Secrets of Active Transport Become Visible
Active transport — the ability to move molecules against a concentration gradient — is one of the key distinguishing features between life and non-life. Passive transport, as with osmosis, we know by experience: a fluid will naturally spread through a semipermeable membrane from a region of high concentration to one of low concentration until the concentration is equalized. That’s why bromine tablets in a spa will spread from the filter out into the water. It’s why wildfire smoke will leak into a room through any cracks in the wall, but not out. It would take Maxwell’s Demon to combat this natural tendency which follows from the Second Law of Thermodynamics.
Life cannot operate on the principle of osmosis. A cell with a passive osmotic membrane will die. Cells need to actively bring in or expel substances, often forcing them against a strong concentration gradient. They need to maintain pH homeostasis regardless of conditions outside, often pumping in cations like Na+, K+, Ca2+and Mg2+ or anions like chloride Cl– even when the interior already has a much higher concentration than the exterior. By osmosis, these concentrations would quickly equalize and life would stop. In a real sense, life involves a constant battle against thermodynamic entropy, using energy to combat what natural forces would do.
Unnatural Selection
Biochemists have long known about the existence of specialized membrane channels where active transport takes place, and knew they were highly efficient, but how they operated was long a mystery. Roderick MacKinnon was one scientist who began to figure out the mechanisms of active transport in the 1990s. He won the Nobel Prize in 2003 for his discoveries about “selectivity filters” within ion channels that permit some molecules to pass but not others. Since then, advances in super-resolution microscopy have been revealing details at near-atomic scales about what might be dubbed “unnatural selection” inside these channels.
Membrane channels are often named according to the molecules they transport: anion or cation channels, sodium channels, potassium channels, chloride channels, aquaporins (water channels), and others. Let’s examine the inner workings of one chloride channel, about which scientists’ knowledge has been updated recently. We can share the excitement of discovery about how its “selectivity filter” determines which ions are allowed to pass. As a teaser, consider that the selectivity filter of a potassium channel is much smaller than the width of a potassium ion, yet it can transport 100 million ions per second!
The CFTR Chloride Channel
Last month in PNAS, Levring and Chen announced the “Structural identification of a selectivity filter in CFTR,” a chloride channel responsible for fluid homeostasis in epithelial tissues. It’s called CFTR (Cystic Fibrosis Transmembrane conductance Regulator) because of the fatal disease that occurs when genetic defects hinder passage of chloride ions. At the other extreme, cholera makes the channel too indiscriminate, leading to the diarrhea that causes dehydration and death. Kidney disease can also result from defective CFTR channels. This is not a part to mess with!
The shape of CFTR looks like a curved cornucopia with a narrow constriction inside. Notice how the authors identify precise amino acid residues (indicated by a letter and a position number) along the channel path that interact with the chloride ions passing through:
In this study, we identify a chloride-binding site at the extracellular ends of transmembrane helices 1, 6, and 8, where a dehydrated chloride is coordinated by residues G103, R334, F337, T338, and Y914. Alterations to this site, consistent with its function as a selectivity filter, affect ion selectivity, conductance, and open channel block. This selectivity filter is accessible from the cytosol through a large inner vestibule and opens to the extracellular solvent through a narrow portal. The identification of a chloride-binding site at the intra- and extracellular bridging point leads us to propose a complete conductance path that permits dehydrated chloride ions to traverse the lipid bilayer.
Diagrams of the interior show a chloride ion making electrostatic contacts with amino acid residues on its traverse, as if running the gauntlet through armed guards that each ensure it has a valid permit to pass. The structure “encloses a continuous conduit across the membrane for chloride to permeate down its electrochemical gradient.” Each chloride ion is hydrated with a water jacket but must remove its jacket on the way through:
Hydrated chloride enters the inner vestibule from the cytosol through a lateral portal between TMs [transmembrane domains] 4 and 6…. Chloride remains hydrated in the inner vestibule and is stabilized by a positive electrostatic surface potential. The width of the vestibule tapers down and converges at a selectivity filter, where only dehydrated chloride can enter. Dehydrated chloride moves into this selectivity filter, stabilized by interactions with G103, R334, F337, T338, and Y914 and rehydrates upon exit into the epithelial lumen through a narrow lateral exit between TMs 1 and 6.
