KCNE Peptides Differently Affect Voltage Sensor Equilibrium and Equilibration Rates in KCNQ1 K+ Channels

KCNQ1 voltage-gated K+ channels assemble with the family of KCNE type I transmembrane peptides to afford membrane-embedded complexes with diverse channel gating properties. KCNQ1/KCNE1 complexes generate the very slowly activating cardiac IKs current, whereas assembly with KCNE3 produces a constitutively conducting complex involved in K+ recycling in epithelia. To determine whether these two KCNE peptides influence voltage sensing in KCNQ1 channels, we monitored the position of the S4 voltage sensor in KCNQ1/KCNE complexes using cysteine accessibility experiments. A panel of KCNQ1 S4 cysteine mutants was expressed in Xenopus oocytes, treated with the membrane-impermeant cysteine-specific reagent 2-(trimethylammonium) ethyl methanethiosulfonate (MTSET), and the voltage-dependent accessibility of each mutant was determined. Of these S4 cysteine mutants, three (R228C, G229C, I230C) were modified by MTSET only when KCNQ1 was depolarized. We then employed these state-dependent residues to determine how assembly with KCNE1 and KCNE3 affects KCNQ1 voltage sensor equilibrium and equilibration rates. In the presence of KCNE1, MTSET modification rates for the majority of the cysteine mutants were ∼10-fold slower, as was recently reported to indicate that the kinetics of the KCNQ1 voltage sensor are slowed by KCNE1 (Nakajo, K., and Y. Kubo. 2007 J. Gen. Physiol. 130:269–281). Since MTS modification rates reflect an amalgam of reagent accessibility, chemical reactivity, and protein conformational changes, we varied the depolarization pulse duration to determine whether KCNE1 slows the equilibration rate of the voltage sensors. Using the state-dependent cysteine mutants, we determined that MTSET modification rates were essentially independent of depolarization pulse duration. These results demonstrate that upon depolarization the voltage sensors reach equilibrium quickly in the presence of KCNE1 and the slow gating of the channel complex is not due to slowly moving voltage sensors. In contrast, all cysteine substitutions in the S4 of KCNQ1/KCNE3 complexes were freely accessible to MTSET independent of voltage, which is consistent with KCNE3 shifting the voltage sensor equilibrium to favor the active state at hyperpolarizing potentials. In total, these results suggest that KCNE peptides differently modulate the voltage sensor in KCNQ1 K+ channels

Secondary Structure of a KCNE Cytoplasmic Domain

Type I transmembrane KCNE peptides contain a conserved C-terminal cytoplasmic domain that abuts the transmembrane segment. In KCNE1, this region is required for modulation of KCNQ1 K+ channels to afford the slowly activating cardiac IKs current. We utilized alanine/leucine scanning to determine whether this region possesses any secondary structure and to identify the KCNE1 residues that face the KCNQ1 channel complex. Helical periodicity analysis of the mutation-induced perturbations in voltage activation and deactivation kinetics of KCNQ1-KCNE1 complexes defined that the KCNE1 C terminus is α-helical when split in half at a conserved proline residue. This helical rendering assigns all known long QT mutations in the KCNE1 C-terminal domain as protein facing. The identification of a secondary structure within the KCNE1 C-terminal domain provides a structural scaffold to map protein–protein interactions with the pore-forming KCNQ1 subunit as well as the cytoplasmic regulatory proteins anchored to KCNQ1–KCNE complexes

Xenopus laevis oocytes infected with multi-drug-resistant bacteria: implications for electrical recordings

The Xenopus laevis oocyte has been the workhorse for the investigation of ion transport proteins. These large cells have spawned a multitude of novel techniques that are unfathomable in mammalian cells, yet the fickleness of the oocyte has driven many researchers to use other membrane protein expression systems. Here, we show that some colonies of Xenopus laevis are infected with three multi-drug-resistant bacteria: Pseudomonas fluorescens, Pseudomonas putida, and Stenotrophomonas maltophilia. Oocytes extracted from infected frogs quickly (3-4 d) develop multiple black foci on the animal pole, similar to microinjection scars, which render the extracted eggs useless for electrical recordings. Although multi-drug resistant, the bacteria were susceptible to amikacin and ciprofloxacin in growth assays. Supplementing the oocyte storage media with these two antibiotics prevented the appearance of the black foci and afforded oocytes suitable for whole-cell recordings. Given that P. fluorescens associated with X. laevis has become rapidly drug resistant, it is imperative that researchers store the extracted oocytes in the antibiotic cocktail and not treat the animals harboring the multi-drug-resistant bacteria

Beyond forest cover: Land use and biodiversity in rubber trail forests of the Chico Mendes Extractive Reserve

Among the strategies to promote sustainable tropical forest development around the world, the Federal Extractive Reserve System of Brazil is widely cited as an exemplary model. It is designed to protect rubber tapper communities, their forests, and their livelihoods while preventing deforestation and conserving biodiversity. In response to changing markets and policies, rubber tappers in the Chico Mendes Extractive Reserve have recently diversified production to include market agriculture and cattle production, precipitating deforestation in the reserve, with the implication of increased ecological degradation compared to the extraction of nontimber forest products (NTFPs). Our remote sensing and forest inventory analyses yield different insights about the environmental consequences of distinct land-use mixes in two extractive communities, one of which emphasizes cattle and the other, NTFPs. Remote sensing results show a predictably greater impact on forest cover in the cattle-oriented community. This preliminary study is based on nested household- and community-level forest inventory and biodiversity analyses in two communities. Surprisingly, we found higher tree biodiversity in the rubber trail forests of the cattle-oriented community, and significantly lower tree species richness, tree density, total basal area, and number of trees of commercial size in the same land-use unit in the NTFP-focused community. Land-use surveys indicate lower levels of game consumption and hunting in the cattle-oriented community, and strong support for the development of sustainable timber extraction in both communities. The distinct type and degree of forest impact in the two communities exposes the problem of single-impact assessment as the sole means of performance and categorical land-use prohibitions as an effective mode of regulation in conservation areas.Peer reviewedGeograph

Social likeability, conformity, and body talk: Does fat talk have a normative rival in female body image conversations?

Fat talk, dialogues among women involving negative body-focused discussions, was studied as a function of conformity and social likeability through the use of four vignettes depicting young women in conversation. Using a 2 (body presentation style of the group: negative or positive) × 2 (body presentation style of the target, Jenny: negative or positive) factorial design, 215 college women (92.1% non-Hispanic Caucasian) read one of four vignettes in a classroom setting and made ratings on a social likeability scale. Participants’ personal ratings of Jenny's likeability were higher when she spoke positively about her body, whereas they expected the other group members in the vignette to like Jenny more when she conformed to the group's body presentation style. This study is the first to support two competing norms for women's body image—the existing norm to fat talk versus a newly documented norm that some women like others who express body acceptance