The zebrafish represents a powerful system to rapidly determine gene function during embryogenesis and early development. Although a number of ion channels have been investigated in zebrafish, such as KCNH2 and NCX1h (heart) (62), KCNQ2 (epilepsy) (63), gemini (synapses) (64) and Kir7.1 (pigmentation) (65), there was not a comprehensive study of the role of different ion channels play during zebrafish development. CJ team intends to perform a morpholino screen to rapidly target a variety of ion channels proposed by the different members of the ICST consortium. CJ team has 17 years of experience working with zebrafish. In particular it has an in-depth knowledge of all aspects of zebrafish development, a huge experience of handling morpholinos and have been involved in a large scale ENU mutagenesis screen at the Hubrecht Lab (Netherlands), giving the required expertise to rapidly distinguish interesting phenotypes.
The understanding of ion channel signalling relies on the analysis of electrical and chemical activity from multiple cellular sites. This concept also applies to the study of functional deficits in animal models with the ultimate goal of understanding the mechanisms of human diseases. Here, the aim is to study electrical and chemical signals in multiple subcellular compartments, particularly in neuronal dendrites and spines. MC is focusing on obtaining single-trial measurements with a spatial resolution on the order of 1 micron and a temporal resolution of less than 1 millisecond. To this end, he is developing advanced optical methods to achieve high-resolution membrane potential, Ca2+ and Na+ imaging and photostimulation. He records from several types of cells, both in acute tissue slices and in culture, to explore the role of specific ion channels, the integration of their signalling and the functional consequence of, either pharmacological blockade, or impairment caused by a genetic mutation. An example of a current achievement is the simultaneous channelrhodopsin stimulation and membrane potential imaging (in collaboration with Prof Leslie Loew, University of Connecticut) using novel voltage sensitive dyes excitable at wavelengths that are inert for channelrhodopsin.
Optogenetic tools have been originally designed to target specific neurons in the brain to remotely control their activity by light. During a sabbatical stay in the lab of Ehud Isacoff in Berkeley, GS has designed a strategy to control mammalian ion channel activity with light. He has designed a TREK1 channel that is controlled by light via a tethered photoisomerizable pore-blocker. This photo-switchable channel, called TREKlight, has a single point mutation and behaves like a wild-type channel in visible light but is closed by an UV pulse. Coexpressed with native TREK1 channels, it behaves like a dominant negative. A role of TREK1 currents in the hippocampal GABAB response has been demonstrated by this original approach (submitted for publication). This technique will be extended in vivo by injecting virus (lentivirus and/or AAV2) for maximal transfection efficiency into mouse neuronal structures. This approach offers a powerful strategy for identifying unknown currents and will next be applied for obtaining a pharmacological foothold in others multi-subunit signaling proteins (other K2P channels with FL, Kir channels and ASIC with EL). The next step is to generate a Knock-In (KI) mouse where TREK1 will be replaced by TREKlight (StarTREK mouse). StarTREK will have the same characteristic as a wild-type mouse with the unique advantage to control TREK channels by light with both temporal and spatial precision. This mouse will be used to precisely measure the TREK1 current component in cells of interest and to study the role of TREK1 in different neuronal preparations (synaptic plasticity in the hippocampus, 5HT release form the neurons of dorsal raphe nucleus…). These StarTREK KI mice will be compared to TREK1 KO mice for depression, anxiety, and locomotion behavior (with CH). This will constitute the first example of an instantaneous and reversible gene KO in the mouse, an approach that will be applicable to other channels lacking specific pharmacological tools. New optical tools can also be used to study structure-function of channels, for example to monitor association of the regulatory C terminus of TREK1 to the plasma membrane in real time. This technique has been used to decipher the molecular mechanisms of TREK1 regulation by G-protein coupled receptors and fluoxetin (Prozac) (66) and to develop a high-throughput screening test for drugs that alter TREK1 channel function (patent pending). This technique will be applied to others channels notably Kir1.2 and P2X receptors with FR.