1, and FGF19 and upregulated the epithelial sodium channel in response to known biologically active agents, suggesting intact secretory and absorptive functions. A screen of over 2,000 pharmacologically active compounds for inhibition of potassium ion transport in the mouse distal colon cultures led to the identification of a tool compound.

The cardiac-delayed rectifier K ؉ current (IKS) is carried by a complex of KCNQ1 (Q1) subunits, containing the voltage-sensor domains and the pore, and auxiliary KCNE1 (E1) subunits, required for the characteristic IKS voltage dependence and kinetics. To locate the transmembrane helix of E1 (E1-TM) relative to the Q1 TM helices (S1-S6), we mutated, one at a time, the first four residues flanking the extracellular ends of S1-S6 and E1-TM to Cys, coexpressed all combinations of Q1 and E1 Cys-substituted mutants in CHO cells, and determined the extents of spontaneous disulfide-bond formation. Cys-flanking E1-TM readily formed disulfides with Cysflanking S1 and S6, much less so with the S3-S4 linker, and not at all with S2 or S5. These results imply that the extracellular flank of the E1-TM is located between S1 and S6 on different subunits of Q1. The salient functional effects of selected cross-links were as follows. A disulfide from E1 K41C to S1 I145C strongly slowed deactivation, and one from E1 L42C to S6 V324C eliminated deactivation. Given that E1-TM is between S1 and S6 and that K41C and L42C are likely to point approximately oppositely, these two cross-links are likely to favor similar axial rotations of E1-TM. In the opposite orientation, a disulfide from E1 K41C to S6 V324C slightly slowed activation, and one from E1 L42C to S1 I145C slightly speeded deactivation. Thus, the first E1 orientation strongly favors the open state, while the approximately opposite orientation favors the closed state.arrhythmias ͉ cardiac repolarization ͉ electrophysiology ͉ atrial fibrillation ͉ S1T he slow, outwardly rectifying K ϩ current (I KS ) is one of two delayed rectifier K ϩ currents critical for repolarization of the heart, particularly during sympathetic nervous system stimulation (1, 2). The I KS channel is composed of four pore-forming KCNQ1 (Q1) subunits and two auxiliary KCNE1 (E1) subunits (3-5). Several human mutations in Q1 and E1 cause variants of long QT syndrome (6), short QT syndrome (7), or atrial fibrillation (8, 9).Although a tetramer of Q1 subunits alone forms a voltagegated channel, only Q1 and E1 together form a channel with the slow activation and deactivation kinetics and the minimal inactivation characteristics of I KS (10, 11). Furthermore, E1 is necessary for sympathetic modulation of I KS (12). How E1 exerts its effect on Q1 is not yet fully understood.There have been a number of conclusions about Q1-E1 interactions in the I KS channel, not all of which are compatible. There is evidence for (13) and against (14, 15) the contribution of E1 to the pore wall and its accessibility from the pore. There is also evidence that E1 interacts with the pore domain, although not necessarily exposed in the pore (16,17), that the E1 TM helix (E1-TM) interacts directly with Q1 S4 helix (18), that E1 modulates Q1 through its C terminus (19-21), and that E1 interacts with the cytoplasmic Q1 S4-S5 linker (22).More recently, a site of possible Q1-E1 interaction was suggested by the association of mutations in Q1 S...

