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Security Gates in the Cell

A key characteristic of life is active transport: control over what enters and exits the cell. Closer looks reveal exquisite “selectivity filters” with moving parts that make active transport work.

The simplistic portrayals of cells taught in high school biology classes of the 1970s (let us say) stressed general functions, not detailed structures. These included terms like active transport, metabolism, reproduction, and the like, described in broad terms with simple diagrams. Ions go in; waste products go out. No wonder students were bored. Memorize; repeat on test. What if they had been shown the images at the nanometer scale now available to us through advanced imaging technologies like cryo-EM microscopy? They would be texting the images to friends with “Wow!” emojis. Some might have been inspired to become molecular biologists.

Scientists have learned much in the last fifty years about the channels that enable active transport. They have learned that multiple channels exist that control specific molecules. Some aquaporin channels let water in or out. Some voltage-gated channels control the flow of electrically charged molecules, like ions of potassium, sodium, and chloride. Some mechanosensitive channels respond to touch. They have also subdivided these channels into families with similar functions that differ in particular applications, and noted that some channels are so specific, they can distinguish between molecules that differ only minutely in size. 

In recent decades, the nature of selectivity filters has been elucidated at the atomic level. Often it is the specific placement of amino acids in the filter that validates and regulates the passage of molecules. Other exciting discoveries explore the nature of conformational changes (i.e., moving parts) in the proteins surrounding the channel pores. Some of these conformational changes mimic the action of slots in a vending machine that will accept dimes but not pennies. 

Channel structure is a lively field in molecular biology. Here are some recent discoveries about diverse channels and how they work. To emphasize their importance, the papers usually describe dire health consequences when they don’t work.

Voltage-Gated Channels

Prokaryotic microbes have channels, too, making the “origin of life” all the more difficult to explain. One of the most intensely studied is the bacterial potassium channel KcsA. This is one of the channels that Roderick MacKinnon studied that earned him a Nobel Prize in Chemistry in 1998. It contains a ring of proteins that extend through the lipid membrane, selectively permitting potassium ions but not others. The analogues to KcsA in humans are important for regulation of the heart and conduction of nerve impulses.

Research on KcsA continues, as shown in a paper in PNAS by Sun, Xu et al. KcsA activates rapidly, allowing ions in, but inactivates slowly (on the order of a millisecond to a second), blocking the channel. This team wanted to learn how inactivation occurs. Is it due to the pH gate or the selectivity filter?

Transmembrane allosteric coupling is a feature of many critical biological signaling events…. Activation of KcsA is initiated by proton binding to the pH gate upon an intracellular drop in pH. Numerous studies have suggested that this proton binding also prompts a conformational switch, leading to a loss of affinity for potassium ions at the selectivity filter and therefore to channel inactivation. [Emphasis added.]

It turns out that the pH gate, although 30 Angstroms away from the selectivity filter, changes potassium affinity at the filter by four orders of magnitude at neutral pH. This “dramatic effect” implies that the two parts are allosterically coupled. The finding shows that even for the best-studied channel in a lowly microbe, there is still much to learn.

Related: A paper by Zhao et al. in Science Advances investigates how potent neurotoxins, such as those in sea anemones, tether themselves to membranes and try to hitchhike onto K+ ions to block the KcsA channels. For scientists, this kind of research helps them understand the mechanisms of ion channels.

Chloride Channel: “Hold On, Don’t Rush”

“Metal ions such as zinc (Zn2+), iron and copper are a subset of nutrients called micronutrients, and act as cofactors for proteins that have roles in growth and development,” write Citron and Zoncu in a news item in Nature. Here’s why zinc is important:

A plethora of proteins rely on Zn2+ to carry out their functions. As a result, extensive cellular resources are devoted to ensuring that Zn2+ concentrations in cells are kept within an optimal range. Notably, many DNA-binding proteins require Zn2+, including some that coordinate the production of proteins that themselves help to balance metal levels. Thus, a cellular feedback loop keeps Zn2+ levels in check. 

Citron and Zoncu report on the discovery of a “zinc sensor” in Nature by Redhai et al. that allows the lowly fruit fly to respond to shortages in zinc, which can hinder growth and metabolism. The study included two dozen co-authors. The main actor is a transmembrane channel they named Hodor (for “Hold on, don’t rush”). 

