Henry Colecraft

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Interviews

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Engineering and reverse engineering ion channels with Henry Colecraft

Henry M. Colecraft, PhD is the John C. Dalton Professor and Associate Vice Chair in the Department of Physiology and Cellular Biophysics, and Professor in the Department of Molecular Pharmacology & Therapeutics at Columbia University Irving Medical Center. Originally from Ghana, West Africa, he got his first degree at King’s College London, UK. After a gap year in Ghana, where he taught in a secondary school, he went to the USA to start his Ph.D. project (that project was about signal transduction in cardiac muscle cells and muscarinic receptors) with Shey-Shing Sheu at the University of Rochester, New York.

“Shey-Shing was a great mentor – he really allowed me to explore and basically come up with my own project and struggle along and bring it along. That really helped me out in terms of gaining the confidence that I could come up with a project and see it through. And I think it’s a bit of a slower progression in terms of getting publications and things like that, but it certainly helps mature you as a scientist, even at that early stage,” says Henry Colecraft.

Henry started studying ion channels when he embarked on his postdoc with David Yue at Johns Hopkins. And that’s where (as he said) he had his love affair with ion channels. Later on, he was hired onto the faculty at John Hopkins as an assistant professor, and then recruited to Columbia where he’s working till now as a full professor. He also leads the lab studying ion channel structure-function relationship and developing novel genetically encoded ion channel modulators.


I had the pleasure to speak with Henry Colecraft about his career, ion channel engineering, genetically encoded calcium channel inhibitors, nanobodies, targeted (de)ubiquitination, CACNA1A channelopathies and more. Henry is an amazing teacher and storyteller, so talking to him was an absolute delight. I’ve learned so many things from Henry Colecraft and I’m happy to share this interview with you. Enjoy.


So, what was that amazing postdoctoral project that made you fall in love with ion channels at John Hopkins, Henry?

Well, the project involved voltage-gated calcium channels, particularly neuronal type of calcium channels (N- and P/Q-type). Those channels are regulated by a number of intracellular molecules and other proteins. And I was interested in understanding how those channels are inhibited by heterotrimeric G proteins, particularly by the Gbeta/gamma subunit. This mechanism is really important for how opiates, for example, have their analgesic effects, because they inhibit these presynaptic calcium channels, and, in a way, that prevents transmission. So, I learned whole-cell patch clamp, as well as single channel analysis from that study. And also, that project has really helped me realize that calcium channels are something that I would enjoy continuing to explore. 

And so, you decided to focus on calcium channels in your own lab.

Yeah, when I started my lab, I started out in a very focused way on what I knew best, which was calcium channels. And I was really interested in the fundamental understanding of structure-function mechanisms and trying to tease out how these channels are regulated by the auxiliary proteins, the beta subunits. And we made certain progress on that aspect of it, really understanding how the beta subunit interacts with the channel and how it regulated its traffic onto the cell surface.

But now, we expanded our research to other areas, including ion channel engineering, the development of genetically encoded ion channel modulators, and the study of how mutations in ion channels cause molecular and cellular dysfunctions that lead to disease (channelopathies).

I can’t wait to learn more about each of these topics you are working on now. But first, could you tell me more about the beta? Why did you make it the focus of your work?

The short answer is that this particular class of ion channels that we’re studying – they just don’t form functional channels without the beta.

As you know, every channel is born inside the cell. And they have to get to the cell surface in order for them to be functional. And when they don’t get to the cell surface, bad things happen, you get all kinds of diseases that erupt from that. And so, understanding the mechanism by which that happens, and how it goes wrong in disease is a very important aspect of what we normally think about.

Every channel is born inside the cell. And many of them have to get to the cell surface in order to be functional. And when they don’t get to the cell surface, bad things happen.

