Abstract
The status of lactate has evolved from being considered a waste product of cellular metabolism to a useful metabolic substrate and, more recently, to a signaling molecule. The fluctuations of lactate levels within biological tissues, in particular in the interstitial space, are crucial to assess with high spatial and temporal resolution, and this is best achieved using cellular imaging approaches. In this study, we evaluated the suitability of the lactate receptor, hydroxycarboxylic acid receptor 1 (HCAR1, formerly named GPR81), as a basis for the development of a genetically encoded fluorescent lactate biosensor. We used a biosensor strategy that was successfully applied to molecules such as dopamine, serotonin, and norepinephrine, based on their respective G-protein-coupled receptors. In this study, a set of intensiometric sensors was constructed and expressed in living cells. They showed selective expression at the plasma membrane and responded to physiological concentrations of lactate. However, these sensors lost the original ability of HCAR1 to selectively respond to lactate versus other related small carboxylic acid molecules. Therefore, while representing a promising building block for a lactate biosensor, HCAR1 was found to be sensitive to perturbations of its structure, affecting its ability to distinguish between related carboxylic molecules.
Highlights
Since its discovery at the end of the 18th century and for almost two centuries, lactate has been viewed as a waste product of the metabolism generated during hypoxia, and having several adverse effects, such as muscle soreness and fatigue
We investigated whether the strategy used previously for several neurotransmitters based on their respective specific G-protein-coupled receptors (GPCRs) [17] could be deployed to develop a lactate-sensitive fluorescence biosensor, and constructed an intensiometric fluorescent lactate-sensitive biosensor based on the hydroxycarboxylic acid receptor 1 (HCAR1) lactate receptor coupled to circularly permuted GFP (cpGFP)
Rapid fluorescence changes were observed with kinetics that are compatible with the expected kinetics of physiological lactate variations found in tissues, for instance in response to neuronal activity or to intense physical exercise
Summary
Since its discovery at the end of the 18th century and for almost two centuries, lactate has been viewed as a waste product of the metabolism generated during hypoxia, and having several adverse effects, such as muscle soreness and fatigue. Several studies have indicated the valuable contribution of lactate as a metabolic substrate in tissues, including muscle [2], as a neuroprotective agent, and more recently as a signaling molecule, for reviews, see, e.g., [3]. In the central nervous system, lactate was proposed to play an important role as energy substrate for neurons [4]. Extracellular brain lactate levels are estimated to be in the low millimolar range at resting state [5,6] and to undergo a two-fold increase during synaptic activity [7]. Plasma lactate, which can cross the blood–brain barrier, can rise to 10–20 mM [8]
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