10.5: Active Transport

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Learning Goals (ChatGPT o3-mini)

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Principles and Classification of Active Transport

• Distinguish primary from secondary active transport based on the energy source used, classify transporters as uniporters, symporters, or antiporters based on the directionality of solute and co-solute movement, and identify whether a given transport event is electrogenic or electroneutral, using the Na⁺/K⁺ ATPase, SGLT1, and the sodium-calcium exchanger as specific examples.

Major Facilitator Superfamily (MFS) Transporters

• Describe the shared structural features of MFS transporters — two pseudo-symmetrical six-helix bundles forming a central ligand-binding cavity — and explain the "clamp-and-switch" alternating-access mechanism by which conformational transitions between inward-open, occluded, and outward-open states drive solute transport, using the human monocarboxylate transporter (MCT2) and lactose permease as examples of proton-coupled symport.

P-type and ABC ATPases

• Explain the Post-Albers model for P-type ATPase function, describing how ATP-dependent phosphorylation of a conserved Asp residue to form a mixed anhydride intermediate drives the conformational transition between the high-affinity inward-facing E1 state and the low-affinity outward-facing E2 state, and apply this model to the Ca²⁺ ATPase (SERCA1) and Na⁺/K⁺ ATPase as specific examples.
• Explain how vanadate acts as a transition-state analog inhibitor of P-type ATPases by mimicking the trigonal bipyramidal geometry of the phosphate transfer transition state, and describe why 70% of neural ATP turnover is devoted to maintaining the Na⁺/K⁺ gradient across neuronal membranes.
• Describe the structural organization of ABC transporters — transmembrane domains (TMDs) and nucleotide-binding domains (NBDs) — and explain how ATP binding drives NBD dimerization and the conformational switch between inward-open and outward-open states that powers drug and lipid efflux, using P-glycoprotein (ABCB1) as the example and connecting its overexpression in tumor cells to multidrug resistance.

Nuclear Import and Export: A GTP-Driven Selectivity Mechanism

• Explain how nuclear localization sequences (NLS) and nuclear export sequences (NES) on cargo proteins are recognized by importins and exportins respectively, describe how the RanGTP/RanGDP gradient across the nuclear envelope determines the directionality of importin:cargo and exportin:cargo:RanGTP complex formation and dissociation, and explain why GTP hydrolysis — rather than ATP hydrolysis — is the thermodynamic driver of nuclear transport directionality.
• Describe how FG-repeat nucleoporins (FG-Nups) in the inner ring of the nuclear pore complex form a selective hydrophobic meshwork that excludes molecules above ~40 kDa by passive diffusion while allowing nuclear transport receptors (karyopherins) carrying cargo to transit through transient hydrophobic FG:receptor interactions, and summarize the experimental evidence from designer FG-Nup nanopores that reconstitutes this selectivity in a synthetic system.

In the previous sections, we explored facilitated diffusion, diffusion through channels, and diffusion through larger pores. In each case, once a carrier/permease protein was available, a channel (gated by ligand binding, changes in membrane potential, lipid binding, or mechanical forces) or a pore formed, solute flows down a chemical gradient (facilitated diffusion) or an electrochemical gradient in a thermodynamically favored process. But what about moving solutes from low to high concentration against a concentration gradient, which would be necessary to capture the last bit of a vital nutrient or energy source? Active transport does just that, but it requires an energy source. Many different types of chemical species are actively transported across the cell and organelle membranes, including sugars, amino acids, (deoxy)nucleotides, metabolites (like carboxylic acids),

Type of Active Transport

A membrane receptor is required for active transport to occur, which recognizes the ligand to be transported. However, the energy source driving transport against a concentration gradient is of major interest to us. The biological world has adapted to use almost any available energy source.

ATP hydrolysis: One would expect this ubiquitous carrier of free energy to drive active transport. This is one of ATP's predominant roles in the biological world. 70% of all ATP turnover in the brain is used to generate and maintain a Na⁺/K⁺ ion gradient across nerve cell membranes via the membrane protein Na⁺/K⁺-ATPase.

Energy released by oxidation: You may have encountered this in previous biology courses. Active transport of protons driven by oxidative processes is exergonic. In electron transport in respiring mitochondria, NADH is oxidized as it passes electrons to a series of mobile electron carriers (ubiquinone, cytochrome C, and eventually dioxygen) using protein complexes in the inner membrane of the mitochondria. The energy lost in this thermodynamically favored process is coupled to conformational changes in the complex, which eject protons from the matrix into the inner membrane space. One can imagine a series of conformation-sensitive pKa shifts in various side chains within the complexes, leading to vectorial proton discharge.

Light: Photosynthetic bacteria have a membrane protein called bacteriorhodopsin, which contains retinal, a conjugated polyene derived from beta-carotene. It is analogous to the visual pigment protein rhodopsin in retinal cells. Light absorption by retinal induces conformational changes in retinal and bacteriorhodopsin, leading to the vectorial discharge of protons.

The collapse of an ion gradient: The favorable collapse of an ion gradient can drive the transport of a different species against a concentration gradient. We have already observed that the collapse of a proton gradient across the inner mitochondrial membrane (through F₀F₁ATPase) can drive the thermodynamically unfavored synthesis of ATP. The collapse of a proton gradient provides a proton-motive force that can drive the active transport of sugars. Likewise, a sodium-motive force can drive the active transport of metal ions. Since the energy to make the initial ion gradients usually comes from ATP hydrolysis, ATP indirectly powers the transport of the other species against a gradient.

Active transport can be divided into two classes:

• primary - driven by "primary" energy sources, which for us means ATP or photons
• secondary - driven by coupled transport (also called cotransport) of solutes down a concentration gradient, which provides the thermodynamic force to move a solute "uphill."