HENRY COLECRAFT

So, for the voltage-gated calcium channels, it was found that the channel is made up of the main subunit, which actually forms a protein at the surface of the cell. That protein can close and open and allow the calcium ions to flow into the cell, because the calcium can’t pass through the cell membrane, which is sort of fatty and hydrophobic. But in order for those channels to get to the surface, they need this accessory beta subunit, which is a cytosolic subunit. And we were interested in understanding how that beta subunit helps that channel to get to the surface. And if you think about a neuron or a heart cell, for example, it turns out that the calcium channel has to go to various specialized regions within those cells. And we’re also interested in understanding the signals that direct these channels to these particular localizations.

Is it the beta subunit that determines where the calcium channel will go?

It’s one of the determinants but the beta subunit is really important. Without the beta, the channel is typically trapped inside the cell, and we don’t really know what happens inside the cell with those channels. Those are things that we are still studying. We still need to develop tools that allow us to be able to dissect out the signals.

So, given the fact that the beta is so important for calcium channels, do you consider it a therapeutic target?

Yes. One of the aspects of the voltage-gated calcium channels we’ve been interested in is the fact that they are very important therapeutic targets. Calcium channels are targeted for the treatment of hypertension or cardiac arrhythmias, and they’re also very important targets for the treatment of things like pain, stroke, and potentially Parkinson’s disease. And there is a bunch of calcium channel blockers on the market today. But one of the issues with small molecule blockers of the calcium channels, is that once you take a small molecule, it goes everywhere inside the body. And since these channels are widely distributed, there’s a high propensity for off-target effects.

FASEB-Conference-2019
FASEB Conference, Portugal, 2019

Therefore, one program that we’ve been developing is genetically encoded calcium channel inhibitors or blockers. And we think those would be advantageous, because they’re genetically encoded, so you can express them in a very restricted manner in the target population of cells or tissue that you’re interested in. As such, you can get a greater specificity than you can get with a small molecule.

And so, while we were thinking about that, a few years ago, there was a paper published from the Seino Lab in Japan, where they identified a small G protein, which bound the calcium channel beta subunit (see here). And they fished it out using a yeast two-hybrid approach. And when they coexpressed that protein with the channel, the channel went away, it was wiped out. So, it’s a very strong calcium channel inhibitor. And we’ve reproduced that work in our own lab. And what we’ve done since then, is really trying to understand how those proteins were inhibiting the calcium channel by binding the beta subunit and then developing tools or methods to mimic that with our own approach. And that has been a very successful approach because we’ve developed intracellular-acting antibodies, or nanobodies, which target the calcium channel beta subunit. We published this in eLife back in 2019 (see here). We generated a nanobody to a beta subunit and hooked it up to a ubiquitin ligase. So, this ubiquitin ligase is what adds ubiquitin to the target protein complex. And ubiquitin is a signal that helps to remove channels from the membrane and also potentially leads to their degradation. So, what we’ve found was that we can use this approach to remove channels from the membrane. And that creates a powerful beta-targeted inhibitor of the calcium channels.

So, that’s one approach that we’ve taken, and we think that can become a very powerful approach because these beta subunits have different isoforms. And it’s been impossible to really inhibit a channel based on the isoform of the beta. But if you’re able to do that in a beta isoform-specific manner, then you have a really powerful and very targeted calcium channel inhibitor, which you cannot achieve with a small molecule.

And so, this is what we’ve done in our recent study (Nat Commun 2022). We identified a nanobody that selectively binds beta1 subunit and partially inhibits beta1-associated calcium channels but shows no effect on beta2/beta3/beta4-associated calcium channels. Fusing this nanobody to a ubiquitin ligase produced a construct that we called Chisel-1 (calcium channel inhibitor via selective targeting of beta1) – a tool that potently and selectively inhibits beta1-associated calcium channel by promoting targeted ubiquitination of the channel, decreasing channel surface density, and reducing single-channel open probability.

So, that’s one of the aspects of how we’ve been thinking about these genetically encoded calcium channel inhibitors.

Well, the advantages of genetically encoded ion channel inhibitors over small molecules are quite evident. So, these intracellular-acting antibodies, or nanobodies, are they any different from “classical” antibodies?

