Inward rectifier potassium channels in the HL‐1 cardiomyocyte‐derived cell line

HL‐1 is a line of immortalized cells of cardiomyocyte origin that are a useful complement to native cardiomyocytes in studies of cardiac gene regulation. Several types of ion channel have been identified in these cells, but not the physiologically important inward rectifier K⁺ channels. Our aim was to identify and characterize inward rectifier K⁺ channels in HL‐1 cells. External Ba²⁺ (100 µM) inhibited 44 ± 0.05% (mean ± s.e.m., n = 11) of inward current in whole‐cell patch‐clamp recordings. The reversal potential of the Ba²⁺‐sensitive current shifted with external [K⁺] as expected for K⁺‐selective channels. The slope conductance of the inward Ba²⁺‐sensitive current increased with external [K⁺]. The apparent Kd for Ba²⁺ was voltage dependent, ranging from 15 µM at ?150 mV to 148 µM at ?75 mV in 120 mM external K⁺. This current was insensitive to 10 µM glybenclamide. A component of whole‐cell current was sensitive to 150 µM 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS), although it did not correspond to the Ba²⁺‐sensitive component. The effect of external 1 mM Cs⁺ was similar to that of Ba²⁺. Polymerase chain reaction using HL‐1 cDNA as template and primers specific for the cardiac inward rectifier K_ir2.1 produced a fragment of the expected size that was confirmed to be K_ir2.1 by DNA sequencing. In conclusion, HL‐1 cells express a current that is characteristic of cardiac inward rectifier K⁺ channels, and express K_ir2.1 mRNA. This cell line may have use as a system for studying inward rectifier gene regulation in a cardiomyocyte phenotype. J. Cell. Physiol. 225: 751–756, 2010. © 2010 Wiley‐Liss, Inc.     

Triosephosphate Isomerase: N and C Chemical Shift Assignments and Conformational Change upon Ligand Binding by Magic-Angle Spinning Solid-State NMR Spectroscopy

Microcrystalline uniformly ¹³C,¹⁵N-enriched yeast triosephosphate isomerase (TIM) is sequentially assigned by high-resolution solid-state NMR (SSNMR). Assignments are based on intraresidue and interresidue correlations, using dipolar polarization transfer methods, and guided by solution NMR assignments of the same protein. We obtained information on most of the active-site residues involved in chemistry, including some that were not reported in a previous solution NMR study, such as the side-chain carbons of His95. Chemical shift differences comparing the microcrystalline environment to the aqueous environment appear to be mainly due to crystal packing interactions. Site-specific perturbations of the enzyme's chemical shifts upon ligand binding are studied by SSNMR for the first time. These changes monitor proteinwide conformational adjustment upon ligand binding, including many of the sites probed by solution NMR and X-ray studies. Changes in Gln119, Ala163, and Gly210 were observed in our SSNMR studies, but were not reported in solution NMR studies (chicken or yeast). These studies identify a number of new sites with particularly clear markers for ligand binding, paving the way for future studies of triosephosphate isomerase dynamics and mechanism.     

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane

β₂-Adrenergic receptors (β₂-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β₂-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β₂-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β₂-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β₂-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β₂-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals.     

Genome sequences of five Salmonella enterica serovar Heidelberg isolates associated with a 2011 multistate outbreak in the United States

Salmonella enterica serovar Heidelberg has caused numerous outbreaks in humans. Here, we report draft genomes of five isolates of serovar Heidelberg associated with the recent (2011) multistate outbreak linked to ground turkey in the United States. Isolates 2011K-1110 and 2011K-1132 were recovered from humans, while isolates 2011K-1138, 2011K-1224, and 2011K-1225 were recovered from ground turkey. Whole-genome sequence analysis of these isolates provides a tool for studying the short-term evolution of these epidemic clones.     

Witnessing Phenotypic and Molecular Evolution in the Fruit Fly

This multi-day exercise is designed for a college genetics and evolution laboratory to demonstrate concepts of inheritance and phenotypic and molecular evolution using a live model organism, Drosophila simulans. Students set up an experimental fruit fly population consisting of ten white-eyed flies and one red-eyed fly. Having red eyes is advantageous compared to having white eyes, allowing students to track the spread of this advantageous trait over several generations. Ultimately, the students perform polymerase chain reaction and gel electrophoresis at two neutral markers, one located in close proximity to the eye color locus and one located at the other end of the chromosome. Students observe that most flies have red eyes, and these red-eyed flies have lost variation at the near marker but maintained variation at the far marker hence observing a ??selective sweep?? and the ??hitchhiking?? of a nearby neutral variant. Students literally observe phenotypic and molecular evolution in their classroom!