Active transporters can also be divided into classes based on the direction of movement of the solute and any cotransported solute into uniport (no cotransported solute), symport (solute and cosolute transported in the same direction), and antiport (solute and cosolute transported in the opposite direction) as shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): Uniport (no cotransported solute), symport (solute and cosolute transported in the same direction), and antiport (solute and cosolute transported in the opposite direction) transporters

Examples of these mechanisms include facilitated diffusion and active transport. Examples of these types include:

• uniport: An often-used example is GLUT 1, which catalyzes glucose's FACILITATED DIFFUSION (not active transport) down a concentration gradient. A somewhat complicated example of a uniport active transporter is the Na⁺/K⁺ ATPase, which moves Na⁺ ions out of a cell and K⁺ ions into a cell against concentration gradients. The movement of one ion does not provide the chemical potential thermodynamic driving force to move the other. Rather, ATP hydrolysis is required. So, this protein can be called a uniport active transporter for two different ions.
• symport: Another glucose transporter, the glucose symporter (SGLT1), found in the small intestines, heart, and brain, cotransports one glucose or galactose for every two Na⁺ ions that move into the cells down a concentration gradient.
• antiport: The sodium-calcium exchanger pumps out one Ca²⁺ ion (low to high concentration) driven by the influx of three Na⁺ into the cell. This keeps cytoplasmic Ca²⁺ low.

If the species moved is charged, two other terms are used:

• electrogenic - a net electrical imbalance is generated across the membrane by symport or antiport of charged species
• electroneutral - no net electrical imbalance is generated across the membrane by symport or antiport of charged species

In this chapter, we will explore examples of several active transporters. All are polytopic with alpha-helical transmembrane domains that, through a series of conformational transitions, can move chemical species bound to the receptor or channel protein across the membrane by creating transient openings in the protein. We will end with another look at the nuclear pore complex and see how it allows the movement of large proteins and RNA molecules through its pore through a very different type of mechanism. In that case, it is not active transport since the relative concentration of the transported species on either side of the pore is less important than the mechanism by which certain species are targeted to move into and out of the nucleus.

Major Facilitator Superfamily (MFS) Transporters

There are about 74 families of active transporters in the Major Facilitator Superfamily (MFS), the largest family of secondary transporters that use symport or antiport mechanisms. There are also uniporters within this family. Different members of the MFS move a diverse array of solutes, including sugars, nucleotides, peptides, and drugs, across membranes against a concentration gradient. All have two membrane domains consisting of six transmembrane helix bundles that pack to give an apparent two two-fold rotational axis of symmetry (also called pseudo C₂ axis). The ligand to be transported binds between the two domains in a central cavity, potentially open to either side of the membrane. Figure \(\PageIndex{2}\) panel A (top) below shows a cartoon of the 12 helices and 6-helix bundles. Helices 1 and 4 in domain one and the analogous ones in domains 7 and 10 are involved in ligand movement through the membrane. Helices 2, 5, 8, and 11 are involved in domain-domain interactions. The bottom figure (B) shows two views of the protein with the ligand in spacefill rendering.

Figure \(\PageIndex{2}\): Structure of the Major Facilitator Superfamily (MFS). Images from Lee J, Sands ZA and Biggin PC (2016) A Numbering System for MFS Transporter Proteins. Front. Mol. Biosci. 3:21. doi: 10.3389/fmol.2016.00021 Creative Commons Attribution License (CC BY)

As structure mediates function, they all display a similar "clamp and switch" mechanism, in which solute binds to the protein in an open (to binding) conformation, leading to conformation changes forming a closed form, followed by further conformational changes that cause the complex to open to the other side of the membrane. Figure \(\PageIndex{3}\) shows hands opening, closing, and reopening in the other direction as an analogy for this "clamp-switch" mechanism.

Figure \(\PageIndex{3}\): Hands opening, closing, and reopening in the other direction as an analogy for this "clamp-switch" mechanism of the Major Facilitator Superfamily (MFS)

We will describe two different transporters in this family.

Human monocarboxylate transporter (7BP3)

These proton-linked monocarboxylate transporters (MCTs) move many monocarboxylates (including lactate, pyruvate, and the ketone bodies acetoacetate, beta-hydroxybutyrate, and acetate) out of the cell, driven by the flow of protons across the membrane in the same direction (symport). In cells engaging in glycolysis, a fundamental energy utilization pathway, it catalyzes the high-affinity transport of pyruvate, the end product of glycolysis, and lactate, a reduced derivative of pyruvate, out of the cell. Their activity is effectively turned off in cells not engaged in glycolysis. The flux of pyruvate/lactate has a steep dependence on their concentrations, allowing the protein to act as an on-off switch for transport. A step dependency of activity on concentration is a hallmark of cooperative interactions. Structural analyses reveal conformational changes at the transporter's dimeric interface.

We will see when we study glycolysis that the conversion of the oxidized pyruvate to its reduced form, lactate, requires a reducing agent, in this case, NADH, which is oxidized back to NAD+, an oxidizing agent required for glycolysis to continue. When pyruvate is high, NADH is also high, allowing for the conversion of pyruvate to lactate, which can be exported out of the cell. This reaction describes the interconversion:

Pyruvate + NADH ↔ Lactate + NAD⁺

Using MCT 2 to remove lactate from the cell pulls the above reaction to the right, regenerating NAD⁺ and allowing glycolysis to continue.

Figure \(\PageIndex{4}\) shows an interactive iCn3D model of the inward open human monocarboxylate transporter 2 (7BP3).

Figure \(\PageIndex{4\): Inward open human monocarboxylate transporter 2 (7BP3). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...yq5U8v4BvdDA57

Several key residues are shown. Aspartic acid 293 (side chains in red sticks) is conserved in all MCTs studied and is likely involved in H⁺ transport. It is surrounded by Val 156, Met 289, Ala 290, and Phe 351 (side chains shown in cyan sticks), all nonpolar amino acids, which would elevate the pKa of Asp 293, allowing it to remain protonated until a conformational change alters its environment, leading to proton release. Two positively charged residues, Lys 38 and Arg 297 (side chains in blue sticks), are involved in substrate interactions. Lys 38 faces outward from the cell in the inward-open conformation. Figure \(\PageIndex{5\) below shows the same amino acids in a zoomed view. The amino acids are labeled.