Yes, they are smaller, and they can be expressed within the cell. We express them using viruses. So, we deliver the genetic material, and then the cell itself makes a protein. The problem with normal heavy chain light chain antibodies is that they aren’t going to work within the cell because the inside of the cell it’s a reducing environment. And those things are held together by disulfide bonds, so they don’t work. And they’re also too large. The nanobodies, on the other hand, are on the order of 12 to 15 kilodaltons, and they are well-behaved inside the cell and recognize the antigens inside the cell.

OK. By looking at your publication profile it becomes clear that ion channel engineering and the development of genetically encoded ion channel modulators is an important direction in your lab. It’s interesting to see how you first develop new techniques and then apply them to your scientific problems.

Well, I think one of the reasons I got into science really was trying to figure out how things work, and I have always loved the mechanistic aspect of it. But during my evolution as a scientist, I figured out that sometimes the tools to answer the questions you wanted to answer, were not available. And so, I stumbled into trying to develop novel tools to allow us to be able to ask us questions in a way that we hadn’t been able to do before.

We, biophysicists, are very good at measuring the activity of the channels once they arrive on the cell surface… But the reality is that most of the lifetime of the channel is spent inside the cell. And for us, it’s like looking at a hidden life of an ion channel.

HENRY COLECRAFT

For example, if you think about it as a biophysicist, we are very good at measuring the activity of the channels once they arrive on the cell surface. Therefore, we have a very particular view of those ion channels at the cell surface. But the reality is that most of the lifetime of the channel is spent inside the cell. And for us, it’s like looking at a hidden life of an ion channel. And we don’t have really good tools to see that, we don’t have good tools to manipulate that hidden aspect. And so, that’s an important part of what we are trying to develop right now – the tools to allow us to truly spy on ion channels throughout their lifecycle and not just when they are at the cell surface.

The hidden life of an ion channel… spying on ion channels… – you really know how to drive interest to your research. So, tell me about some other cool techniques that you’ve developed in your lab. For example, I’d love to know more about how you came up with the ChIMP method.  

Okay. So, the ChIMP also came out of the Seino Lab work with the small G proteins that inhibit the calcium channels. I mentioned it earlier. So, we were interested in understanding how this worked and then trying to mimic that with different approaches. And you can argue that those small G proteins themselves are genetically encoded calcium channel inhibitors or blockers. So, why can’t you just use those for your applications? And the answer is that they have several drawbacks. First, they are totally non-selective and inhibit different calcium voltage-gated channels. Second, they have other things that they do inside the cell beyond inhibiting calcium channels: they regulate the cytoskeleton and bind the proteins. So, they would be a very dirty drug if you were trying to use them that way. That was one of the rationales for trying to really understand how they do what they do and then trying to mimic that.  

So, we studied the mechanisms for how these small G proteins inhibit the channel, and what we’ve found was that they inhibit the channel in three different ways. The first way was that they bind the beta subunit of the channel and somehow get the channel to be removed from the cell surface. And that’s what we mimicked by adding the ubiquitin ligase to the beta subunit to get channel removal from the surface.

But on top of that, we also found a second mechanism that was interesting as well, and that was the inhibition of channels that still stayed at the cell surface. We know that voltage-gated calcium channels have voltage sensors that move when you depolarize the membrane, right? That’s how they sense voltage. And then there’s an opening step at which they open, and then calcium comes in. So, what we found was that the small G proteins could inhibit that final opening step and so the channels don’t open. And what was very interesting is that there were two things that were necessary for that inhibition to occur. First, these small G proteins had to have a membrane-binding tail – that was absolutely required. And the second thing was binding to the beta subunit. And that really got us thinking, why is it that you need these two things, beta binding and membrane targeting? And we came up with a very simple-minded hypothesis – I remember my student was like: “Oh, no, I don’t think that can be right.” So, we knew that the beta subunit binds a cytosolic part of the channel. And our idea was that when these small G proteins bind the beta and hook it up to the membrane, that causes a conformational change in the I-II cytosolic loop, and that somehow closes the pore.

