The Crucial Decade That Ion Channels Were Proven to Exist

“The Crucial Decade That Ion Channels Were Proven to Exist: The Vision of Bertil Hille and Clay Armstrong and How It Came Through.”

Recently I stumbled upon a nice article in the Pflügars Archiv (the European journal of physiology) that is part review and part history. Its title is “The Crucial Decade That Ion Channels Were Proven to Exist: The Vision of Bertil Hille and Clay Armstrong and How It Came Through.” Ion channels are an important topic in Intermediate Physics for Medicine and Biology, being discussed in Chapter 6 (Impulses in Nerve and Muscle Cells) and Chapter 9 (Electricity and Magnetism at the Cellular Level). The authors are Luigi Catacuzzeno, Antonio Michelucci, and Fabio Franciolini, all with the University of Perugia in Italy.

The article begins with a discussion of Hodgkin and Huxley’s research on a nerve axon. Russ Hobbie and I describe this work in Section 6.13 of IPMB (“The Hodgkin–Huxley Model for Membrane Current”). We focus on their 1952 papers in the Journal of Physiology, and especially the fifth one which developed their mathematical model in detail. It’s a wonderful paper, and when I used to teach my graduate bioelectricity class at Oakland University the students were assigned to read it. I thought I was familiar with the story behind Hodgkin and Huxley’s research, but I learned something new from Catacuzzeno et al. They write (with citations removed)

We wish to recall, in Hodgkin’s words, how in the summer of 1949, in about a month, they managed to complete all the experiments used in the five papers published in 1952, as a special lesson for today’s times, when everything seems to move so fast and often with little thought behind it: “I think that we were able to do this so quickly and without leaving too many gaps because we had spent so long thinking and making calculations about the kind of system which might produce an action potential of the kind seen in squid nerve. We also knew what we had to measure in order to reconstruct an action potential.”

One of the interesting features of the Hodgkin and Huxley work is that they did not know about ion channels. I find it hard to even begin teaching the subject without talking about ion channels, yet they presented all their results without referring to them. Catacuzzeno et al. seem to share my surprise.

Notably, in none of their papers did Hodgkin and Huxley ever mention “ion channels,” only ion currents and conductance. In fact, the concept of an ion channel, as we know it today, did not even exist at the time. Carriers [now known as “transporters”] were more in vogue in the scientific community, also in association with membrane excitation. In the last of their 1952 papers, commenting on the Na⁺ inward current, Hodgkin and Huxley wrote that it could not be excluded “the possibility that Na⁺ ions cross the membrane in combination with a lipoid solubile carrier.” This shows how strongly rooted the concept of the carrier was at the time, and how far removed the concept of the ion channel was.

Ion Channels of Excitable Membranes, by Bertil Hille.

The main purpose of Catacuzzeno et al.’s article is to explain how the existence of ion channels was established. The heroes of the story are Bertil Hille and Clay Armstrong. Russ and I refer to Hille in IPMB when we cite his wonderful book Ion Channels of Excitable Membranes. I’m embarrassed to say that we don’t mention Armstrong at all. The “crucial decade” in the title of the article is the ten years from roughly 1965 to 1975. 

One nice thing about this review is that it really helps the reader see the scientific method in action. It presents the hypotheses that Hille and Armstrong introduce, and then explains how they designed experiments to test them. Sometimes this perspective gets lost in textbooks like IPMB, but I like how it’s highlighted in Catacuzzeno et al.’s more qualitative and historical review.

Catacuzzeno et al. claim that one of the key pieces of evidence supporting the idea of ion channels that are selective for different ions is the existence of chemicals that block a particular type of channel: tetrodotoxin (TTX) for a sodium channel and tetraethylammonium (TEA) for a potassium channel. Selectivity became a key issue. They write