Figure \(\PageIndex{5\): Key amino acid side chains in inward open human monocarboxylate transporter 2 (7BP3)

Lactose Permease (Lactose-proton symport)

This E. Coli protein is also a symporter, which uses the movement of H⁺ down a concentration gradient to drive lactose into E. Coli to capture as much lactose - an energy source - as possible. Deprotonation of a protonated Glu 269 leads to ligand binding, as the protein has no binding site in the protonated state. Lactose induces the formation of its binding site. Figure \(\PageIndex{6\) below shows the change in conformation going from a more acidic pH (5.6, PDB 2CFP) to a more neutral pH (6.5, PDB 2CFQ). One lactose is transported from one H⁺, which moves down a concentration gradient. Lactose moves from the outside periplasm to the cytoplasm.

Figure \(\PageIndex{6\): Conformation changes in lactose permease from a more acidic pH (5.6, PDB 2CFP) to a more neutral pH (6.5, PDB 2CFQ)

Figure \(\PageIndex{7}\) shows an interactive iCn3D model of lac permease (1pv7), which highlights three residues essential for lactose binding (Glu 126, Arg 144, and Glu 269 in stick) and three involved in proton transfer (Arg 302, His 322, and Glu 325 in spacefill).

Figure \(\PageIndex{7}\): Lac permease (1pv7). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...9mt2juToECQPg6

It has a bound nonhydrolyzable lactose analog (beta-D-galactopyranose-(1-1)-1-thio-beta-D-galactopyranose), shown in spacefill, in the center hydrophilic cavity. The two 6-helix bundles, which exhibit pseudo-2-fold symmetry, are evident. The protein is in the open-to-inward conformation. (The blue dummy atoms represent the inner leaflet.)

Figure \(\PageIndex{8}\) is a video of a molecular dynamics simulation showing lactose moving through lactose permease.

Figure \(\PageIndex{8}\): Molecular dynamics simulation showing lactose moving through lactose permease. https://www.ks.uiuc.edu/Gallery/Movi...annelProteins/

As with other members of the MFS transporters, lactose permease has 12 transmembrane helices that form two bundles. The lactose binding site is between the two bundles. When the protein is in the open-to-out conformation, it can bind protons, likely at an exposed His. This enables lactose binding, which is followed by a conformational change to the open-to-in form. When the protonated His loses a proton, lactose dissociates into the cytoplasm. The movement of both H⁺ and lactose must occur together for transport.

Structural work by Singh et al. on the Leu Transporter (LeuT), a member of the solute carrier 6 or sodium-coupled transporters, which is an active transporter requiring movement of Na⁺ ions into the cells to power the uptake of Leu, shows an "open-to-out" and occluded binding state for ligand (Leu). Tryptophan, a competitive nontransportable inhibitor, binds to the open-to-out state but is too large for the obligate occluded state, so it is not transported. Figure \(\PageIndex{9}\) shows inward open, closed, and outward conformations of different major facilitator superfamily transporters.

Figure \(\PageIndex{9}\): Inward open, closed, and outward conformations of different major facilitator superfamily transporters

Another member of the MFS Transporter family is LeuT, the leucine transporter, which is similar to the neurotransmitter sodium symporter. Figure \(\PageIndex{10}\) shows the transition between two conformational states. The first is the outward open leucine transporter (LeuT). This initial state has bound tryptophan, a competitive inhibitor too big to transport, and traps the bound state in the open conformation. The second state has bound Leu and is closed. The conformational changes are subtle but sufficient to allow transient binding, occlusion, and expulsion to the other side of the membrane (not shown). Active transport through this transporter is driven by Na⁺ ions (purple). In the open state, the ions are farther apart, allowing access to the amino acid for transport.

Figure \(\PageIndex{10}\): Transition between two conformational states of the Leu transporter

Membrane ATPases

Many active transporters power the uphill transport of solutes through coupled exergonic cleavage of ATP. The largest family is the P-type ATPase, which is used to transport a host of different solutes, including ions (Cu, Zn, Mn, Mg, Ca, Na, K, Cd, Co, Pb, Ni, and H) and phospholipids (from one leaflet to another by translocases like flippases). They convert chemical energy (from cleavage of phosphoanhydride bonds) to electrochemical potential energy. Over 160,000 P-type ATPases from prokaryotes and eukaryotes are known, so they are highly prevalent and evolved early. They should be understood at both a structural and thermodynamic level.

How is energetic coupling achieved? An early model proposed by Albers and Post suggested that energetic coupling occurred by the covalent transfer of a terminal phosphate (P_i) from ATP to an Asp in the protein channel to form a covalent Asp-Pi intermediate, which is an example of a mixed anhydride. This is illustrated in Figure \(\PageIndex{11}\).

Figure \(\PageIndex{11}\): Formation of a high energy (compared to its hydrolysis products) mixed anhydride of Asp in membrane ATPase. (Remember, there is no such thing as a "high energy" bond.)

This reaction is thermodynamically disfavored, since hydrolysis of ATP to form ADP has a less negative ΔG⁰ than the same reaction with a mixed anhydride. Ultimately, hydrolysis of the mixed anhydride in the next step would favor the overall coupled reactions.

Another feature of the Post-Albers model is that the phosphorylated channel, P-ATPase, has two conformations:

• a high-affinity cation inward (cytoplasm) facing cation binding site called E₁
• a low-affinity cation outward (extracellular or luminal) facing cation binding site called E₂

Figure \(\PageIndex{12}\) shows a more detailed (A) and simpler (B) set of chemical equations to represent the different states of E₁ and E₂ in the Post-Albers model.