So, when we thought about that, there was a prediction from that hypothesis, and that prediction is this: you don’t really need small G proteins to do that. You could potentially put a membrane-targeting motif on the beta subunit itself. If you can recruit that to the membrane, then you can inhibit the channel. And so, we did that experiment. And it turned out that that was actually right. We could now use the beta subunit itself by putting in a motif or a module that you can recruit to the membrane with a small molecule. And that converted the beta into an inhibitor. And then we found out that we could expand that to other cytosolic proteins that bound to different parts of the calcium channel – we could convert them into inhibitors by hooking them up to the membrane. And that’s what came up with the approach called ChIMP – channel inactivation induced by membrane-tethering of an associated protein. And the benefit of that is this: for the small G proteins, or for the nanobody targeted to the ubiquitin ligase, the inhibition of the channel is constitutive, so, you have no control of overhead from a temporal perspective. But with a ChIMP you could induce that acutely with a small molecule. So, it’s like a chemogenetic version of it that allows us to have temporal control of the inhibition.

I remember seeing something like this in papers from György Hajnóczky group studying ER-mitochondria contacts and calcium transfer. This small molecule that you use to induce binding to the plasma membrane, is it something like rapamycin?

Yeah, we use this. So, this is a very strong approach developed by Stuart Schreiber and colleagues quite a few years ago (see here), where rapamycin is a naturally occurring substance that’s actually utilized as an immunosuppressant drug clinically. Rapamycin functions by binding two proteins simultaneously, one of which is a kinase called mTOR and the other is FKBP protein. And so, that approach has now been utilized to heterodimerize two proteins. So, you can have two proteins, one with an FKBP binding module, and one with the module from mTOR, called FRB. And if you have two proteins linked to those two things and express them inside the cell – under basal conditions there’ll be separate but once you add rapamycin they come together. And so, it’s a way to recruit two proteins together in an acute manner.

Yes, I love this technique. And also both the ChIMP and the targeted ubiquitination are really cool approaches to inhibit calcium channels. Now, I have to tell you that I was thrilled to discover your Nature Methods 2020 paper where you talk about the deubiquitination as a way to selectively upregulate desired ion channels. It’s essentially using the same approach that you used before, but in the reverse direction. Could you tell me more about this?

Sure. Let me introduce that by saying what we know of several diseases like cardiac arrhythmia, epilepsy, cystic fibrosis, etc. So, the cardiac arrhythmia occurs in the heart, epilepsy in the brain, cystic fibrosis in the lungs. On the surface, they seem like very different diseases, and you can’t think of a way where you can try to treat all these diseases with the same approach. But there is a way, and that’s what this paper addresses.

Colecraft_Kanner
Henry Colecraft and Scott Kanner.

So, the premise of the paper is that for those particular examples that I’ve given you, cardiac arrhythmias, cystic fibrosis, and epilepsy, a subset of those diseases is really mediated by a common biological mechanism. There are inherited or de novo mutations in an ion channel that occur in families, and many of those mutations prevent the ion channel from trafficking to the cell surface.

And so, when you think about it that way, you can now appreciate that the fundamental biology is the same. And if you can figure out a way where you can rescue channels that are having a hard time to get to the cell surface, then you can have an approach that can be utilized for many different diseases.

So, that was the idea. And what we’ve known from the literature, and from my own work, is that ubiquitination plays a very critical role in the surface density of ion channels. For a normal channel, the surface density is determined by the rate of delivery to the cell surface, the rate of removal, and also the rate of degradation of the protein. And it turns out that ubiquitination plays a key role in all those three steps. So, the more ubiquitin you have on a wild-type channel, the more it’s retained inside the cell, and the more is degraded. And so, the lightbulb moment for us really came from the realization that many of the mutant ion channels that cause disease they behave like a channel that is ubiquitinated in that the channels are retained.

