A Sensitive Membrane-Targeted Biosensor for Monitoring Changes in Intracellular Chloride in Neuronal Processes

Background: Regulation of chloride gradients is a major mechanism by which excitability is regulated in neurons. Disruption of these gradients is implicated in various diseases, including cystic fibrosis, neuropathic pain and epilepsy. Relatively few studies have addressed chloride regulation in neuronal processes because probes capable of detecting changes in small compartments over a physiological range are limited. Methodology/Principal Findings: In this study, a palmitoylation sequence was added to a variant of the yellow fluorescent protein previously described as a sensitive chloride indicator (YFPQS) to target the protein to the plasma membrane (mbYFPQS) of cultured midbrain neurons. The reporter partitions to the cytoplasmic face of the cellular membranes, including the plasma membrane throughout the neurons and fluorescence is stable over 30-40 min of repeated excitation showing less than 10% decrease in mbYFPQS fluorescence compared to baseline. The mbYFPQS has similar chloride sensitivity (k 50 = 41 mM) but has a shifted pKa compared to the unpalmitoylated YFPQS variant (cytYFPQS) that remains in the cytoplasm when expressed in midbrain neurons. Changes in mbYFPQS fluorescence were induced by the GABA A agonist muscimol and were similar in the soma and processes of the midbrain neurons. Amphetamine also increased mbYFPQS fluorescence in a subpopulation of cultured midbrain neurons that was reversed by the selective dopamine transporter (DAT) inhibitor, GBR12909, indicating that mbYFPQS is sensitive enough to detect endogenous DAT activity in midbrain dopamine (DA) neurons. Conclusions/Significance: The mbYFPQS biosensor is a sensitive tool to study modulation of intracellular chloride levels in neuronal processes and is particularly advantageous for simultaneous whole-cell patch clamp and live-cell imaging experiments. © 2012 Watts et al

Fluorescence images of cultured midbrain neurons expressing either mbYFPQS or cytYFPQS.

<p>Single Z-stack images (1 µm) at mid-soma level taken 3 days post-transfection. Note that the fluorescence of the processes of the cytYFPQS-expressing cell is difficult to observe without saturating the fluorescence of the soma.</p

Minimal photobleaching of mbYFPQS occurs with repeated photo-stimulation.

<p>A. A cultured midbrain neuron expressing mbYFPQS was imaged with an exposure of 100 ms every 5 s for 35 min with less than 6% decrease in baseline fluorescence. B. A neuron imaged with the same parameters (100 ms exposure, every 5 s) that was whole-cell patch-clamped with potassium gluconate internal solution (12.6 mM Cl⁻). A decrease in fluorescence was observed with break-in to whole-cell mode (denoted by dotted line) but after reaching a new stable baseline, there was less than 5% decrease in fluorescence over 35 min of imaging. C. Brightfield and epifluorescence image of cell imaged in <i>B</i>. D. Bar graph of % increase in fluorescence of typical internal solutions (CsCl (134 mM Cl⁻); KGluconate (121 mM Gluconate/12.6 mM Cl⁻); KMeSO4 (121 MeSO4/12.6 mM Cl⁻)) compared to fluorescence in high intracellular chloride concentrations (158 mM Cl⁻). Experiments were done in perforated mbYFPQS-expressing neurons after incubation in ionophores used for chloride and pH calibration experiments. The results show that gluconate and methylsulfate ions do not appreciably quench the mbYFPQS fluorescence.</p

Chloride and pH calibration of mbYFPQS expressed in midbrain neurons.

<p>Cultured midbrain neurons expressing mbYFPQS were incubated with the following ionophores for 20–30 min, tributyltin-Cl (1 µM), nigericin (1 µM) and valinomycin (1 µM) prior to superfusion of a high potassium extracellular solution containing different final chloride concentrations. A. Plot of fluorescence versus chloride concentration for the soma and a process in a representative cell. A solution substituting nitrate (NO₃) for chloride was use to quench the mbYFPQS fluorescence at 158 mM NO₃ to verify that measured fluorescence was due to mbYFPQS. Concentrations of chloride are denoted by numbers and arrows. Responses are averages of 4–6 points just prior to next chloride concentration. B. The mbYFPQS fluorescence was plotted versus chloride concentration for the soma (n  =  18) and processes (n  =  9). Data were fit with the equation Y  =  F0/(1 + ([Cl⁻_int]/k₅₀)). There was no significant difference in k₅₀ (F(1, 10)  =  1.710, p > 0.05). R square values for fits were 0.993 and 0.985 for the soma and process, respectively. C. pH calibration of mbYFPQS and cytYFPQS expressed in midbrain neurons. Following incubation of neurons in ionophores as in A & B, neurons were exposed to high potassium extracellular solutions containing either 8 mM or 30 mM Cl⁻ at various pH (n  =  5–8/group). Fluorescence of mbYFPQS was normalized to the fluorescence at pH 8.3 and plotted versus pH. Data were fit with sigmoidal curves to estimate apparent pK_a.</p