Single-molecule measurements give insight into how pacemaker ion channels interact with cAMP

By Meranda Masse
Department Communications & Graduate Student (Cavagnero)

Randall Goldsmith
Prof. Randall Goldsmith

If you’ve ever been on an amusement park ride, been scared suddenly, or maybe even had to address a large crowd when you were nervous; you’ve likely felt your heart start to thump faster even though you weren’t exerting a lot of energy. What happened was a certain chemical compound called cyclic adenosine monophosphate (cAMP) activated pacemaker ion channels, which caused an increase in electrical activity that led to your heart beating faster.

Ion channels are found in many different cells throughout the body and can control many different types of bodily functions, such as muscle contractions. Some examples of where these channels can be found are within the nervous system, and within the heart. Within the heart and brain specifically, there are pacemaker ion channels. Pacemaker ion channels are responsible for delivering electrical currents to these areas. In this way they are similar to pacemaker devices, which deliver electrical pulses, but rely on a biochemical pathway rather than a physical one.

For many years, researchers have tried to study how these channels are activated by the chemical compound cAMP. Unfortunately, their efforts have yielded opposing results, with some suggesting that the binding is cooperative (meaning that when one cAMP binds it affects the binding of the second cAMP), and others suggesting that binding is noncooperative (binding of one cAMP is not affected by the binding of another cAMP). The discrepancies of these results are likely due to the nature of bulk measurements and their inability to resolve complex mixtures with multiple interconverting species. So, to answer this question, single-molecule measurements are a must.

A key difference between single-molecule and bulk measurements is the type of information they can yield. In the case of single-molecule measurements, unsurprisingly, individual molecules are observed. This is different from bulk measurements because in bulk, there will be lots of molecules that may be doing entirely different things, which all become averaged together to obtain a final measurement. Another key difference is that single molecule measurements require very small concentrations, whereas bulk measurements can be performed at higher concentrations.

To explore how pacemak­er ion channels are activated by cAMP, a team consisting of lead graduate student David White, Prof. Randall Goldsmith, and Prof. Baron Chanda, de­cided to team up and look at pacemaker ion channels and how they interact with cAMP at the single-molecule level. Their work was recently published in Nature (Vol 595, pp 606–610 (2021)).

In general, single-molecule measurements require that anything fluorescently labeled be at a very low concentration. In the body, cAMP is in relatively high concentrations when compared to the pacemaker ion channels that it interacts with. At these concentrations, it is very difficult to perform single-molecule measurements. Thankfully, the team knew of a technology that could alleviate this issue called Zero Mode Waveguides.

Goldsmith described Zero Mode Waveguides as a way to specifically observe an individual in a crowd, while still seeing how the crowd can affect them and their actions. Using this method, the group could study how physiologically relevant concentrations of cAMP interact with pacemaker ion channels, allowing for much more accurate studies of their interactions with one another.

Specifically, Zero Mode Waveguides are tiny holes (often around 150 nm) that focus light to an exceedingly small volume, far beyond the theoretical limit attainable by using optical lenses.

By limiting the zone of excitation, this method is able to monitor single binding events even at a much higher concentration because the freely diffusing labels in the solution do not contribute to background noise.

Thanks to the work of students from both labs, the two groups were able to use this method to observe how cAMP binding occurs at the single-molecule level. Goldsmith commented on how these measurements really give us insight into the dynamics of the interactions taking place. Overall the groups found that the more cAMP was added, the more the channels opened up.

“The cAMP activation of these channels seems to be noncooperative, which means it’s a linear response. As more and more cAMP binds, these channels tend to open more and more,” commented Chanda.

First author on the paper, Dr. David White, mentioned that while there are some limitations to the study, the pinnacle of their achievement really was being able to observe binding and conformational changes of cAMP to functional pacemaker ion channels. Their future work will be looking at how these conformational changes may be different with added complexity such as the addition of potential to mimic membranes, or other cellular components.

These researchers were able to answer very fundamental questions about how pacemaker ion channels operate. They were also able to showcase the power and beauty of Zero Mode Waveguides as a technique for studying single molecules at physiologically relevant concentrations. Future single-molecule work will likely utilize this technique due to its ability to overcome concentration limitations.

This work is published in Nature, titled: cAMP binding to closed pacemaker ion channels is non-cooperative. (Nature volume 595, pages606–610 (2021))