And so, the simple hypothesis we had was this: if we remove the ubiquitin from those mutant channels, can we make them get to the cell surface and have some function? So, when we’re thinking about how to do it, it’s actually a very formidable challenge. And that is because ubiquitin, as the name implies, is ubiquitous – every single protein inside the cell utilizes ubiquitin as a way to regulate it. And so, the challenge becomes how do you remove ubiquitin from a target protein specifically, without affecting the ubiquitination of all the other proteins inside the cell, which would otherwise give you side effects that would make it essentially undruggable. That’s why we came up with this approach we refer to as engineered deubiquitinases. So, inside the cell, there are these natural erasers of ubiquitin, called deubiquitinases. And what we did was harness that catalytic activity and fuse it to a nanobody that targets a particular ion channel of interest – exactly like we did before with ubiquitin ligase. So, when we did that, we found that we could deubiquitinate ion channels specifically without affecting the ubiquitination of other proteins inside the cell.

We tested this approach on mutant channels that cause long QT syndrome (KCNQ1) or cystic fibrosis (CFTR). Specifically, it’s loss-of-function mutations in those channels that caused these diseases. And it has been shown previously, for both channels, that the primary mechanism behind this loss of function is impaired channel trafficking to the cell surface. So, these mutated channels couldn’t basically get to the cell surface. And what we found was that we could use the targeted deubiquitination approach to rescue the surface density and function of these mutant ion channels.

But in order for this technique to be effective, the trafficking-deficient channels must retain the ability to function after being incorporated into the membrane.

That’s right. This would work for channels, which if you got them to the membrane, will still be functional. Of course, there are some other mutations where the channel is hopelessly destroyed. In that case, you wouldn’t be able to rescue the channel’s function. But for many mutations, the channel is functional if you’re able to get it to where it needs to go. So, we think of this technique as a general approach applicable to diverse ion channels and amenable to therapeutic development.

Yes, I see what you mean. And I want to congratulate you on cofounding a new company, Stablix, which as shown on its website “is pioneering an entirely new field of targeted protein stabilization”. It’s clear that Stablix’s protein stabilization technology originated from your lab. What diseases will Stablix address in the first place?

stablix

Yes, it’s really encouraging to see that our targeted deubiquitination approach is now being translated into new therapies. So, the company will primarily develop programs to treat rare diseases, cancer and immunological disorders. And I am eager to collaborate with Stablix to make new treatments available to patients.

OK. So, we’ve been talking about ion channel engineering approaches so far. But in some of your papers you talk about reverse engineering. So, how are these things different, ion channel engineering vs reverse engineering?  

Well, all our approaches, we refer to them under the broad umbrella of ion channel engineering, because we’re trying to engineer new tools to study ion channels in different ways. And the reverse engineering approach is something that came out in the earlier part of my career. So, one of the really fabulous things about ion channel research is that in many cases we’re able to take the genes for these ion channels and then put them in a very simplified system, a reduction system, where there are no other ion channels present, for example. And then we can study them in detail in terms of their biophysical mechanisms. So, one of the cells we use are called HEK293 cells, which are easy to transfect, they’re very easy to work with. So, we’ve done a lot of our work in that environment, and many other ion channel labs do the same. But you know, like people, proteins are social creatures, they act differently depending on the context. And one of the things we’re interested in is trying to not only understand the biology of these channels in a simplified system, but also in real cells, such as heart cells and neurons.

And the tools to do that were quite challenging, because one of the reasons we prefer the reduction systems is that they’re simple, easy to use, the cells are robust, and you can do a lot of things with them. But if we go on to something like the heart cell, an adult cardiac myocyte – you can’t transfect an adult myocyte. But what you can do is you can use a virus to infect them. And so, while we were trying to study calcium channels in heart cells, we wanted to put these channels into viruses, but we ran into a problem. The calcium channel is a big protein, and viral vectors have a packaging capacity. So, you can’t just put these channels into the virus. And so, we had to come up with ways to allow us to overcome that barrier. And that was part of what we call reverse engineering. So, we were working on it, and one approach that was really neat, came out to us quite by chance.