The next three figures are from the following reference: Zhang, X.C., Zhang, H. P-type ATPases use a domain-association mechanism to couple ATP hydrolysis to conformational change. Biophys Rep 5, 167–175 (2019). https://doi.org/10.1007/s41048-019-0087-1. Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/

Figure \(\PageIndex{12}\): Chemical equations for conversion of states E₁ and E₂ for membrane ATPases in the Post-Albers mode

• The top line in both shows the reactions of E₁ (high affinity) with the substrate (ions) and ATP. Since E₁ is (auto)phosphorylated by ATP, it is considered a kinase.
• The bottom line in both shows the reactions of E₂ (low affinity) with the substrate (ions). Since E₂ is dephosphorylated, it is considered a phosphatase.

S₁ and S₂ differ for a specific P-type ATPase.

Detailed Structures

Now let's look at the structures of a specific P-type ATPase, the sarcoplasmic/endoplasmic reticulum calcium ATPase 1 (or more simply a Ca²⁺ pump), which catalyzes the active uphill transport of Ca²⁺ from the cytoplasm back into the lumen of the sarcoplasmic reticulum (SR) in muscle cells and acts in the regulation of striated muscle contraction. The actual free concentration of Ca²⁺ in the SR is about 390 μM compared to 0.1-0.2 μM in the cytoplasm. On muscle excitation, Ca²⁺ floods into the cytoplasm but must be actively transported back into the SR by the Ca²⁺ pump.

Here is the simplified chemical reaction: ATP + Ca²⁺(cyto) + H₂O → ADP + Ca²⁺(s. rect) + H⁺ + P_i.

We'll stick with a cartoon representation of each of the various structural states of this calcium pump, as illustrated in Figure \(\PageIndex{13}\) (also from Zhang et al.).

Figure \(\PageIndex{13}\): States of this calcium pump

Again, different states of E₁, the kinase state, are shown in the top line, while E₂, the phosphatase state, is shown in the bottom line. PDB IDs are shown for each state. Ca²⁺ is shown as a cyan sphere. Note how the cytoplasmic N (nucleotide-binding), P (phosphorylation), and A (actuator) domains and the single transmembrane domain, colored to show the C-terminal (C_TM) and N-terminal (N_TM). are represented in the cartoon version to the right. P represents the phosphorylated Asp 351 mixed anhydride. The asterisk * denotes a transition state for the phosphorylation of Asp 351 (top line) and its dephosphorylation (bottom line).

Ca²⁺ moves from the cytoplasm to the lumen of the SR. Note that the first E₁ state is open inward (to the cytoplasm) while the first E₂ state is open outward (to the lumen of the SR, where the bulk of the Ca²⁺ ions are stored. This mimics the open-in and open-out states of the Major Facilitator Superfamily (MFS) channels discussed above.

Detailed Thermodynamics

Zhang et al. provide two different views of the thermodynamics of Ca²⁺ ion pumping into the sarcoplasmic reticulum, as shown in A (domain dissociation) and B (classic) in Figure \(\PageIndex{14}\). A quick view of the two "energy landscapes" shows:

• As expected, both go from higher to lower free energy since ATP hydrolysis powers the process.
• They differ mostly in the energetic coupling steps

Figure \(\PageIndex{14}\): Thermodynamics of Ca²⁺ ion pumping into the SR as shown in A (domain dissociation) and B (classic)

In A, the energy released in ATP hydrolysis occurs when the Asp 351-P_i mixed anhydride is dephosphorylated (ΔG_deph), which is coupled to the dissociation of the P and A (* actuator) domains. This is shown in the structural cartoon near the last step in the complete cycle.

In the cartoon diagram, six states are represented. Any mechanism can be broken down into more and more states on a more complete description of the actual mechanism. For example, ATP cleavage to ATP can be represented by three states, ATP, ADP, and P_i, or many more if intermediates and transition states are included.  The complete Ca²⁺ transport cycle in the thermodynamics diagram is broken down into 12 states, represented by 8 horizontal solid lines ^_____ and 4 dashed ------ lines. Vertical lines with arrows show transitions between states.

• Green arrows → show changes in free energy due to the relative stability (chemical potential) of ATP.
• Red arrows → show changes in free energy due to the relative stability (chemical potential) of the substrate ions.
• Cyan arrows → show changes in free energy due to the electrical potential of the substrate ions.

ΔG_L is for steps involving the loading of reactants, while ΔG_R is for the release. In both diagrams, the conversion of state E₁^.S^.ATP to E₁P^.S is uphill, as we predicted for the formation of the

On the far right of each graph are identical sets of large unfilled arrows (since ΔG is a state function), as the beginning and ending free energies of free E₁ must be independent of the pathway (i.e., mechanism). The two sets of upward unfilled arrows (red for the change in the chemical potential of the substrate ions and cyan for the change in the electric potential of the substrates are both positive as the ions move uphill from a lower to high electrochemical potential. The unfilled downward green arrow shows the change in free energy (or chemical potential) for ATP hydrolysis. Since energy cannot be created or destroyed, the narrow black arrow (Q_x) represents energy that is dissipated in the reaction cycle. The starting and ending states are identical (i.e., E₁), only differing by the energy dissipation (denoted as Q_X) of the P-ATPase transporter during one functional cycle.

There are other membrane ATPase types whose structures and functions differ from those of the P-type described above. Some run in reverse to synthesize ATP. They also vary in substrate ions. Here are several different types.

• F-ATPases (ATP synthases, F₁F₀-ATPases): These are used to synthesize ATP and are powered by the collapse of a H⁺ gradient. They are found in mitochondria, chloroplasts, and bacterial cell membranes. In the chapters on mitochondrial oxidative phosphorylation and chloroplast photosynthesis, we will discuss them further.
• V-ATPases (V₁V₀-ATPases): These pump protons into organelles to acidify them (ex. lysosome) and are also found in bacteria.
• A-ATPases (A₁A₀-ATPases): These are used to synthesize ATP in Archaea.
• E-ATPases: These are found on the cell surface and hydrolyze extracellular nucleotide triphosphate.

Let's look at some special features of one P-type ATPase that transports Na⁺ and K⁺ ions. It is important for establishing intracellular and extracellular concentrations in neurons, so they are critical for neuron function.

