Comparative performance of a genetically-encoded voltage indicator and a blue voltage sensitive dye for large scale cortical voltage imaging.

Traditional small molecule voltage sensitive dye indicators have been a powerful tool for monitoring large scale dynamics of neuronal activities but have several limitations including the lack of cell class specific targeting, invasiveness and difficulties in conducting longitudinal studies. Recent advances in the development of genetically-encoded voltage indicators have successfully overcome these limitations. Genetically-encoded voltage indicators (GEVIs) provide sufficient sensitivity to map cortical representations of sensory information and spontaneous network activities across cortical areas and different brain states.

Exploration of genetically encoded voltage indicators based on a chimeric voltage sensing domain.

Deciphering how the brain generates cognitive function from patterns of electrical signals is one of the ultimate challenges in neuroscience. To this end, it would be highly desirable to monitor the activities of very large numbers of neurons while an animal engages in complex behaviors. Optical imaging of electrical activity using genetically encoded voltage indicators (GEVIs) has the potential to meet this challenge.

IKs channels open slowly because KCNE1 accessory subunits slow the movement of S4 voltage sensors in KCNQ1 pore-forming subunits.

Human I(Ks) channels activate slowly with the onset of cardiac action potentials to repolarize the myocardium. I(Ks) channels are composed of KCNQ1 (Q1) pore-forming subunits that carry S4 voltage-sensor segments and KCNE1 (E1) accessory subunits. Together, Q1 and E1 subunits recapitulate the conductive and kinetic properties of I(Ks).

Transfer of Kv3.1 voltage sensor features to the isolated Ci-VSP voltage-sensing domain.

Membrane proteins that respond to changes in transmembrane voltage are critical in regulating the function of living cells. The voltage-sensing domains (VSDs) of voltage-gated ion channels are extensively studied to elucidate voltage-sensing mechanisms, and yet many aspects of their structure-function relationship remain elusive. Here, we transplanted homologous amino acid motifs from the tetrameric voltage-activated potassium channel Kv3.

Optogenetic electrophysiology: a new approach to combine cellular and systems physiology.

Abstract Optogenetics is the latest new subdiscipline in brain sciences that merges optical imaging, protein engineering and genetic dissection of neuronal circuits to optically monitor and control brain activity with high spatial and temporal precision. In this review, the conceptual framework that fueled the development of this methodology is first introduced and the central tools utilized in monitoring and controlling of neuronal circuit elements using light are described. How this innovative approach fosters our understanding of both physiological and pathophysiological properties of brain networks is then discussed.

Oxidative dealkylation DNA repair mediated by the mononuclear non-heme iron AlkB proteins.

DNA can be damaged by various intracellular and environmental alkylating agents to produce alkylation base lesions. These base damages, if not repaired promptly, may cause genetic changes that lead to diseases such as cancer. Recently, it was discovered that some of the alkylation DNA base damage can be directly removed by a family of proteins called the AlkB proteins that utilize a mononuclear non-heme iron(II) and alpha-ketoglutarate as cofactor and cosubstrate.

Direct repair of the exocyclic DNA adduct 1,N6-ethenoadenine by the DNA repair AlkB proteins.

The exocyclic DNA base adduct 1,N6-ethenoadenine (epsilonA) is directly repaired by the AlkB proteins in vitro.

Preparation and characterization of the native iron(II)-containing DNA repair AlkB protein directly from Escherichia coli.

The Escherichia coli AlkB protein was recently found to repair cytotoxic DNA lesions 1-methyladenine and 3-methylcytosine by using a novel iron-catalyzed oxidative demethylation mechanism. This protein belongs to a family of 2-ketoglutarate-Fe(II)-dependent dioxygenase proteins that utilize iron and 2-ketoglutarate to activate dioxygen for oxidation reactions. We report here the overexpression and isolation of the native Fe(II)-AlkB with a bound cofactor, 2-ketoglutarate, directly from E.

Modeling non-heme iron proteins.

Synthetic modeling studies of non-heme iron proteins continue to contribute to our understanding of the mechanism of these proteins. Recently, mononuclear Fe(IV)=O complexes have been prepared and characterized to model the same species that are proposed to be the reactive intermediates in reactions involving mononuclear non-heme iron proteins. Generation of such species for the oxidation of organic substrates has also been demonstrated.

Interaction of human and bacterial AlkB proteins with DNA as probed through chemical cross-linking studies.

The Escherichia coli AlkB protein was recently found to repair cytotoxic DNA lesions 1-methyladenine and 3-methylcytosine by using a novel iron-catalyzed oxidative demethylation mechanism. Three human homologs, ABH1, ABH2 and ABH3, have been identified, and two of them, ABH2 and ABH3, were shown to have similar repair activities to E.coli AlkB.

How do DNA repair proteins locate potential base lesions? a chemical crosslinking method to investigate O6-alkylguanine-DNA alkyltransferases.

O(6)-alkylguanine-DNA alkyltransferases directly reverse the alkylation on the O(6) position of guanine in DNA. This group of proteins has been proposed to repair the damaged base in an extrahelical manner; however, the detailed mechanism is not understood. Here we applied a chemical disulfide crosslinking method to probe the damage-searching mechanism of two O(6)-alkylguanine-DNA alkyltransferases, the Escherichia coli C-Ada and the human AGT.

Probing the structure and function of the Escherichia coli DNA alkylation repair AlkB protein through chemical cross-linking.

Engineering chemical cross-linking groups at the protein/DNA interface provide a powerful method for probing the putative active site and a damage searching mechanism of the Escherichia coli alkylation DNA repair protein AlkB.