The authors showed that mutation of Hodor, a transmembrane protein, leads not only to a reduction in growth of the fruit-fly larvae, but also to a diminished body-fat content and to lower food intake throughout the flies’ development. They demonstrated that Hodor is not a Zn2+ transporter, but instead behaves as a Zn2+-regulated channel that, when activated by Zn2+ binding, allows chloride ions (Cl−) to cross plasma membranes.

The chloride ions, in turn, acidify lysosomes to promote dismantling of spent proteins. The acidified lysosomes then promote the TORC1 pathway (a network that regulates metabolic processes and growth in all animals), and also stimulates feeding by fruit flies both in the brain and in the gut. For these reasons, Hodor is a little player in little insects that can have profound, organism-wide effects.

Related: In Science Advances, Pedemonte et al. (Feb 21) investigate mutations in another chloride channel named CFTR, which opens and closes by ATP-driven conformational changes. Because this channel is essential in epithelial membranes of the gut, lungs, and reproductive organs, defects in this channel are a leading cause of cystic fibrosis. When the chloride channels cannot secrete chloride and bicarbonate, the result is “the production of dense mucus secretions that clog the airways and exocrine gland ducts.” This leads to “progressive loss of respiratory function, pancreatic insufficiency, and infertility.” Here again is a channel that, when its proteins accumulate mutations that break its function, can result in profound, organism-wide effects.

Calcium-Gated Potassium Channel: Ball-and-Chain Mechanism

Some washtubs and toilets have plugs attached to a chain. A sudden change in flow can cause the plug to move and close the drain, depending on the design. Something like that has been discovered in a potassium channel named MthK that works in bacteria around hydrothermal vents. Because this channel is easier to study than the mammalian counterpart named BK, researches at Cornell imaged MthK to study the mechanism in activated and inactivated states. News from Weill Cornell Medicine shares what they found:

With low-temperature electron microscopy (cryo-EM), which bounces electrons instead of light off objects to make atomic-resolution images of them, the scientists obtained pictures of the MthK channel when it was switched open by calcium and switched closed. The pictures revealed that even when the MthK channel is in the calcium-activated, “open” state, the pathway through which ions flow was plugged by a flexible element that sticks into the pore of the channel structure.

The scientists confirmed the function of this plug mechanism by showing that when the ‘ball-and-chain’ was deleted genetically, the flow of potassium ions through the calcium-activated MthK channel was no longer regulated.

A diagram at the top shows the basic idea. The plug is attached to the channel and swings over to plug or “inactivate” the pore. The paper in Nature by Fan et al., “Ball-and-chain inactivation in a calcium-gated potassium channel,” gives the details.

Success by Chance?

It’s important to realize that these channels require directed energy to go “against the flow.” The natural tendency of an opening in the cell membrane is osmosis — flow from high concentration to low concentration. Active transport, therefore, would have been necessary in the earliest presumed “protocell” to let desired molecules in and keep undesirable molecules out. This is another big hurdle for materialist origin-of-life theories. A self-assembling lipid bilayer enclosing hopeful RNA molecules performing simple metabolism is nice to imagine, but it would be a death trap if nutrients could not enter, and if waste products could not exit. Additionally, toxins and harmful molecules would swarm into the protocell by osmosis if simple pores formed in the lipid bilayer “container.” When all the requirements for a functioning protocell are considered, success by chance is beyond unimaginable.

While obeying the second law of thermodynamics in the system, these amazing channels locally act like “Maxwell’s demon” machines, forcing separation of ingredients that would normally diffuse to equilibrium. The reduction of entropy locally is due to the expenditure of molecular energy in ATP, in very precise ways. Sometimes Maxwell’s hypothetical demon is imaged as a little intelligent being directing traffic, but it could be a robotic device. Either way, contradicting natural tendencies requires intelligent design in every mechanism for which we have observed it coming into being. Membrane channels — which this article only begins to explore — are the Rolls-Royces of traffic-controlling mechanisms.

Photo: A traffic cop directing traffic, by Calebrw / CC BY-SA.