Na⁺/K⁺ ATPase

This protein keeps K⁺_in and Na⁺_out high relative to their respective concentrations on the other side of the neural cell membranes. ATP and 3 Na⁺ ions bind to the cytoplasmic domain of the enzyme in the E₁ conformation. As described more generally above, in the presence of Na ions, the bound ATP is cleaved in a nucleophilic attack by an Asp side chain of the protein. Hence, the protein is a Na⁺-activated ATPase or kinase. The phosphorylated enzyme changes conformation to the E₂ form, in which Na⁺ ions are now on the outside of the cell membrane, from which they dissociate. The phosphorylated protein in conformation E₂ now binds 2 K⁺ ions on the outside, which activates hydrolysis of the Asp-PO₃ mixed anhydride link. The dephosphorylated protein is more stable in the E1 conformation, to which it changes as it brings K⁺ ions into the cell. Hence, this protein is an electrogenic antiporter. P-type transporters are inhibited by vanadate (VO₄^3-), a transition state analog of phosphate. In the lab, transport mediated by P-type membrane proteins can drive ATP synthesis.

A detailed kinetic analysis of ATP and VO₄^-3 interactions shows low-affinity and high-affinity sites for each on Na/K ATPase. The high-affinity vanadate site appears to be identical to the low-affinity ATP site, suggesting that vanadate binds tightly to the E2 form of the enzyme. The low-affinity vanadate site appears to be the same (based on competition assays) as the ATP site, which is probably the E₁ form. Hence, vanadate binds preferentially to the E2 form, thereby inhibiting the transition to the E1 form. Vanadate also inhibits phosphatases, enzymes that cleave the phosphoesters of Ser, Thr, and Tyr residues in proteins. This supports the notion that vanadate binds preferentially to the E2 form, which has a phosphoanhydride link (Asp-O-phosphate) that is hydrolyzed, promoting the conversion of E₂ back to E₁. Vanadate is probably a transition-state analog inhibitor. It can readily adopt a trigonal bipyramidal structure, mimicking the transition state for cleavage of the tetrahedral anhydride bonds of ATP and Asp-O-PO₃.

Figure \(\PageIndex{15}\) is a YouTube animation of the Na⁺/K⁺ ATPase from xx. Permission Question?

Figure \(\PageIndex{15}\): Animation of the Na⁺/K⁺ ATPase pump

These interactions are depicted in Figure \(\PageIndex{16}\)below (after Stryer 4th ed)

Figure \(\PageIndex{16}\): Steps of the Na⁺/K⁺ ATPase pump

ABC Transporters

The proteins comprise one of the largest families of membrane proteins with seven main families (ABCA to ABCG). Up to 3% of all bacterial proteins encode proteins associated with the ABC transporters. Different ABC transporters move a variety of required chemical species, from small (ions, sugars, amino acids, nucleosides, vitamins) to large (peptides, lipids, oligonucleotides, and polysaccharides) into the cell. They also remove toxic (to the cell) molecules such as xenobiotics (molecules foreign to the cell, like drugs, toxins, etc) and potentially toxic metabolites. All of these require ATP hydrolysis. Moving toxic molecules out of the cell is beneficial to the cell's health, but in the case of a tumor cell, not to the organism's benefit. TAs in other active transporters using ATP hydrolysis lead to two distinct conformations: open-outward and open-inward. All ABC transporters have an LSGGQ amino acid sequence in the NBD. They also have a phosphate-binding loop (P-loop or Walker A motif). Most eukaryotic ABC transporters move solutes from the inside to the outside of the cell.

Figure \(\PageIndex{17}\) shows the various structures within the ABC transporter superfamily. The transmembrane domain (TMD, in green) and the nucleotide-binding domain (NBD, blue) are present in most gene versions. PK represents prokaryotic and EK eukaryotic organisms (shown at the very right for each).

Figure \(\PageIndex{17}\): Variations in the structures within the ABC transporter superfamily ng, J., Feng, J., Yuan, D., et al. Tracing the structural evolution of eukaryotic ATP-binding cassette transporter superfamily. Sci Rep 5, 16724 (2015). https://doi.org/10.1038/srep16724. Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/

The ABC transporter genes denoted as full structures have 2 TMDs and 2 NMDs, whereas half-structures have 1 of each. Some genes encode either a single NBD or a single TMD (prevalent in prokaryotes), along with half structures. The ABC2 structure has only two NBDs. The single structure represents the ABC transporter gene with a single TMD or NBD; ABC2 structure represents the ABC transporter gene with only two NBDs. The families possessing certain structures are listed on the right. TMDs typically have 6-10 transmembrane α-helices. Those involved in the export of chemical species have six.

Structural cartoons representing domain and ligand binding for various ABC transporters are shown in Figure \(\PageIndex{18}\) below.

Figure \(\PageIndex{18}\): Domain and ligand binding for a variety of ABC transporters. Structure and mechanism of ABC transporters. Stephan Wilkens. F1000Prime Reports 2015, 7:14 (doi:10.12703/P7-14). Creative Commons Attribution-Non Commercial License (http://creativecommons.org/licenses/.../3.0/legalcode).

(A) shows an inward-open drug (D) exporter. The binding of 2 ATPs causes dimerization of the two NBD domains, resulting in a conformational change to an outward-facing state, allowing the drug to dissociate. Hydrolysis of ATP enables the release of ADP/P_i and dissociation of the 2 NBD domain, which results in a conformational change to the inward-open form.

(B) shows the delivery of a substrate (S) from its binding protein (ex., part of the ABC transporter complex MalEFGK involved in maltose/maltodextrin import) in the periplasm of E. Coli for delivery into the cell.

(C) shows an outward-open variant of (B) (ex., part of the Vitamin B12 import system permease protein BtuC)

The mammalian protein multidrug resistance (MDR), also known as P-gp, Phospholipid transporter ABCB1, or ATP-dependent translocase ABCB1, is an example of an ABC Transporter. It moves both drugs across the membrane and phospholipids, including phosphatidylcholine, phosphatidylethanolamine, ceramides, and sphingomyelins, from the inner to the outer leaflet. The gene for this protein is often mutated in tumor cells, which reduces the effectiveness of chemotherapeutic drugs.

