PubMed Health⌕ Search

Biomedical subjects

Gail A Robertson

Publications and source records attributed to Gail A Robertson.

8 recordsLinked to original sources
PubMed2005

Specific serine proteases selectively damage KCNH2 (hERG1) potassium channels and I(Kr).

KCNH2 (hERG1) encodes the alpha-subunit proteins for the rapidly activating delayed rectifier K+ current (I(Kr)), a major K+ current for cardiac myocyte repolarization. In isolated myocytes I(Kr) frequently is small in amplitude or absent, yet KCNH2 channels and I(Kr) are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and I(Kr) are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed K+, Na+, and L-type Ca2+ channels, and in ventricular myocytes I(Kr), slowly activating delayed rectifier K+ current (I(Ks)), and inward rectifier K+ current (I(K1)), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current (I(KCNH2)) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished I(Kr); and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of I(KCNH2) and its mature channel protein over several hours. Thus the channel protein for I(KCNH2) and I(Kr) is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low I(Kr) density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells.

Animals↗
PubMed2005

Gating and assembly of heteromeric hERG1a/1b channels underlying I(Kr) in the heart.

Until recently, ion channels generating cardiac IKr were thought to comprise four identical alpha subunits encoded by the ERG1a transcript. Despite studies identifying another transcript, ERG1b, failure to identify the corresponding protein in native tissue led to the conclusion that the ERG1b subunit is not a constituent of cardiac IKr channels. Interestingly, hERG1b subunits coexpressed in heterologous systems preferentially form heteromultimers with hERG1a and modify the deactivation gating properties previously attributed to the hERG1a N-terminus. The two subunits are identical except for their divergent N-termini. Moreover, IKr kinetic properties are more closely mimicked by currents from heteromeric, compared to homomeric, channels. Studies with a new generation of antibodies now show that ERG1b subunits also contribute to IKr channels in vivo, likely in heteromeric assemblies with ERG1a. Bidirectional coimmunoprecipitation of ERG1a and 1b subunits from canine and human ventricle indicates that the subunits associate in native tissue, where they are also found by immunocytochemistry to localize to the same subcellular compartment. These new findings raise questions as to the role of the respective N-termini in deactivation gating and assembly in vivo, as well as the disease mechanisms of mutations causing hERG-linked long QT syndrome, approximately 20% of which reside in the hERG1a N-terminus and have previously been evaluated only in the context of the hERG1a homomers.

Animals↗
PubMed2004

Cardiac IKr channels minimally comprise hERG 1a and 1b subunits.

Previous studies suggest native cardiac IKr channels are composed of alpha subunits encoded solely by the 1a transcript of the ERG1 gene. Using isoform-specific ERG1 antibodies, we have new evidence that subunits encoded by an alternate transcript, ERG1b, are also expressed in rat, canine, and human heart. The ERG1a and -1b subunits associate in vivo where they localize to the T tubules of ventricular myocytes. These data indicate native ventricular IKr channels are heteromers containing two alpha subunit types, ERG1a and -1b. The hERG1b-specific exon thus represents a novel target to screen for mutations causing type 2 long QT syndrome. These findings also suggest phenotypic analyses of existing type 2 long QT syndrome mutations, especially those exclusive to the hERG1a amino terminus, should be carried out in systems expressing both subunits.

Animals↗
PubMed2004

Identification and interrogation of highly informative single nucleotide polymorphism sets defined by bacterial multilocus sequence typing databases.

A unified, bioinformatics-driven, single nucleotide polymorphism (SNP)-based approach to microbial genotyping has been developed. Multilocus sequence typing (MLST) databases consist of known variants of standardized housekeeping genes. Normally, seven fragments are defined; a sequence type (ST) consists of the variants of these fragments that are found in a particular isolate. A computer program that can identify highly informative sets of SNPs in entire MLST databases has been constructed. The SNPs either define a particular user-specified ST or provide a high value for Simpson's index of diversity (D), and may thus be generally applicable to that species. SNP sets that are diagnostic for Neisseria meningitidis ST-11 and ST-42, and high-D SNP sets for N. meningitidis and Staphylococcus aureus, were identified and real-time PCR methods to interrogate these SNPs were demonstrated. High-D SNP sets were also identified in other MLST databases. This widely applicable approach allows rapid genetic fingerprinting of infectious agents.

Algorithms↗
PubMed2003

International Union of Pharmacology. XLI. Compendium of voltage-gated ion channels: potassium channels.

This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels. The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/.

Phylogeny↗
PubMed2002

Interaction with GM130 during HERG ion channel trafficking. Disruption by type 2 congenital long QT syndrome mutations. Human Ether-à-go-go-Related Gene.

Many mutations in the Human Ether-à-go-go-Related Gene (HERG) cause type 2 congenital long QT syndrome (LQT2) by disrupting trafficking of the HERG-encoded potassium channel. Beyond observations that some mutations trap channels in the endoplasmic reticulum, little is known about how trafficking fails. Even less is known about what checkpoints are encountered in normal trafficking. To identify protein partners encountered as HERG channels are transported among subcellular compartments, we screened a human heart library with the C terminus of HERG using yeast two-hybrid technology. Among the proteins isolated was GM130, a Golgi-associated protein involved in vesicular transport. The interaction mapped to two non-contiguous regions of HERG and to a region just upstream of the GRASP-65 interaction domain of GM130. GM130 did not interact with the N or C terminus of either KvLQT1 or Shaker channels. LQT2-causing mutations in the HERG C terminus selectively disrupted interactions with GM130 but not Tara, another HERG-interacting protein. Native GM130 and stably expressed HERG were co-immunoprecipitated from HEK-293 cells using GM130 antibodies. In rat cardiac myocytes and HEK-293 cells, confocal immunocytochemistry showed co-localization of GM130 and HERG to the Golgi apparatus. Overexpression of GM130 suppressed HERG current amplitude in Xenopus oocytes, as if by providing an excess of substrate at the Golgi checkpoint. These findings indicate that GM130 plays a previously undefined role in cargo transport. We propose that the cytoplasmic C terminus of HERG participates in the tethering or possibly targeting of HERG-containing vesicles within the Golgi via its interaction with GM130.

Animals↗
PubMed2002

Mapping the receptor site for ergtoxin, a specific blocker of ERG channels.

We show here that ergtoxin (ErgTx) is a bona fide, specific blocker of the human ether-a-go-go-related gene (HERG) channels. It does not affect the function of either M-eag or M-elk channels. A chimeric construction containing a segment of the P-region of M-eag channel inserted into the HERG channel drastically diminished or completely abolished the inhibitory effect of ErgTx, whereas chimeras of the P-region of HERG channel into M-eag channels recovered the inhibitory effect. From the P-region point mutants of HERG channel assays, only the mutant N598Q shows about 25% decrement of the ErgTx inhibitory effect. ErgTx recognizes the P-region of HERG channels, blocking the channel function with a K(d) in the order of 12 nM.

Amino Acid Sequence↗