So, when I first got to Columbia, I was sitting in a seminar by a guy called Tom Muir. I considered myself a pretty good molecular biologist at that time, but he spoke about something that sort of simply blew me away. And that is that in things like cyanobacteria there are protein splicing elements called inteins (in this particular case, something called split inteins) which basically are peptides, that aren’t fully mature proteins. But when they are coexpressed, and come together, they form a full intein. And that thing is able to stitch together the two flanking parts of the protein and excise itself out of the resulting protein. I was like: “Wow, that is amazing!” And I was really excited by that, because it immediately showed me how I could potentially use that to overcome the size limit capacity that we’re encountering with respect to expressing calcium channels in the adenovirus. So, the idea is that if we cut the channel into two, then those two parts are now within the packaging capacity of the virus. Next, we can hook up the inteins to the two parts, and the idea is that when we coexpress them together the two inteins will come together, stitch together my channel, and then take themselves out. And so, we did that, and that was a paper published in PNAS back in 2013 (see here). And that then allowed us to be able to start engineering these proteins inside live cells. And so, that was one aspect of the reverse engineering that we did.

What great tools you’ve developed in your lab. Amazing things. So, of all your discoveries and developments, what is the most important or the most satisfying for you?

Oh, that’s like asking: “Who’s your favorite child?” It’s a tough one. In terms of what I think ultimately is going to have the largest impact, I think I’ll have to say our work on the ubiquitin engineering of ion channels is going to have the biggest impact. I believe that that is going to have translational value that will ultimately end up in treating human disease. And so, I would say the targeted deubiquitination and the targeted ubiquitination aspect of what we’ve done, I think is so far going to be the biggest impact thing.

It’s interesting that you talk about impact and translational value as a measure of the importance of your work. You know, many basic scientists insist that their job is to extend the knowledge about a specific phenomenon, without any particular application or use in view. At the same time, patients and families are not interested in research for the sake of research. They are looking for translational value, for a cure.

Yes, I understand, and I think what we really need is a hybrid approach. And this is where we need a concerted effort to make the bridge between the basic research and the translational. This is what I’m trying to push for, because there has been some basic research already done on multiple ion channels, for example. And you know how basic scientists are: we study the thing and then a lot of times we just study it. And so, the push is to really try to not just leave it at the basic science, not just do my stuff in HEK cells and publish my paper on how this mutation affects this gating, but actually to make the concerted effort to now try to bridge that to the translational aspect. And that’s where I think the push is, at least from my perspective. And that’s how I’m trying to approach it in our CACNA1A project, for example. As you know, we collaborate with CACNA1A Foundation with the goal to accelerate the understanding, diagnosis and treatment of CACNA1A-linked diseases.

Yes, CACNA1A Foundation is doing amazing job connecting patients, families, clinicians and scientists. Could you tell me more about your CACNA1A project?

Sure. So, a couple of years ago, I was on vacation in Africa. And I got an email from one of my colleagues who is a neurologist at Columbia, Wendy Chung. And she told me about a patient of hers that had been born with ataxia and some other neurological problems. And they’ve done the whole exome sequencing, and it turned out to be a mutation in a voltage-gated calcium channel CACNA1A. And at that time, she wondered whether we could figure out what the mutation did, because typically, when people get this kind of information (sequence of a mutant protein), it’s usually referred to as a variant of unknown significance, because nobody knows whether that mutation is pathological or not. So, you have to sort of figure out what it does to the channel.

And so, we started a project trying to understand first of all that mutation, but then we quickly realized that there’s actually a whole range of mutations that occur in CACNA1A that are linked to disease. Therefore, we’ve started a project direction, that’s really trying to understand how these mutations affect the channel in terms of biophysical activity. We were able to make the mutation in the lab, put it in a HEK cell, and very quickly determine whether the mutation is a loss-of-function, a gain-of-function or something else. So that’s one aspect of that approach.

But when you think about the problem of trying to come up with a therapy for this kind of thing, it’s actually quite complicated. You can understand what the mutation does at the level of a single protein, but you really need to widen the perspective to really see how that ultimately translates into the phenotype in a whole person. And we’re doing this in a stepwise manner. So, the second aspect of what we’re doing is to generate iPSC neurons from the patients so that we can grow their own neurons in a dish and ask how the mutation affects the biology of the neurons within that patient’s specific background. And the advantage of that is that we can now also use CRISPR genome editing methods to correct the mutation and see whether that is sufficient to correct any deficiencies that we observe. Alright, so that’s the second aspect of that.