Figure \(\PageIndex{19}\) shows an interactive iCn3D model of the structure of the mouse P-glycoprotein (4M2T) bound to a cyclic peptide inhibitor in the inward open conformation is shown below. Most proteins bind substrates specifically, but P-glycoprotein binds them indiscriminately, which makes it so useful for pumping drugs out of the cell.

Figure \(\PageIndex{19}\): Mouse P-glycoprotein bound to a cyclic peptide inhibitor in the inward open conformation (4M2T). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...1bHUEmryErK5PA

The iCn3D model below shows the conserved (mouse and human) transmembrane-domain aromatic amino acids (H60, F71, T114, F299, Y303, Y306, F332, F339, F724, F728, F766, F938, Y949, F953, F974, F979) involved in the transport pathway, colored in sticks. The inhibitor (cyclic-tris-(S)-valineselenazole; QZ59-SSS) is shown in spaceiflll. The consensus LSGGQ (527-531 and 1172-1176) sequences in the nucleotide-binding domain are shown in colored spheres.

Here is a view showing again the transmembrane domain with the conserved aromatic amino acids.

Figure \(\PageIndex{20}\) shows an interactive iCn3D model of just the transmembrane domain with the conserved aromatic amino acids of the mouse P-glycoprotein bound to a cyclic peptide inhibitor in the inward open conformation (4M2T).

Figure \(\PageIndex{20}\): Transmembrane domain of the mouse P-glycoprotein bound to a cyclic peptide inhibitor in the inward open conformation (4M2T). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...PdHtggbLFWuCo7

Another look at the Nuclear Pore Complex

In the previous section, we examined the incredibly complicated structure of the nuclear pore. Both small molecules and large proteins (synthesized in the cytoplasm) and RNAs (synthesized in the nucleus) move through their large pores. The movement of solutes through its gaping pore does not require ATP cleavage in a primary active transport or the collapse of a chemical gradient in a secondary active transport process. Then why study it in this chapter section? It turns out that the regulation of the process requires GTP cleavage and a gradient of a particular protein called Ran.

What's fascinating about the nuclear pore is its selectivity for protein transfer across the pore. What proteins are allowed in and out? What is the origin of the specificity? The specificity is partly determined by a protein family, the karyopherin-βs receptors (22), which bind and transport nuclear proteins. There are two types of these nuclear transport receptors or transport factors:

• Importins facilitate the movement of proteins into the nucleus (ex., nuclear proteins like histones, DNA and RNA polymerases, etc).
• Exportins facilitate the export of RNA (except mRNA) from the nucleus.

Large proteins destined to be moved through the nuclear pore are called cargo proteins. They have a molecular signal that differentiates them from molecules that should stay in the cytoplasm, move into organelles, or be secreted from the cell. The signals are called:

• NLS or nuclear localization sequences
• NES or nuclear export sequences

There is no obvious NLS consequence, and different cargos use different motifs. A classical NLS is enriched in Lys and Arg residues. Others appear enriched in Pro-Tyr or Ile-Lys. Large domain structures might also participate in the NLSs. Prediction programs are used to identify clusters of lysines and arginines, with gaps between them. Classical NESs appear rich in leucines in an 8-15 amino acid sequence with regularly spaced conserved hydrophobic amino acids.

A third player, RAN (Ras-related nuclear protein), determines which direction the transport receptor:cargo complex moves. Ran is a small GTP-binding protein that can bind and hydrolyze GTP to GDP. It is a member of the small G protein family of GTPases.

Ran itself can run (a pun) or move across the nuclear membrane and is found in the cytoplasm and nucleus. What determines which way a Cargo:receptor complex goes? It depends on whether GTP or GDP is bound to RAN! Higher concentrations of RanGTP are found in the nucleus, and lower concentrations in the cytoplasm.

• Importins bind cargo proteins with a NLS in the cytoplasm where RanGTP is low, move into the nucleus, and release the cargo protein when the abundant RanGTP displaces the bound cargo protein.
• Exportins bind both a cargo protein with a NES and Ran:GTP to form a ternary Exportin:Cargo:RanGTP complex in the nucleus. This moves into the cytoplasm, where Ran-bound GTP is hydrolyzed to GDP, causing the complex to dissociate and freeing the exported cargo protein.

Importins are dimeric structures consisting of an alpha and a beta subunit. The alpha subunit binds to the cargo protein via the cargo protein's NLS sequence. The beta subunit interacts with the nucleoporin proteins (Nups) in the

These interactions are shown in Figure \(\PageIndex{21}\).

Figure \(\PageIndex{21}\): Model of nuclear import and export. Cargo containing NLS (Nuclear localization signal) is imported with the help of Importin α and importin β heterodimer. Nuclear export of cargo containing NES (nuclear export signal) is mediated by exportins. Ran GTP is also required during that process. Khan Asmat Ullah, Qu Rongmei, Ouyang Jun, Dai Jingxing. Front. Physiol., 03 April 2020 | https://doi.org/10.3389/fphys.2020.00239. Creative Commons Attribution License (CC BY).

Figure \(\PageIndex{22}\) shows an interactive iCn3D model of the Delpi electrostatic surface potential map of the putative NLS from the carboxy-terminal of a cargo protein, the W protein of the Hendra virus (4M2T), which is imported into the nucleus.

Figure \(\PageIndex{22}\): Delphi electrostatic surface potential map of the putative NLS from the carboxy-terminal of a cargo protein, the W protein of the Hendra virus (6BW9) which is imported into the nucleus (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...mUkDXG1KoiieTA

The virus originated in bats and recently emerged. The peptide sequence containing the NLS is: 419 CLGRRVVQPGMFADYPPTKKARVLLR 444. The red surface indicates negative potential, and the blue surface indicates positive potential, both associated with the Lys and Arg side chains in the consensus sequence. It is bound to the human importin-α3 subunit (molecular surface shown in white), which binds the NLS sequence with high affinity.

