Design and characterization of a constitutively open KcsA

Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL 60637, USA.
FEBS letters (Impact Factor: 3.34). 02/2010; 584(6):1133-8. DOI: 10.1016/j.febslet.2010.02.015
Source: PubMed

ABSTRACT The molecular nature of the structure responsible for proton sensitivity in KcsA has been identified as a charge cluster that surrounds the inner helical bundle gate. Here, we show that this proton sensor can be modified to engineer a constitutively open form of KcsA, amenable to functional, spectroscopic and structural analyses. By combining charge neutralizations for all acidic and basic residues in the cluster at positions 25, 117-122 and 124 (but not E118), a mutant KcsA is generated that displays constitutively open channel activity up to pH 9. The structure of this mutant revealed that full opening appears to be inhibited by lattice forces since the activation gate seems to be only on the early stages of opening.

1 Follower
  • [Show abstract] [Hide abstract]
    ABSTRACT: Heritability is the proportion of phenotypic variation in a population that is attributable to genetic variation among individuals. Many ophthalmic disorders and biometric traits are known to have a genetic basis and consequently much work has been published in the literature estimating the heritability of various ocular parameters. We collated and summarized the findings of heritability studies conducted in the field of ophthalmology. We grouped the various studies broadly by phenotype as follows: refraction, primary open-angle glaucoma, age-related macular degeneration (AMD), cataract, diabetic retinopathy, and others. A total of 82 articles were retrieved from the literature relating to estimation of heritability for an ocular disease or biometric trait; of these, 37 papers were concerned with glaucoma, 28 with refraction, 4 with AMD, 5 with diabetic retinopathy, and 4 with cataract. The highest reported heritability for an ophthalmic trait is 0.99 for the phenotype ≥ 20 small hard drusen, indicating that observed variation in this parameter is largely governed by genetic factors. Over 60% of the studies employed a twin study design and a similar percentage utilized variance components methods and structural equation modeling (SEM) to derive their heritability values. Using modern SEM techniques, heritability estimates derived from twin subjects were generally higher than those from family data. Many of the estimates are in the moderate to high range, but to date the majority of genetic variants accounting for these findings have not been uncovered, hence much work remains to be undertaken to elucidate fully their molecular etiology.
    Survey of Ophthalmology 11/2010; 55(6):561-83. DOI:10.1016/j.survophthal.2010.07.003 · 3.51 Impact Factor
  • Article: Biology.
    Advances in Experimental Medicine and Biology 01/2014; 794:7-40. DOI:10.1007/978-94-007-7429-2_2 · 2.01 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: To examine the function of ligand-gated ion channels in a defined membrane environment, we developed a robust sequential-mixing fluorescence-based stopped-flow assay. Channel activity is determined using a channel-permeable quencher (e.g., thallium, Tl(+)) of a water-soluble fluorophore (8-aminonaphthalene-1,3,6-trisulfonic acid) encapsulated in large unilamellar vesicles in which the channel of interest has been reconstituted, which allows for rapid solution changes. To validate the method, we explored the activation of wild-type KcsA channel, as well as it's noninactivating (E71A) KcsA mutant, by extravesicular protons (H(+)). For both channel types, the day-to-day variability in the reconstitution yield (as judged from the time course of fluorescence quenching) is <10%. The activation curve for E71A KcsA is similar to that obtained previously using single-channel electrophysiology, and the activation curves for wild-type and E71A KcsA are indistinguishable, indicating that channel activation and inactivation are separate processes. We then investigated the regulation of KcsA activation by changes in lipid bilayer composition. Increasing the acyl chain length (from C18:1 to C22:1 in diacylphosphatidylcholine), but not the mole fraction of POPG (>0.25) in the bilayer-forming phospholipid mixture, alters KcsA H(+) gating. The bilayer-thickness-dependent shift in the activation curve is suggestive of a decrease in an apparent H(+) affinity and cooperativity. The control over bilayer environment and time resolution makes this method a powerful assay for exploring ligand activation and inactivation of ion channels, and how channel gating varies with changes in the channels' lipid bilayer environment or other regulatory processes.
    Biophysical Journal 03/2014; 106(5):1070-1078. DOI:10.1016/j.bpj.2014.01.027 · 3.83 Impact Factor


Available from