Genetically Encoded Indicators of Cellular Calcium Dynamics Based on Troponin C and Green Fluorescent Protein

Abstract

Genetic calcium probes offer tremendous potential in the fields of neuroscience, cell biology, and pharmaceutical screening. Previously, ratiometric and non-ratiometric indicators of cellular calcium dynamics have been described that consist of mutants of the green fluorescent protein (GFP) as fluorophores and calmodulin as calcium-binding moiety in several configurations. However, these calmodulin-based types of probes have a series of deficiencies, such as reduced dynamic ranges, when expressed within transgenic organisms and lack of calcium sensitivity in certain targetings. We developed novel types of calcium probes based on troponin C variants from skeletal and cardiac muscle. These indicators have ratio changes up to 140%, K d s ranging from 470 nM to 29 M, and improved subcellular targeting properties. We targeted the indicators to the plasma membrane of HEK293 cells and primary hippocampal neurons. Upon long lasting depolarization, submembrane calcium levels in hippocampal neurons were found to be in equilibrium with bulk cytosolic calcium levels, suggesting no standing gradient persists from the membrane toward the cytosol. We expect that such novel indicators using specialized calcium sensing proteins will be minimally interacting with the cellular biochemical machinery.Genetically encoded fluorescent indicators used to visualize cellular calcium levels have many advantages over other fluorescent dyes that have to be applied externally. They are generated in situ inside cells after transfection, do not require cofactors, can be specifically targeted to cell organelles and cellular microenvironments, and do not leak out of cells during longer recording sessions. Furthermore, they can be expressed within intact tissues of transgenic organisms and thus solve the problem of loading an indicator dye into tissue, while allowing the labeling of specific subsets of cells of interest (for review, see Ref. 1). Two classes of green fluorescent protein (GFP) 1 -based calcium indicators have been described so far: (i) ratiometric indicators (termed "cameleons") consisting of a pair of fluorescent proteins engineered for fluorescence resonance energy transfer (FRET) which carry the calcium-binding protein calmodulin as well as a calmodulin target peptide sandwiched between the GFPs (2-4); and (ii) various non-ratiometric indicators with calmodulin directly inserted into a single fluorescent protein (5-8). However, calmodulin-based indicators show deficiencies in certain applications, e.g. they display only a reduced dynamic range in transgenic invertebrates compared with in vitro data of the purified indicator proteins and acute transfections (9 -11) and fail to show calcium responses when targeted to certain sites within cells. No successful transgenic expression in mammals has been reported yet. Calmodulin is a ubiquitous signal protein in cell metabolism and thus under stringent regulation involving a plethora of calmodulinbinding proteins (12). It activates numerous kinases and phosphatases, modulates ion channe...

Chemical tags: Applications in live cell fluorescence imaging

“…The particular disadvantage of chemical Ca 2þ -sensors is the difficulty of controlling cellular localization, for example, the targeting to specific organelles. As an alternative to the chemical sensors, the Tsien lab and others developed genetically encoded Ca 2þ -indicators (GECIs) based on fusions of FPs with calcium responsive elements like calmodulin or troponin [75][76][77][78]. These indicators allow for the dissection of the spatial and temporal control of calcium signaling processes in cells because they are genetically encoded and therefore specifically localized.…”

Reporting neural activity with genetically encoded calcium indicators

“…Most GECI recognition elements are based on naturally evolved calcium-binding proteins with large Ca 2+ -dependent conformational changes such as calmodulin (CaM) (Miyawaki et al, 1997;Nakai et al, 2001) or troponin-C (TnC) (Heim and Griesbeck, 2004). Wild-type recognition elements have been engineered to improve binding and response properties Garaschuk et al, 2007).…”

“…Single-FP reporter elements typically comprise a circularly permuted or split FP whose fluorescence properties (emission wavelength or intensity) are modulated by recognition element-dependent changes in chromophore protonation state or strain (Baird et al, 1999;Nagai et al, 2001;Nakai et al, 2001). In 2-FP GECIs, conformational changes in the recognition element modulate FRET transfer efficiency between the FP pair, altering the emission intensity of both donor and acceptor (Miyawaki et al, 1997;Heim and Griesbeck, 2004). The molecular architecture of several representative GECIs is shown in Fig.…”

“…Early attempts to target yellow cameleons to the plasma membrane resulted in non-functional indicators (Heim and Griesbeck, 2004), while generation of transgenic mice expressing yellow cameleons (YC3.0 and YC3.12, Hires et al…”

Visualizing circuits and systems using transgenic reporters of neural activity

“…Because action potential firing induces large Ca ++ transients in neuronal cell bodies, Ca ++ indicators provide excellent cellular resolution. Although there are a large number of protein Ca ++ -sensors, including Camgaroo-1 and 2 [19], Cameleon [20], GCaMP1.3 [21], Flash-and Inverse Pericam [22], and the troponin C-based sensor TN-L15 [23], the majority of these sensors have not been widely employed in vivo because Ca ++ sensitivity is reduced when these proteins are expressed in the mammalian CNS.…”