An astute reader might ask why GTP remains bound rather than being cleaved to GDP in the nucleus, given Ran's intrinsic GTPase activity. There is yet another protein found only in the cytoplasm (i.e., it lacks a nuclear import signal) that binds Ran:GTP and promotes the GTP → GDP exchange. So oddly, hydrolysis of a nucleotide triphosphate (GTP, not ATP, as in the case of most transporters discussed above) is required for nuclear import. A different but very fascinating mechanism!

The cargo complexes described above must still pass through the nuclear pore. In section 11.3, we discussed the structure of the nuclear pore, which consists of many nucleoporin proteins (NUPs). The inner ring is called the FG Nups layer. The disordered parts of the inner ring Nups have repetitive sequences enriched in phenylalanine and glycine (hence the name FG Nups) and stick out into the central pore. The FG repeats can take multiple forms, including Phe-Gly (FG), Gly-Leu-Phe-Gly (GLFG), or Phe-any-Phe-Gly (FXFG). These are intrinsically disordered proteins, and the disordered regions in the pore act as a filter, allowing certain molecules to pass while excluding those with molecular weights greater than 40 kDa. Large molecules complexed to importins/exportins move through the pore. This disordered mesh prevents the passive diffusion of molecules but allows protein complexes carrying cargo, such as the exportin/importin complex, through. It's a bit like electrophoresis of small protein complexes through the pores of an acrylamide or agarose gel polymerized matrix, only without the "push" of an electric field. Presumably, the FG-Nups make transient hydrophobic (induced-dipole:induced dipole) interactions between the FGs on the Nups and the nuclear transport receptors.

Figure \(\PageIndex{23}\) shows an export complex (5XOJ) from yeast of Ran (cyan with bound GTP in spacefill) bound to an exportin (Xpo1p, white surface) and 3 Nup42p peptides containing SxFG/PxFG repeats (spacefill, side chains, which are mostly nonpolar). To bind 3 Nup peptides, the exportin Xpo1 also contains repeating binding sites on domains called HEAT repeats 14–20 of Xpo1p. The exportin Xpo1p is a toroidal protein with 21 HEAT repeat domains, each consisting of two antiparallel sheets connected by loops of varying sizes.

Figure \(\PageIndex{23}\): Yeast export complex (5XOJ) with Ran (cyan with bound GTP in spacefill) bound to an exportin (Xpo1p, gray surface) and three Nup42p peptides containing SxFG/PxFG repeats (spacefill, side chains))

Some believe the unfolded mesh of FGs in the core might condense upon binding motifs on importins/exportins or through FG:FG domain associations. But what is the basis for importins/exportins interactions with the FG structures? To study it in more detail, Fragasso et al. designed and constructed an artificial FG-Nup that binds to the importin transport receptor Kap95, which interacts with a cargo protein containing a nuclear localization sequence (NLS). They attached them to the inside of solid-state nanopores and demonstrated the rapid movement of Kap95 through the derivatized nanopore, whereas a control "non cargo" lacking an NLS (BSA) was blocked. Underivatized nanometer-sized pores are made in a silicon nitride synthetic membrane (SiN) by using an ion or electron beam to tune the size of the hole.

Figure \(\PageIndex{24}\) (top image) shows colored code structures of the inner ring (top of figure), snapshots of different yeast GLFG NUPs. These are named after their Gly-Leu-Phe-Gly repeating motifs and are especially cohesive. The GLFG-Nups, shown in red, are mostly found in the inner ring compared to FxFG/FG-Nups in green.

Figure \(\PageIndex{24}\): Inner ring (top of figure) snapshots of different yeast GLFG nups. Fragasso, A., de Vries, H.W., Andersson, J., et al. A designer FG-Nup that reconstitutes the selective transport barrier of the nuclear pore complex. Nat Commun 12, 2010 (2021). https://doi.org/10.1038/s41467-021-22293-y. Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/.

The bottom part of Figure \(\PageIndex{24}\) shows snapshots of molecular dynamics simulations of various yeast GLFG-Nups compared to the NupX synthetic one. Three yeast GLFG Nups and the synthetic NupX exhibit compact and extended domains. Each has a collapsed cohesive domain at one end (characterized by a low charge to hydrophobic amino acid residue ratio (C/H ), lots of alternating FG and GLFG repeats, with light green showing non-FG/GLFG/charged residues ) and an extended domain at the other end (high ratio of charged/hydrophobic amino acids, no FG repeats, pink-red showing non-FG/GLFG/charged residues). Bright green shows the FG repeats, red the GLFG repeats, and white the charged groups.

Figure \(\PageIndex{25}\) shows the pore in the silicon nitride synthetic membrane (SiN) membrane before (left) and after derivitization with NupX.

Figure \(\PageIndex{25}\): Pore in the SiN silicon nitride synthetic membrane before (left) and after derivatization with NupX

Figure \(\PageIndex{26}\) shows increasing size derivatized pores in the SiN membrane. In the smallest particle, the NupXs don't fit and are expelled from the pore. In the 30 nm particle, the NupX cohesive domains plug the hole. In the 45 nm pore, a hole appears.

Figure \(\PageIndex{26}\): Derivatized pores in the silicon nitride (SiN) membrane of increasing size

Lastly, Figure \(\PageIndex{27}\) shows snapshots of 30 nm pores lined with NupXs. In panel c, an importin, Kap95 (spheres with orange binding spots), is shown forming a transient interaction with and translocating through the NupX-lined hole. BSA (sphere without binding sites) may interact weakly but does not translocate through the pore.

Figure \(\PageIndex{27}\): Snapshots of 30 nm pores lined with NupXs and interacting with an importin, Kap95 (spheres with orange binding spots, panel C) and BSA (sphere, panel D)

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

This chapter addresses active transport — the movement of solutes from low to high concentration against an electrochemical gradient — which requires an external energy source and underpins fundamental cell biological processes from nutrient capture to neural signaling to gene regulation.