Classic biophysical experiments beginning in the mid 1960s, which showed distinct conduction properties for different ions, began to provide the first clues as to the architecture and basic physico-chemical properties of the conduction pores and the mechanisms underlying ion permeation and selectivity. Hille focused his efforts on investigating the selectivity properties of these membrane pores with the idea that it would perhaps lead to something instructive regarding their structural and chemical properties. A few studies had already addressed this topic but not in a systematic way as Hille had in mind. By selectively blocking either pathway [sodium or potassium], his studies showed that at least 10 cations could easily permeate through the Na⁺ pores and four through the K⁺ pores of Ranvier’s node [in a myelinated nerve axon]. Considering the size of the ions tested and the length of hydrogen bonds, Hille estimated the Na⁺ pores to have a size, in its most constricted portion, which he called the “selectivity filter,” of 3.1 × 5.1 Å and assumed to be lined by oxygen dipoles that would establish hydrogen bonds with the permeating cations… Most interesting was another observation: all cations with a methyl group were impermeant, regardless of their size. In other words, large hydroxy guanidinium could go through the Na⁺ pore, while small methylammonium could not. These results provided experimental support for previous proposals that permeant ions interact with the pore wall and that this interaction contributes to the membrane’s permeation properties; in other words, the membrane or the pores in it do not merely select by ion size, as if they were simple physical sieves.

Bertil Hille received his PhD from the Rockefeller University in 1967. It was during his graduate studies that he began his collaboration with Clay Armstrong (both of them were young during the crucial decade when ion channels were established). He did a post doc with Alan Hodgkin of Hodgkin and Huxley fame. He then became a professor for many years at the University of Washington’s School of Medicine.

Clay Armstrong, who was six years older than Hille, is a former student of Andrew Huxley. He received his MD degree from the Washington University School of Medicine in 1960. He is currently an emeritus professor of physiology at the University of Pennsylvania. Catacuzzeno et al. describe his research in their review.

Other findings of that period, in particular Armstrong’s experiments with TEA⁺ derivatives on the outward K⁺ current of the squid giant axon, strengthened the notion that the membrane pores were at least partly made up of protein. Years earlier [Ichiji] Tasaki and [Susumu] Hagiwara had obtained action potentials with a long-lasting plateau, like the cardiac action potential, when they perfused internally the squid giant axon with TEA⁺. These data were interpreted as being due to a TEA⁺-dependent block of the outward K⁺ current (which they called anomalous rectification) and resulting failure of K⁺ current-dependent repolarization. Armstrong and [Leonard] Binstock continued their investigation with TEA⁺ by probing the drug on the K⁺ current under voltage clamp, thinking that these compounds could disclose new mechanisms and the pore architecture. First, they found that internal TEA⁺ eliminated the outward K⁺ current, whereas it was totally ineffective when applied from the outside. However, the most interesting results came when Armstrong began probing a series of TEA⁺ derivatives made by replacing one of the four ethyl groups by a progressively longer hydrophobic chain and found that the efficacy of block increased with the chain length. Using C9⁺ (nonyl triethylammonium ion) from the inside, he found that the K⁺ current no longer reached a steady state level during the voltage step but inactivated in a manner quite like the Na⁺ current.

I love how Catacuzzeno et al. include anecdotes that highlight the human side of science. For instance, they demonstrate the initial resistance to the idea of ion channels with this story:

To further represent the general sentiment on the subject at that time, it may also be helpful to recount what happened at the 1966 Biophysical Society meeting, when Armstrong and Hille presented two separate abstracts, both with the word “channel” in the title. As Hille recalls in a recent retrospective “the Chair of the session, Toshio Narahashi, began by announcing that the word ‘channel’ could not be used in the session. After our vigorous objection, he allowed us to use the word ‘provided it did not imply any mechanism!’”.

I highly recommend the article by Catacuzzeno et al. as ancilliary reading when studying from Intermediate Physics for Medicine and Biology. It’s wonderfully written, informative, and fascinating. They conclude

Asked why a skeptical medical student would take an interest in the study of ion channels, Clay Armstrong, upon receiving the Albert Lasker Basic Medical Research Award in November 1999 [along with Hille and Roderick MacKinnon], gave the following answer: “I think that ion channels are the most important single class of proteins that exist in the human body or any body for that matter” Undoubtedly, Armstrong knows well that all proteins of the body are crucial and that we cannot do without most of them; undoubtedly, Armstrong is biased in favor of ion channels after a lifetime spent with them. Yet, if he says that ion channels are of outstanding importance, then there must be something very special around them.

Bertil Hille

 https://www.youtube.com/watch?v=2MmUkaWUbyQ

Clay Armstrong

https://www.youtube.com/watch?v=r47ChajRftg