The Meaning of pH in Small Spaces

Kiran working at wet lab bench

Students

2020
College

Faculty

Professor of Pharmacology

Project Summary

A postsynaptic membrane contains receptors that trigger an action potential when the synaptic cleft drops below pH 5. A vesicle of pH 3 releases its contents into the cleft. What factors should be considered when determining whether a postsynaptic action potential will occur?

 

None of my chemistry, biology, or neuroscience classes taught me how to even go about answering this question. On the nanoscale of biologically important environments, the concept of pH becomes ambiguous—the addition of a single proton can alter the pH by four or five units due to the limited amount of water present. My ongoing project has been to investigate the meaning of pH in such small spaces. I use reverse micelles—lipid membranes that encapsulate water and are dissolved in organic solvents—to mimic these nanoenvironments. Employing fluorescence spectroscopy and the fluorophore HPTS, I am able to compare the percentage of HPTS protonated at different pH values in bulk aqueous solution and in reverse micelle solution.

 

My results indicate that at the same pH values, HPTS is less protonated in reverse micelles than in bulk solution. For example, at pH values 3.0 through 5.6, HPTS drops from 100% protonated in bulk solution to partially protonated in reverse micelle solution. Because HPTS cannot be “partially” protonated, HPTS is reporting that two distinct pH environments exist in reverse micelle solutions: reverse micelles that encapsulate a proton and reverse micelles that do not. But what does that mean with regards to our scenario? Given that an action potential requires the simultaneous activation of receptors, the question is not “What is the pH of the synaptic cleft?”, but rather “How many protons are readily available to bind to receptors?” Confined, crowded environments prevent protons from diffusing freely, creating two discrete pH environments, say pH values 3 and 7, depending on whether or not the local environment contains a proton. Regardless of what bulk pH we say the cleft is, it will be a mixture of local environments at pH values 3 and 7. Thus, for an action potential to occur, a number of receptors must bind a proton, thereby recognizing a local pH of 3.

 

As the only permanent undergraduate in the Axelsen lab, I was given almost complete control and independence with this project. I designed the experiments, researching published literature and going through the process of trial and error before defining what buffer, reverse micelles, fluorophore, concentrations, and fluorescence spectrometer settings I would stick with. I developed the mind of a researcher, figuring out what the data was telling me and determining how to change the parameters of the experiment to account for all possible variables. I delved into the true meanings of pH and pKa, trying to understand from my results what the interactions amongst buffer, reverse micelle, fluorophore, and protons were at every pH value. I realized the values of patience and precision, and I developed an understanding of how research into even the simplest of subjects pertains to much broader and more complicated biological processes.