There are a number of ways this can be done. If you mean receptor mediated internalisation, the first ways this was accomplished was to monitor total binding using radioactive ligand on live cells, the authors had noticed that the amount of total binding sites over time went into soluble endosomes instead of insoluble membrane. This can be done as well by looking at how much of the receptor was not dissassociable from repeated washing steps or after the use of extracellular digestive enzymes (or biotinylating the receptor and monitoring how much was not biotinylated before and after agonist administration for example). Otherwise more contemporary fluorescent methods might be easier, however less quantifiable. There are a number of fluorescent ligands that could be used, but I don't recommend using this approach since all live cells under imaging will internalise non specifically anything that is in the medium to some degree. Rather I would recommend looking at fluorescent receptor localisation. ImageJ can be used to look at punctated signals within cells of a certain dimension, while a receptor at the membrane would fluoresce around the plasma membrane, internalised receptors are seen as variable sized endosomes and lysosomes (if receptor goes towards recycling or degradation). Thus quantification of full endocytic agonist can be your comparative agonist readout, albeit it is difficult to determine the amount of molecules going inside your cell. Concerning reuptake (as in neurotransmitter restocking), you could use radioactive analogues to be incubated with the cells in question for a period of time, after a few washes all that is kept by the cells could be your amount uptaken. This is often used to measure neurotransmitter release following agonist stimulation, since these uptaken radioactive analogues will be included in the released vesicles.

My PhD thesis was based on temperature effects on receptor pharmacology, I can thus tell you a few things. Firstly, since binding is a factor of Koff and Kon, temperature affects both of these factors, nevertheless, when all other variables are taken into account, the temperature factor on binding affinity somewhat cancels itself out, meaning overall binding should be around the same. However, the rate at which equilibrium is reach could be drastically changed (normally this is proportional to temperature). Since the time your ligands are binding to the receptor could be crucial to receptor conformational stability (denaturation, protease activity, pH changes, oxydation and so on) longer incubation times are generally correlative to failed experiments. Temperature has an effect on all buffer pH so higher temperatures often mean acidifying effects, that could also affect your binding. As for cellular functions, they are generally slowed down at lower temperatures, this is true for activation and internalisation. Higher temperatures most often makes things go faster, up to the point of non functionality. Since a wide range of cellular mechanism work in concert during internalisation higher temperatures (over 37oC) often lead to reduced internalisation rate from denaturation of critical components (however the critical temperature where this tendency is maximised and then drastically reduced, I do not know). Conformational dynamics are also elevated as temperatures increase, but again as this goes over 37oC the receptor and its binding proteins can attain, non natural conformations and subsequently loose important structural functions. Since these proteins have all evolved to work around 37oC, evidently the maximal response of pharmacological characteristics is somewhat close to that temperature.

There are a number of ways this can be done. If you mean receptor mediated internalisation, the first ways this was accomplished was to monitor total binding using radioactive ligand on live cells, the authors had noticed that the amount of total binding sites over time went into soluble endosomes instead of insoluble membrane. This can be done as well by looking at how much of the receptor was not dissassociable from repeated washing steps or after the use of extracellular digestive enzymes (or biotinylating the receptor and monitoring how much was not biotinylated before and after agonist administration for example). Otherwise more contemporary fluorescent methods might be easier, however less quantifiable. There are a number of fluorescent ligands that could be used, but I don't recommend using this approach since all live cells under imaging will internalise non specifically anything that is in the medium to some degree. Rather I would recommend looking at fluorescent receptor localisation. ImageJ can be used to look at punctated signals within cells of a certain dimension, while a receptor at the membrane would fluoresce around the plasma membrane, internalised receptors are seen as variable sized endosomes and lysosomes (if receptor goes towards recycling or degradation). Thus quantification of full endocytic agonist can be your comparative agonist readout, albeit it is difficult to determine the amount of molecules going inside your cell. Concerning reuptake (as in neurotransmitter restocking), you could use radioactive analogues to be incubated with the cells in question for a period of time, after a few washes all that is kept by the cells could be your amount uptaken. This is often used to measure neurotransmitter release following agonist stimulation, since these uptaken radioactive analogues will be included in the released vesicles.