Precision Authentication
How does the channel filter out other anions? Fluorine (atomic number 9) is smaller than chlorine (atomic number 17), so why doesn’t it slip through? The authors tested the authentication ability of CFTR by means of amino acid substitutions. They confirmed that four residues in the channel perform a qualification test on incoming anions as they dehydrate:
As was previously reported, and consistent with permeating anions having to dehydrate, wild-type CFTR exhibits a lyotropic permeability sequence, with relative permeabilities inversely related to the enthalpy of dehydration … Upon R334A, F337A, T338A, or Y914F substitution, the relative anion permeabilities were all altered, albeit to different degrees, consistent with previous work.
Figure 5 in the paper shows a chloride ion being inspected by four amino acid residue “cops” on four sides. A rogue molecule is not going to pass! The precision of this filter is astonishing. How much mutation could the system tolerate without breakdown? And how many accidents built this filter by chance in the first place? Details in the following quote will not be on the quiz, but to get a feel for the complexity involved, look at how many residues participate in authenticating chloride ions as they run the gauntlet:
Previous mutational work has identified a plethora of residues, many are arginine and lysine, that influence CFTR ion selectivity and/or conductance. Mapping these residues onto the CFTR structure indicates that basic residues, including K95, R104, R117, K190, R248, R303, K335, R352, K370, K1041, and R1048… are positioned along the cytosolic and extracellular vestibules, with their side chains exposed to solvent. Different from the residues that directly coordinate chloride [the selectivity filter], the function of these arginine and lysine residues is to stabilize the partially hydrated anions through electrostatic interactions and to discriminate against cations.The side chains of Q98 and S341 also face the cytosolic vestibule to form anion–dipole interactions with chloride and contribute to ion selectivity. R334, positioned at the extracellular mouth of the pore, plays a dual role in forming the selectivity filter and attracting anions into the pore through electrostatic interactions. Many other functionally important residues, including P99, L102, I106, Y109, I336, S1118, and T1134…, do not directly interact with chloride. Instead, they form a second coordination sphere of [the selectivity filter] that likely contributes to structuring [selectivity filter] residues with the appropriate geometry to coordinate chloride.
Airport Analogy
Think of these other “important residues” as part of the “coordination sphere” at an airport. The entire structure serves the purpose of narrowing down the flow of passengers to the “selectivity filter” of X-ray machines that inspect passengers and their baggage. The entire superstructure is necessary and must have been planned with the appropriate geometry and personnel to guide the passengers to the inspection site, even though the X-ray machine is as narrow as a human.
TSA workers at airports could never boast of this much quality control in their authentication protocols. And human workers have eyes and minds to think about what they are doing! The CFTR channel operates automatically in the dark, by the delicate “touch” of electrostatic interactions within a precisely structured narrow passageway within the coordination sphere. One source says that CFTR conducts millions of chloride ions per second! The TSA could learn something about efficiency here, as many of us airline passengers could attest.
Speaking of touch, my next article will discuss some other channels that respond when contacted — the so-called mechanosensitive channels.
Useless Darwinese
Did CFTR evolve? Because the CFTR channel has some similarities to other chloride channels like CLC, the authors glibly surmise that it “uniquely evolved from a family of active transporters,” assuming that “unrelated ion channels have evolved to select and conduct chloride using common chemical strategies.” Such a narrative gloss is not only useless, it makes no sense. A strategy implies foresight: seeing a need and designing a solution. While some frequent flyers might be tempted to smirk that TSA strategies seem mindless and unguided, elaborate structures like CFTR channels that operate extremely efficiently and accurately with low tolerance for alteration look engineered. They had to work from the start. Without those precisely placed amino acid residues already present at the right spot within a larger coordination sphere, there would be no authentication, and active transport would stop. The alternative is disease and death. Our uniform experience confirms that elaborate, efficient strategies that work — employing irreducibly complex structures with multiple coordinating parts supporting the function — are always products of intelligent design.
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