Achromatopsia is an autosomal recessive disorder characterized by cone photoreceptor dysfunction. We recently identified activating transcription factor 6 (ATF6) as a genetic cause of achromatopsia. ATF6 is a key regulator of the unfolded protein response. In response to endoplasmic reticulum (ER) stress, ATF6 migrates from the ER to Golgi to undergo regulated intramembrane proteolysis to release a cytosolic domain containing a basic leucine zipper (bZIP) transcriptional activator. The cleaved ATF6 fragment migrates to the nucleus to transcriptionally up-regulate protein-folding enzymes and chaperones. ATF6 mutations in patients with achromatopsia include missense, nonsense, splice site, and single-nucleotide deletion or duplication changes found across the entire gene. Here, we comprehensively tested the function of achromatopsia-associated ATF6 mutations and found that they group into three distinct molecular pathomechanisms: class 1 ATF6 mutants show impaired ER-to-Golgi trafficking and diminished regulated intramembrane proteolysis and transcriptional activity; class 2 ATF6 mutants bear the entire ATF6 cytosolic domain with fully intact transcriptional activity and constitutive induction of downstream target genes, even in the absence of ER stress; and class 3 ATF6 mutants have complete loss of transcriptional activity because of absent or defective bZIP domains. Primary fibroblasts from patients with class 1 or class 3 ATF6 mutations show increased cell death in response to ER stress. Our findings reveal that human ATF6 mutations interrupt distinct sequential steps of the ATF6 activation mechanism. We suggest that increased susceptibility to ER stress-induced damage during retinal development underlies the pathology of achromatopsia in patients with ATF6 mutations.cone photoreceptor | achromatopsia | endoplasmic reticulum stress | ATF6 | unfolded protein response A chromatopsia is a heritable blinding disease caused by cone photoreceptor dysfunction that spares the rod system. Using next-generation whole-exome sequencing, we recently discovered autosomal recessive mutations in the activating transcription factor 6 (ATF6) gene in patients with achromatopsia (1). ATF6 mutations span the entire coding region and include missense, nonsense, splice site, and single-nucleotide deletion and duplication changes (1-3). We previously showed that a missense mutation that introduced an arginine-to-cysteine substitution at amino acid residue 324 of the ATF6 protein compromised ATF6 activity in patient fibroblasts obtained from an achromatopsia family (1). However, the functional consequences of the other ATF6 mutations found in patients with achromatopsia remain unknown.In humans, ATF6 is a 670-amino acid glycosylated transmembrane protein found in the endoplasmic reticulum (ER) (4). In response to protein misfolding in the ER or other forms of ER stress, ATF6 migrates from the ER to the Golgi apparatus, where the site 1 protease (S1P) and site 2 protease (S2P) cleave ATF6 in the transmembrane domain to liberate the cyt...

Disruption of the cellular pathway modulating endogenous 24-hour rhythms, referred to as 'the circadian clock', has been recently proven to be associated with cancer risk, development and progression. This pathway operates through a complex network of transcription-translation feedback loops generated by a set of interplaying proteins. The expression of core circadian clock genes is frequently dysregulated in human tumors; however, the specific effects and underlying mechanisms seem to vary depending on the cancer types and are not fully understood. Additionally, specific oncogenes may differentially induce the dysregulation of the circadian clock in tumors. Pharmacological modulation of clock components has been shown to result in specific lethality in certain types of cancer cells, and thus holds great promise as a novel anti-cancer therapeutic approach. Here we present an overview of the rationale and current evidence for targeting the clock in cancer treatment.

Tauopathies are neurodegenerative diseases characterized by tau protein pathology in the nervous system. EIF2AK3 (eukaryotic translation initiation factor 2 alpha kinase 3), also known as PERK (protein kinase R-like endoplasmic reticulum kinase), was identified by genome-wide association study as a genetic risk factor in several tauopathies. PERK is a key regulator of the Unfolded Protein Response (UPR), an intracellular signal transduction mechanism that protects cells from endoplasmic reticulum (ER) stress. PERK variants had previously been identified in Wolcott-Rallison Syndrome, a rare autosomal recessive metabolic disorder, and these variants completely abrogated the function of PERK's kinase domain or prevented PERK expression. In contrast, the PERK tauopathy risk variants were distinct from the Wolcott-Rallison variants and introduced missense alterations throughout the PERK protein. The function of PERK tauopathy variants and their effects on neurodegeneration are unknown. Here, we discovered that tauopathy-associated PERK alleles showed reduced signaling activity and increased PERK protein turnover compared to protective PERK alleles. We found that iPSC-derived neurons carrying PERK risk alleles were highly vulnerable to ER stress-induced injury with increased tau pathology. We found that chemical inhibition of PERK in human iPSC-derived neurons also increased neuronal cell death in response to ER stress. Our results indicate that tauopathy-associated PERK alleles are functional hypomorphs during the UPR. We propose that reduced PERK function leads to neurodegeneration by increasing neuronal vulnerability to ER stress-associated damage. In this view, therapies to enhance PERK signaling would benefit at-risk carriers of hypomorphic alleles.