My PhD thesis was based on temperature effects on receptor pharmacology, I can thus tell you a few things. Firstly, since binding is a factor of Koff and Kon, temperature affects both of these factors, nevertheless, when all other variables are taken into account, the temperature factor on binding affinity somewhat cancels itself out, meaning overall binding should be around the same. However, the rate at which equilibrium is reach could be drastically changed (normally this is proportional to temperature). Since the time your ligands are binding to the receptor could be crucial to receptor conformational stability (denaturation, protease activity, pH changes, oxydation and so on) longer incubation times are generally correlative to failed experiments. Temperature has an effect on all buffer pH so higher temperatures often mean acidifying effects, that could also affect your binding. As for cellular functions, they are generally slowed down at lower temperatures, this is true for activation and internalisation. Higher temperatures most often makes things go faster, up to the point of non functionality. Since a wide range of cellular mechanism work in concert during internalisation higher temperatures (over 37oC) often lead to reduced internalisation rate from denaturation of critical components (however the critical temperature where this tendency is maximised and then drastically reduced, I do not know). Conformational dynamics are also elevated as temperatures increase, but again as this goes over 37oC the receptor and its binding proteins can attain, non natural conformations and subsequently loose important structural functions. Since these proteins have all evolved to work around 37oC, evidently the maximal response of pharmacological characteristics is somewhat close to that temperature.

Thanks Jason for your effort. I have already looked at radiolabeled substrate assays in uptake, I do have biotinylation results too but was looking for any other approach to go for quantifiable temperature effects on receptor ligand transient but fast dynamics.

Right now I am into imaging to at a base level to see if temperature affects the dynamics and if yes then what best cell line for such a phenotype related to brain and synapse. I ,mean there are good lines but for imaging I could use something that shows a fine membrane stain through fluorescence with either Alexa or any dye.

More so, the fact that I might encounter a defect in uptake rate by these receptors with high temperature..a correlation has to be drawn towards which I am working.

In agreement with what has been said above in regard to ImageJ quantification, and this is just a thought without knowing to much detail on what system you are looking at; is there a fluorescent substrate (e.g., neurotransmitter conjugated to a fluorophore) or a fluorescent analogue of the endogenous substrate of your receptor (e.g., aASP+) that you could use with laser scanning confocal microscopy (LSCM) and an inline heater/cooler perfusion system? Here you could measure relative fluorescent intensity over time in several areas of the cell under the microscope after application of your fluorescent substrate with the ability to make several manipulations to temperature. If you look into this approach, I would take into consideration how fast the dye/fluorophore photobleaches and make sure you are using a vital dye (i.e., a dye/fluorophore that will not break down into things that will hurt or kill your cells).

I have a nice working relationship with Neuro2A cells, they are neuroblastomas, can be transfected with reasonable yields and the fluorescent transfected proteins were very well defined around the membrane. They can have somewhat eccentric morphologies as they sporadically grow neurites as well as sometimes adopt amiboid morphologies. The SH-Sy5Y, are also easy to work with, their shapes are more defined and typically have a bipolar neurite development.

Concerning your experiments, I would proceed using a temperature controlled microscope apparatus, and taking lots of measurements (perhaps even making different buffers for each temperature increments). I would predict that your maximum of rate versus amplitude (kinetics and maximal efficacy) would peak around 37, although its possible the rate would continue to increase at higher temperatures to some degree, the amplitude should taper fast enough (but please try to contradict my hypothesis). You should also be aware that membrane dynamics would continue to increase with temperature indefinitely, meaning that exogenous compounds will continue to gain in cell permeability while temperature increases. This again I would suggest using a fluorescently coupled or labelled receptor. Thus this way internalisation will always be agonist dependant (control experiments would define the constitutive internalisation rates at each temperature point).

During my experiments in the past, I was still able to covalently link (by photolabelling) my ligand (peptide) to my receptor (a GPCR) by affinity interactions at around 50oC. However, my maximum yield was only around 20%. Regarding thermostability (Tm), the ligand-receptor as well as protein-protein interactions inside the cell, are crucially important. As temperature elevates, the rate at which they disassociate would have increased (thus the half life of the functional complexes would have decreased) and subsequently, biological activity as well. As I mentioned above, the affinity should remain close to the same (for the natively folded proteins), but after a complex dissassociates at higher temperatures, the native conformations would be less populace and subsequent reassociation would also be compromised (reducing the concentration dependant Kon rate). Thus the overall amount of functional complex will decrease much faster at higher temperatures. So the time in which the cells are incubated at the above temperatures would be a factor to monitor.

In any event good luck on your experiments, I know from experience that there are numerous factors to take into account while tackling these questions.

HI Joshua, thanks for the comment. I am little curious about what you suggest ..is it actually supposed to be done as live imaging? As only then it would make sense over time and temperature. Right now we have confocal where I have been taking images by fixing cells. My approaches till now has been radioactivity based ligand uptake and imaging and surface expression through biotinylation Immunoblotting.