Pharmaceutical companies are very good at finding molecules that are like a sledgehammer – block a channel or open it… But what you really need is something more subtle, things that will shift the gating back into the normal range.

HENRY COLECRAFT

And then the third aspect is that we found that many of these mutations affect the channel in a very subtle way. You know, the pharmaceutical companies are very good at finding molecules that are like a sledgehammer – block a channel or open it. But for many of the mutations that we are finding is that they don’t just either open or shut down the channel, they cause shifts in the gating of the channel, so the channel can either open too quickly or too easily, or it’s too hard to open. And so, what you really need is something more subtle, things that will shift the gating back into the normal range. Those molecules are not there. So that’s another issue that we’re trying to do – we’re trying to figure out or discover molecules that can correct that basic gating deficit.

And then the final arm of this is to try to model the mutation in a whole organism, and we’re using transgenic knock-in mice to try to do that. And it’s a large problem. It required us to learn a few things that we’d never done in the lab before. But I have a very bold postdoc who’s taking on that project. And we’ve made quite a lot of progress so far.

And apart ataxia, what other neurological disorders are associated with CACNA1A variants, and how are they treated now?

Oh, there are multiple disorders associated with various mutations of that. These include ataxias, migraines, epilepsy, intellectual disability, eye disorders, cerebellar atrophy. And the way these patients are treated now is basically trial and error: we try to give them certain things and see whether they work or not. But what we want to be able to do is to give a neurologist a specific drug and say: “Well, this would be the best treatment for this kind of patient with this kind of mutation.” But that is completely absent right now, and that is the gap that I think we need to bridge.

Cacna1a Foundation meeting 2022
2022 CACNA1A Foundation Annual Meeting

The way to get there is really through the fundamental research. We need to categorize as many of these mutations as possible. I mean, there are hundreds of these mutations, most of which have not been characterized in terms of what they do. And one of the challenges once you have hundreds of mutations: do you have to have hundreds of different therapies for individual people? That would be economically unviable and which drug company is going to figure it out?

So, what we hope to find is that the broad mutations are going to fall into a small subset of categories: you’re going to get some loss-of-function, some gain-of-function, some mixed. And, hopefully, when you find a treatment for one loss-of-function – that will be applicable to a whole bunch of loss-of-function mutations. And so, we need to characterize a lot more of these mutations so that we can fit them into the buckets of what the mutation does to the ion channel. I think we’re going to have a universe of a certain subset of patient derived iPSC neurons, so we can gather the universe of effects that happen with different types of mutations. There’s going to be some drug discovery aspect of this which I think will require some collaboration with pharma. And so, we need to get some buy in from that as a way to do that.

And then the model systems. So, beyond mice, you can think about generating things like zebrafish models, which will allow you to be able to make the mutations in a more rapid way, and potentially have a way to screen molecules. We could possibly do phenotypic screening for small molecules that can correct phenotypes that arise from mutations in CACNA1A, and ultimately try to get small molecules that can help different categories of patients. Then we’ll need to hook up with the neurologists who provide really great information about the clinical disease and its phenotypes, and their subtleties, and how the different mutations come out in different people. So that’s I think what’s going to be needed.

Well, you must be running a big lab if you are working on so many projects in parallel.

Not so big, in fact. Right now, we have about 10 people in the lab. But the people who work there, they are extremely great. You know, one of the pleasures of being a professor is really the possibility to work with outstanding young people. They keep you mentally acute and you try your best to keep up with them. And, my mentorship style, I think, is evolved over the years so that I don’t really have a one size fits all sort of a strategy. I think my most important role in guiding the students is really the choice of project and the choice of question and what they work on, because you can spend a lot of time working on things which can ultimately lead to … nothing. There’s a balance between risk and reward that I tried to strike with them. I think it’s important for them to take on projects that have a certain element of risk. You really want to discover something new and not rehash something that somebody has already done. For me it’s not as exciting to do that.

