Exam 2: Ion and lipid orchestration of secondary active transport

Whats the function of small molecule transporters?

• Small molecule transporters move a wide range of solutes (ions to large organic molecules) across membranes.

• Many belong to the solute carrier (SLC) transporter superfamily, which is the second-largest membrane protein group in humans (after G-protein-coupled receptors)

• SLC transporters are crucial for cell homeostasis, as more than a third are linked to inherited diseases.

• They also affect drug pharmacokinetics and serve as drug targets

What are the types of SLC transporters?

• Ion-coupled SLCs (Secondary Active Transporters):

  • Use existing ion gradients (e.g., Na⁺, H⁺) to drive transport.

• Passive SLCs:

  • Rely on diffusion without energy input.

• Primary Active Transporters:

  • Powered mainly by ATP hydrolysis

Why has interest grown for SLCs?

due to precision medicine and successful drug developments, such as SGLT2 inhibitors for type 2 diabetes, which target sodium-coupled glucose transporters in the kidney

How do transporter mechanisms work?

• Transporters act like enzymes, following Michaelis-Menten kinetics.

• They undergo multiple conformational changes to translocate molecules, allowing access to the binding site one side at a time

How do ion channels work?

• ion channels permit rapid diffusion through open pores without complex conformational cycling.

• Understanding transporters has been slower due to their complex nature compared to ion channels and receptors

What is an ion coupling mechanism? List examples

Transporters use Na+ and H+ gradients for substrate movement

• Strict ion coupling: A fixed ion-to-substrate transport ratio.

• Flexible ion coupling: Variable ion use depending on conditions.

• Allosteric ion-mediated regulation: Ions regulate transporter function beyond mere coupling.

What is the role of lipids in transporter functions?

• Lipids influence oligomerization, transport activity regulation, and conformational changes.

• They can switch transporters on and off and modulate structural dynamics

How many domains do secondary active transporters have?

Two

What are the different mechanistic models that secondary active transporters have?

• Rocker-switch proteins:

  • Contain two symmetric domains that rock around the substrate.

  • Transport occurs by alternating outward-facing and inward-facing conformations.

• Rocking-bundle proteins:

  • Structurally asymmetric, with one domain moving more than the other.

  • Alternating access to substrate-binding sites is achieved by a rocking motion.

• Elevator proteins:

  • One domain physically transports the substrate across the membrane, rather than rocking

What is the alternating access model?

where the substrate-binding site is alternately exposed to the extracellular and intracellular environments

During transport, what is an occluded state?

where the substrate is trapped and not accessible to solvent.

How do gates form in secondary active transport?

• Transmembrane helices and loops near the substrate rearrange to form occlusion.

• Many transporters have non-helical breaks in their transmembrane segments, which create flexible regions.

• These non-helical breaks expose backbone carbonyl oxygens, which help coordinate ion binding

What is the structure of modern transporters?

• The originally separate domains have fused into single polypeptides.

• Many transporters display structural repeats, where multiple transmembrane helices are arranged in a parallel or inverted topology.

• Helices that participate in substrate gating often have inverted symmetry, which enables efficient transport

How did secondary active transporters evolve?

• evolved from smaller precursor proteins with fewer transmembrane segments.

• Over time, these proteins oligomerized to form functional transporter complexes

What do secondary active transporters rely on for movement of molecules?

ion gradients and membrane potentials following the Nernst equation

What electro types can transport reaction be? Define both

• Electrogenic: If net charge moves across the membrane, both concentration gradients and electrical potential drive transport.

• Electroneutral: If no net charge is moved, transport is driven only by concentration gradients

What are the types of coupled ion transport?

• Symport: Coupled ions move in the same direction as the substrate.

• Antiport: Coupled ions move in the opposite direction as the substrate.

• Uniport: Passive transport of a substrate driven solely by its own electrochemical gradient

What are the driving ions?

• H⁺ (protons) and Na⁺ (sodium ions) are the most commonly used to power secondary transport due to their:

  • Inward-directed concentration gradients.

  • Favorable negative-inside membrane potential.

• K⁺ (potassium) and Cl⁻ (chloride) are rarely used as driving ions, since their electrochemical gradients are typically closer to equilibrium across the membrane

How do cell maintain ion gradients?

• Constitutively open ion channels (for passive ion flow).

• Primary active transporters (which use ATP to establish ion gradients)

Describe the deep-sea vent origin theory

• Natural H⁺ gradients in deep-sea hydrothermal vents may have provided the energy for early cellular life (~3.8 billion years ago).

• Early cell membranes were made of fatty acids, which were leaky to H⁺, leading to the need for a more stable energy system.

• Na⁺/H⁺ antiporters likely evolved as an early mechanism to convert proton-motive force (PMF) into sodium-motive force (SMF), which is more stable in fatty acid membranes

What are the prokaryotic transport strategies?

• Organisms in high-salinity or alkaline environments favor SMF.

• Organisms in low-Na⁺ environments rely on PMF

What are the eukaryotic transport strategies?

• Fungi & plants use PMF exclusively.

• Animal cells use both PMF and SMF, depending on tissue type and function

Describe the ion coupling in transport mechanisms of rocker-switch proteins

• Most use H⁺ coupling, where proton binding triggers gating helix rearrangement for substrate occlusion and release.

• Some rocker-switch transporters, like MATE (Multidrug and Toxic Compound Extrusion) transporters, use Na⁺ instead of H⁺ or have promiscuous ion-binding sites that can accommodate either ion

Describe the ion coupling in transport mechanisms of rocking-bundle and elevator transporters

• These transporters exhibit complex ion- and substrate-gated rearrangements.

