Nonconducting W434F mutant varies in structure and conformation

Potassium (K⁺) channels are tiny, highly specialized conduits found in every live cell that transfer K⁺ ions in an exceedingly selective and quick manner across cell membranes. Voltage-gated potassium (Kv) channels are potassium-specific transmembrane channels that are also voltage-sensitive within the cell membrane, where a selectivity filter favors K⁺ ions over sodium (Na⁺).

The selectivity filter, in addition to guiding ion selectivity, uses a technique called C-type inactivation, which enables the selectivity filter to operate as an extra gate that can block the flow of ions. C-type inactivation is a selectivity filter rearrangement that arises when the cell membrane’s activation gate is opened, causing prolonged depolarization.

The question of whether or not a known mutation (W434F) located within the Shaker-IR (inactivation removed) channel epitomizes and functionally expedites the C-type inactivation state was examined in research led by Luis Cuello, PhD, from the Texas Tech University Health Sciences Center (TTUHSC) School of Medicine, and Alain J. Labro, PhD, from the Universities of Antwerp and Ghent in Belgium.

D. Marien Cortes from TTUHSC, Laura Coonen, Evelyn Martinez-Morales, Dieter V. Van De Sande, and Dirk J. Snyders from the University of Antwerp were all members of the Cuello-Labro team. Science Advances published their research.

Each living cell’s proper functional behavior is determined by the particular transport mechanism of K⁺ ions. This allows the K⁺transport system to efficiently regulate a wide range of extremely complicated activities, including the regular electrical activity of brain neurons, the typical immune response of the human body to life-threatening pathogens, and the rhythmic beat of the human heart.

One of these potassium channels, the hERG channel, must go through C-type inactivation in the human heart to maintain the periodicity, or the interval between beats, of the heart. In a typical cell, these potassium channel proteins are in a resting condition, according to Cuello, but they must be triggered for appropriate function. However, for the normal functioning of the human heart, it is vital that they undergo C-type inactivation.

After it does activate, the channel has to undergo inactivation. That is so important for the human heart because it needs to keep its periodicity, and the heart beating is based on that feature; the channel has to inactivate.”

Luis Cuello, Texas Tech University Health Sciences Center

For several years, scientists have examined a specific mutation (W434F) within the Shaker channel, a potassium channel developed from Drosophila melanogaster, a common species of fruit fly, to understand more about Kv channels in humans. The Shaker channel, like potassium channels in the human body, is made up of critical membrane proteins that play a crucial role in the normal functioning of the cell and potassium ion channel operations.

The Shaker channel was found some 50 years ago in the United States and became widely available, particularly to those investigating potassium channels, because it was the only channel available. When a mutation on the channel was discovered—W434F—it was commonly thought to represent the C-type inactivated state of a potassium channel. Cuello stated that his team has demonstrated that this is not the case.

In this paper, we prove that the normal channel that inactivates has a structure and a conformation which is different from that of the mutant.”

Luis Cuello, Texas Tech University Health Sciences Center

Cuello says, “Thanks to our work, we said, ‘Hey, be cautious because that mutant channel is a very different conformation, or you might have a different structure which doesn't have anything to do with the real channel in the heart or in the human body.’ This is important because if you want to design a drug based on this assumption that the normal channel looks like the mutant channel, that's going to be incorrect because it seems to be that in the inactive state, the structure of the W434F is different based on our experimental result.”

The mutant channel was chosen because it produced a channel that does not pass ions. According to some investigations, the mutant does not really conduct ions because it is inactivated like typical potassium channels in the human body, according to Cuello.

The results of the experimental studies performed by his team, however, strongly imply that the mutant channel is confined in a deep inactivated and non-physiologically relevant conformation. The Shaker channel has long been thought of as a structural surrogate to investigate all the channels because the human potassium channels are quite similar to the mutant.

Cuello details, “That particular mutant (W434F) is not a good comparison to the C-type inactivated human channel that we have in the body, so we have to be cautious with it.”

It was also assumed that the channel lacked potassium or sodium selectivity, but Cuello said his team’s findings demonstrated otherwise.

The channel still remains potassium selective; the only thing that it doesn't do is conduct potassium. That’s important when you want to design new drugs because that provides you information about which part of the protein you must tap to tackle it as a therapeutic target. In this case, we are calling attention to our work and saying, ‘Hey, do not establish a structural parallel between the mutant inactivated channel and the inactivation state of a normal channel in the human body.’ Be aware there is no structural equivalent, and that’s extremely important.”

Luis Cuello, Texas Tech University Health Sciences Center

Cuello’s laboratory is shifting its focus away from the fruit fly K⁺ channel and toward three isoforms of human K⁺ channels as the research advances. Two of the channels they are studying are found in the immune system, and one is found in the brain.

Cuello concludes, “We want to study what happens when these channels inactivate. How is that process here, and how we can design specific drugs to tackle a specific state of the channel? Do we need to target the channel when it is in the closed state, or do we need to tackle the channel when it's in the open state? We need to start studying the human isoform of all the potassium channels if we want to design specific drugs for specific diseases.”