“You really want to discover something new and not rehash something that somebody has already done.”

HENRY COLECRAFT

I think all the students need a project which is going to be new, is going to be fundable, and will propel them to go on with their careers elsewhere. And so, that is a fundamental aspect. But then once it comes to how to execute that, my aim is that they all ultimately become the leaders of the project in terms of driving ideas and stuff like that. And you know, it takes some work, and a lot of back and forth, but most of the time, by the time they’re ready to graduate, they are really the drivers and the leaders of their projects. And so that is the way I approach my trainees.

I don’t think you want to ever be limited by what you know… You want to be driven by your questions. And if your question takes you a certain way, the onus is upon you to figure out how to get that done.

HENRY COLECRAFT

And I think one of the things that I stress to the students is that I don’t think you want to ever be limited by what you know. And you don’t want to be limited by technological expertise. You want to be driven by your questions. And if your question takes you a certain way, the onus is upon you to figure out how to get that done. And if it requires you to go and learn a new technique – that’s what you do.

I’ll tell you one story. So, I’ve been interested by the idea about the targeted genetically encoded ion channel inhibitors for a long time. And for a long time, I’ve been trying to figure out a way where we could develop antibodies to these calcium channel subunits. I’ve been digging through a lot of literature and I’ve seen things like darpins or monobodies, but you know, these are all new things and they seemed really hard. And there was a postdoc from Rod MacKinnon’s lab who once came and gave a talk at Columbia. And he had made nanobodies to one of these ion channels that they had solved the structure for. And while I was talking to him, I asked him about how difficult it had been to make that nanobody. And he said: “Oh, not difficult at all.” Once they had a purified protein, they shipped the protein off to a farm in Massachusetts, which has llamas. So, they injected the llamas (because nanobodies are produced by things like llamas and camels and sharks) and provided him the blood. And then you just need to follow the protocol from a nice Nature Protocols article about how to make a phage library and use phage display to isolate nanobodies from immunized llamas.

So, when he said that, I told my student Travis: “Well, we’ve been wanting to do this. So maybe we can bite the bullet and make the beta subunit, ship it off to this farm and then see what happens.” And so, that’s what I mean by “you need a student who’s ready to take that risk.” He didn’t know how to do this, but he went ahead and did it over the protocol, and then he got the nanobodies. But then we had to do the phage display approach to try to isolate binders to the subunit.

Biophys-Journal-Editorial-Board-Dinner-2020
Biophysical Journal editorial board dinner, 2020

And while we were in the middle of that, I’ve gone to a meeting in China. And from the airport, I took a cab with the very interesting guy from Harvard – young scientist called Andrew Kruse. And while we’re chatting, I asked him about what he’s doing. And he said something that really made my ears perk up. He said: “Oh, you know, one of the things we’ve been doing is we’ve made yeast display library of nanobodies, which we can use to select binders to different things.” What? Really? I couldn’t believe my ears. It was unbelievable – quite by chance I took a cab in China with a guy from Harvard who does the things that I need. Wow.

So very quickly, we established contact, I invited him to come to Columbia to give a seminar, we’ve got the yeast library and, you know, one thing I have to commend him for is that they made this library available to the scientific community basically free of charge without strings attached. And so, in the Nature Methods paper, we made nanobodies to the cystic fibrosis channel using that yeast display library that I got from Andy Kruise. That’s the library we’re using to now find binders to different proteins. So, that’s a story about the serendipity of science, right? And it also says that when you don’t know how to do something you shouldn’t be limited by what you know. Go ahead and learn it, and if it doesn’t exist – invent it. That’s one of the philosophies we take in the lab.


  • Watch Henry Colecraft talking about Regulating Ion Channel Function and Expression With Engineered Nanobodies here.
  • Learn about Henry Colcraft’s research projects here.
  • Learn more about CACNA1A from Henry Colecraft here.

Pictures by Henry Colecraft.