The IKs potassium channel, critical to control of heart electrical activity, requires assembly of α (KCNQ1) and β (KCNE1) subunits. Inherited mutations in either IKs channel subunit are associated with cardiac arrhythmia syndromes. Two mutations (S140G and V141M) that cause familial atrial fibrillation (AF) are located on adjacent residues in the first membrane-spanning domain of KCNQ1, S1. These mutations impair the deactivation process, causing channels to appear constitutively open. Previous studies suggest that both mutant phenotypes require the presence of KCNE1. Here we found that despite the proximity of these two mutations in the primary protein structure, they display different functional dependence in the presence of KCNE1. In the absence of KCNE1, the S140G mutation, but not V141M, confers a pronounced slowing of channel deactivation and a hyperpolarizing shift in voltage-dependent activation. When coexpressed with KCNE1, both mutants deactivate significantly slower than wild-type KCNQ1/KCNE1 channels. The differential dependence on KCNE1 can be correlated with the physical proximity between these positions and KCNE1 as shown by disulfide cross-linking studies: V141C forms disulfide bonds with cysteine-substituted KCNE1 residues, whereas S140C does not. These results further our understanding of the structural relationship between KCNE1 and KCNQ1 subunits in the IKs channel, and provide mechanisms for understanding the effects on channel deactivation underlying these two atrial fibrillation mutations.

Rationale Kv1.5 (KCNA5) mediates the IKur current that controls atrial action potential duration. Given its atrial-specific expression and alterations in human atrial fibrillation (AF), Kv1.5 has emerged as a promising target for the treatment of AF. A necessary step in the development of novel agents that selectively modulate trafficking pathways is the identification of the cellular machinery controlling Kv1.5 surface density, of which little is yet known. Objective To investigate the role of the unconventional myosin V (MYO5A and MYO5B) motors in determining the cell surface density of Kv1.5. Methods and Results Western Blot analysis showed MYO5A and MYO5B expression in the heart, while disruption of endogenous motors selectively reduced IKur current in adult rat cardiomyocytes. Dominant negative constructs and shRNA silencing demonstrated a role for MYO5A and MYO5B in the surface trafficking of Kv1.5 and Connexin-43 (Cx43), but not hERG (KCNH2). Live-cell imaging of Kv1.5-GFP and retrospective labeling of phalloidin demonstrated motility of Kv1.5 vesicles on actin tracts. MYO5A participated in anterograde trafficking, while MYO5B regulated post-endocytic recycling. Over-expression of mutant motors revealed a selective role for Rab11 in coupling MYO5B to Kv1.5 recycling. Conclusions MYO5A and MYO5B control functionally distinct steps in the surface trafficking of Kv1.5. These isoform-specific trafficking pathways determine Kv1.5-encoded IKur in myocytes to regulate repolarizing current, and consequently, cardiac excitability. Therapeutic strategies that manipulate Kv1.5 selective trafficking pathways may prove useful in the treatment of arrhythmias.

Photoreceptors are specialized sensory neurons essential for light detection in the human eye. Photoreceptor cell dysfunction and death cause vision loss in many eye diseases such as retinitis pigmentosa and achromatopsia. Endoplasmic reticulum (ER) stress and Unfolded Protein Response (UPR) signaling have been implicated in the development and pathology of heritable forms of retinitis pigmentosa and achromatopsia. We review the role of ER stress and UPR in retinitis pigmentosa arising from misfolded rhodopsins (RHO) and in achromatopsia arising from genetic mutations in Activating Transcription Factor 6 (ATF6).