Hi Jason, we do have SH-SY5Y and my work is in C6 rat glioma cells. Although I am looking at a neuronal phenotype, I use this cell line as the protein I work with is expressed here well and widely cited and used one which ahs neuronal stem cell like properties. We work on genetic disorders like epilepsy, my aim always is to correlate in vitro/in vivo experiments with the patient specific phenotype. Its little tricky when I add a temperature parameter to my assays as there are lot of things that change with temp.

My hypothesis says that there is over-activity of the WT over-expressed protein when I go beyond 37degrees and yes it surely drops after certain increment much the way you would speculate that the whole dynamics changes. My observaion also is that the mutants I have do not increase in that rate compared to WT which shows there inefficiency over the temperature stress and hence a correlation may be to the phenotype.

Now I have to embark on temperature controlled microscopy which right now we donot use and electrophysiology where temperature can be surely manipulated.

What I am doing now is exposing the cells to temperature for 5 mins and then start processing the cells for immunocytochemistry. I donot know if that is the way to go about in this phenotype which is temperature dependent but yes if I am accessing live cell, it would be a different case and more relevant. I give a pulse of my ligand and give temp. pulse for that time and try capturing membrane expression differences in WT and mutants.

Neurotransmitter uptake cannnot be evaluated efectively through receptor function in mammalian cell lines; these are different processes in neurotransmission. The most direct way to measure neurotransmitter uptake is to make "synaptosomes" and monitor uptake of radioactive transmitter. Alternatively, if you were working with brain, slices could be made and voltammetry can be used to monitor uptake. A way to monitor neurotransmitter uptake indirectly is to conduct binding studies using radioligands.

Hi, Dr. William, well the cell lines are not the real systems I agree but when you do not ave access to brain slices as we deal with patient samples and hence the idea is not to grill more into biology of how the system works but more importantly how the normal individual is different from a patient in terms of single gene mutation, cell lines come handy for the first line of work. If I am abl to work with synaptosomes then I will be very glad but right now I have done radioligand binding in glioma cell lines which are established cell lines for the protein expression and they have neuronal stem like properties hence have the required signaling machinery.

May I ask, how easy or difficult is synaptosome prep? My life becomes even tricky as I have a temperature angle to all experiments. The aim is to keep the wild type in place and bring in mutants which are patient specific into the picture.

Dear Kalpita Karan,

My suggestion was to conduct live cell imaging. For example, I use the fluorescent molecule 4-(4-(dimethylamino)styrl)-N-methyl-pyridinium (ASP+) in live rat brain tissue sections to evaluate the transport kinetics of monoamine transporters. I specifically look at uptake of ASP+ in the ciliated ependymal cells lining the cerebral ventricles using relative fluorescent intensity increase over time as my dependent measure.

I have a perfusion chamber and an inline heater on the confocal I am using. I anchor my tissue slice in the perfusion chamber using a small harp, treat separate tissue sections with my drugs or controls in artificial cerebralspinal fluid (aCSF), and then slowly perfuse ASP+ in aCSF into the perfusion chamber while imaging across XYZT. That is, I take one 800x600 (X & Y) image with a 40x water immersion objective every 5 µm through 20 µm if cells (i.e., 5 images total per stack; Z) for ~30 min (T). Using ImageJ, I process the 16 bit hyperstack .tiffs by Z projecting across each of the 5 images in a stack (i.e., one mean gray value for ~1-2 layers of ependymal cells in one 20 µm stack every 11.8 sec) and then look for changes in relative fluorescent units (RFUs) over time in these ependymal cells as they take up the ASP+.

Although I use live tissue sections, many papers use similar methods in vitro (i.e., cell culture; see link to an example paper below). I am not aware of the top of my head if there is a way to evaluate neurotransmitter binding using similar methods, but I see you being able to measure neurotransmitter internalization and receptor dynamics using a similar method.

Keep in mind that RFUs are “relative”. I would not assume a linear relation between amount of uptake of a fluorescent substrate and fluorescent intensity (e.g., due to photo bleaching, autofluorescence, general artifacting…), where you can assume more of a linear relation with radioactivity counts.

I hope this information is of some use to you.

Best,

Josh

http://www-ncbi-nlm-nih-gov.ezproxy1.lib.asu.edu/pubmed/15757904

J Biol Chem. 2005 May 13;280(19):19177-84. Epub 2005 Mar 9.

Substrate binding stoichiometry and kinetics of the norepinephrine transporter.

Schwartz JW, Novarino G, Piston DW, DeFelice LJ.

Thanks Joshua for the extensive input.