Active transport is first classified along two dimensions. Primary active transport is driven directly by ATP hydrolysis or, in specialized systems, by light absorption (bacteriorhodopsin) or oxidation-reduction reactions (mitochondrial electron transport). Secondary active transport couples the thermodynamically favorable collapse of one ion gradient — most commonly Na⁺ or H⁺ — to the uphill movement of a second solute in the same (symport) or opposite (antiport) direction. Transporters are further described as uniporters (single solute, no co-transport), symporters, or antiporters, and as electrogenic (generating a net charge imbalance) or electroneutral. The Na⁺/K⁺ ATPase illustrates several of these concepts simultaneously: it is a primary ATPase-driven, electrogenic antiporter that expels three Na⁺ while importing two K⁺, consuming 70% of all ATP produced in neuronal tissue to maintain the steep ion gradients essential for action potential generation.

The Major Facilitator Superfamily (MFS) is the largest family of secondary transporters, with ~74 subfamilies that collectively move sugars, amino acids, nucleotides, organic acids, and drugs across membranes. All share a conserved architecture of twelve transmembrane helices organized into two pseudo-symmetrical six-helix bundles related by an internal C2 axis, with the transported ligand binding in the central cavity between the two bundles. Transport operates through an alternating-access ("clamp-and-switch") mechanism in which the protein cycles among inward-open, occluded, and outward-open conformations, ensuring that the solute-binding site is never simultaneously accessible from both sides of the membrane. The human monocarboxylate transporter MCT2 illustrates this in the context of metabolic coupling: it exports lactate and pyruvate from glycolytically active cells driven by proton symport, with a conserved protonated Asp residue (Asp 293) embedded in a hydrophobic microenvironment whose elevated pKa delays proton release until a conformational change reexposes the residue to a more polar environment. Lactose permease from E. coli provides a mechanistically complementary example: protonation of an extracellular His enables lactose binding, and deprotonation following the conformational transition to the inward-open state releases lactose into the cytoplasm, with proton and substrate movements obligatorily coupled.

P-type ATPases constitute the largest family of primary active transporters, with over 160,000 members in prokaryotes and eukaryotes. Their mechanism is described by the Post-Albers model, in which ATP phosphorylates a conserved Asp to form a high-energy mixed anhydride intermediate. This phosphorylation stabilizes the high-affinity inward-facing E1 conformation, allowing cytoplasmic substrate binding; subsequent conformational change to the low-affinity outward-facing E2 state releases the substrate to the other side of the membrane, and dephosphorylation of Asp resets the cycle. The Ca²⁺ ATPase SERCA1, which actively pumps Ca²⁺ from the cytoplasm (0.1-0.2 μM) back into the sarcoplasmic reticulum (~390 μM) following muscle contraction, illustrates the thermodynamic coupling in molecular detail: ATP hydrolysis provides the net downhill free energy that drives the otherwise thermodynamically unfavorable uphill Ca²⁺ movement, with the energy landscape tracking through twelve distinct states and energy dissipation (Qx) accounting for the difference between ATP free energy released and electrochemical potential stored. The Na⁺/K⁺ ATPase follows the same E1/E2 cycle, using Na⁺-activated autophosphorylation and K⁺-activated dephosphorylation to couple ATP hydrolysis to the vectorial antiport of three Na⁺ out and two K⁺ in per cycle. Vanadate inhibits P-type ATPases as a transition-state analog, adopting a trigonal bipyramidal geometry that mimics the pentacoordinate phosphate transition state for both the phosphorylation and dephosphorylation reactions, preferentially stabilizing the E2 phosphatase state.

ABC transporters form the largest membrane protein superfamily, with full transporters containing two transmembrane domains (TMDs, typically six helices each) and two nucleotide-binding domains (NBDs) bearing the signature LSGGQ sequence and Walker A/B motifs. ATP binding at the NBD interface drives dimerization of the two NBDs and the conformational switch from inward-open to outward-open, exposing the substrate-binding site to the opposite side of the membrane; ATP hydrolysis and Pi/ADP release then reset the cycle. P-glycoprotein (ABCB1), the mammalian multidrug resistance transporter, uses an unusually promiscuous substrate-binding site lined with conserved aromatic residues to recognize and efflux a broad spectrum of hydrophobic drugs and lipids from the inner to the outer leaflet, contributing to chemotherapeutic resistance in tumor cells whose ABCB1 gene is amplified or overexpressed.

The chapter closes by returning to the nuclear pore complex (NPC) and examining not its structure — addressed in the preceding chapter — but the remarkably sophisticated mechanism by which it achieves selective macromolecular transport. Cargo proteins destined for the nucleus carry nuclear localization sequences (NLS) enriched in Lys and Arg; nuclear export is signaled by hydrophobic-rich nuclear export sequences (NES). These signals are recognized by karyopherin transport receptors: importins bind NLS-bearing cargo in the cytoplasm, where RanGTP concentration is low, and release cargo in the nucleus when abundant RanGTP displaces the NLS from the importin binding site. Exportins operate in the opposite direction, forming a stable ternary Exportin:Cargo:RanGTP complex in the nucleus that dissociates in the cytoplasm when a cytoplasmic GAP protein stimulates GTP hydrolysis to GDP. The RanGTP gradient is thus the thermodynamic compass that specifies transport directionality, with GTP hydrolysis — rather than ATP — serving as the energy currency. Transit through the NPC is mediated by transient hydrophobic interactions between transport receptors and the FG-repeat sequences of the disordered inner ring FG-Nups, which form a selective cohesive meshwork that excludes molecules above ~40 kDa by passive diffusion while allowing rapid transit of importin/exportin:cargo complexes. Elegant experiments using designer FG-Nups grafted onto synthetic silicon nitride nanopores have reconstituted this selectivity in a minimal system, demonstrating that an importin (Kap95) translocates efficiently through FG-Nup-lined pores while an equivalently sized inert protein (BSA) is excluded — providing direct experimental validation of the FG-Nup hydrophobic selectivity model.