• They often feature highly specific ion-binding sites, ensuring tight regulation of transport.

• Structural asymmetry is a defining feature:

  • Labile domain (moves during transport):

    • Called the "bundle" in rocking-bundle proteins.

    • Called the "transport domain" in elevator proteins.

  • Stable scaffold domain (remains stationary):

    • Provides structural integrity to the transporter

What are the advantages of bundle and transport domains?

• More intricate ion coupling mechanisms.

• A greater range of transported substrates.

• More precise regulation of transport activity compared to the simpler symmetric rocker-switch proteins

Why is it important to understand the complexity of secondary active transporters for drug development?

could enhance drug development for various diseases

What provides necessary energy for transport?

Ion gradients (PMF & SMF) with H+ and Na+

Explain H+ transport and the ionizable residues used. Describe how each residue effects pKa

• H⁺ transport requires ionizable residues that can bind and release protons at physiological pH.

• Histidine (pKa ~6.0) is naturally suited for H⁺ transport, but other charged residues (glutamate, aspartate, lysine) can have significantly shifted pKa values when buried in the protein interior.

  • Buried lysine: pKa can shift from 10.4 to 5.3.

  • Buried glutamate: pKa can increase from 4.1 to 7-8.5, making it viable for proton transport.

  • Arginine is unsuitable for H⁺ coupling due to its consistently high pKa

Explain Na+ transport and the ionizable residues used. Describe how each residue effects pKa

• Na⁺ ions are usually coordinated by electronegative oxygen atoms from the side chains of aspartate, glutamate, serine, threonine, asparagine, glutamine, and tyrosine, as well as methionine sulfur and carbonyl oxygens.

• Ionizable residues in transmembrane helices are essential for ion transport, as their placement in the hydrophobic membrane is energetically costly, implying functional necessity

What does symport require before happening. What does H+ sympoters require?

• Symport requires strict coupling of ion and substrate movement to prevent ion leakage.

• In H⁺-coupled symporters, protonation is often required before substrate binding can occur

Where has H+-substrate interactions found?

in plant sucrose transporter SUC1

what occurs in H⁺-coupled oligopeptide transporters?

protonation of an acidic residue is necessary for substrate binding and uptake

What are local electrostatic network reorganization?

• Salt bridge formation and breakage upon ion and substrate binding.

• Protonation of an extracellular histidine, which facilitates proton transfer but is not itself transported

What is LacY (H⁺-Sugar Symporter, Rocker-Switch Model)?

• H+ coupled symporters

• E325 (Glutamate) is the main H⁺ binding site but does not directly coordinate sugar.

• Instead, it positions H322 (Histidine), which binds the sugar

What is XylE (H⁺-Sugar Symporter, GLUT1 Homologue)?

• H+ coupled symporter

• The proton carrier (D27) is 10 Å away from the substrate-binding site.

• Protonation of D27 disrupts a salt bridge with arginine, allowing movement of TM1, which in turn interacts with TM7b to close the substrate-binding cavity

What is DgoT (Galactonate Transporter, VGLUT Homologue)?

• H+ coupled symporters

• Uses a protonation-dependent salt bridge to release a substrate-coordinating arginine

What is Excitatory Amino Acid Transporters (EAATs, Elevator Model)?

• H+ coupled symporter

• TM7 glutamate protonation releases and repositions an arginine residue in TM8 for substrate coordination.

• Protonated glutamate also stabilizes the gate, promoting substrate occlusion and transport

Unlike H+ symporters what interactions do Na+ coupled transporters rely on?

Allosteric interactions

Describe how Archaeal Glutamate Transporters (GltPh & GltTk, Aspartate-Na⁺ Symporters) work

• Three Na⁺ ions bind cooperatively with aspartate, increasing substrate affinity.

• Na⁺ concentration-dependent affinity ensures high substrate binding in the outward-facing state and low affinity in the inward-facing state.

• Prevents ion leakage by favoring Na⁺–substrate co-transport

What is LeuT (Neurotransmitter: Na⁺ Symporter, Rocking-Bundle Model?

• Na+ couple symport

• Na⁺ binds before the substrate at the Na1 site.

• Substrate and Na2 must be bound together to close the transporter gate and allow translocation.

What is SGLTs (Sodium-Glucose Transporters, Rocking-Bundle Model)?

• Na+ coupled symporter

• Na⁺ binding allosterically triggers glucose binding and occlusion

What is NDCBE (Sodium-Driven Cl⁻/Bicarbonate Exchanger)?

• Na+ coupled symporter

• Substrates coordinate Na⁺ ions together with transporter side chains

What is MelB (Rocker-Switch Model)?

• Na+ coupled symporter

• Na⁺-bound outward-facing state remains open until the substrate binds, triggering occlusion and transpor

What is SERT (Serotonin Transporter)?

• Na+ Coupled Symporter

• Serotonin binds independently of Na⁺, but full transport requires two Na⁺ ions and Cl⁻, catalyzing the transition to the inward-facing conformation.

What is pH-Independent Transport in LacY?

• H⁺-Sugar Symporter

• The glutamate proton carrier has a high pKa (~10.5), so it is already protonated in the outward-facing state.

• Lactose binding catalyzes the conformational transition, allowing translocation without requiring pH-dependent protonation

What is Tripartite ATP-Independent Periplasmic (TRAP) Transporters?

• Distinct from other secondary transporters because they require a periplasmic (P-subunit) to bind the substrate.

• Mechanistically similar to elevator Na⁺-dicarboxylate symporters but cannot bind substrates directly.

• The P-subunit binds substrates with nanomolar affinity, ensuring efficient uptake from dilute environments.

• It is unclear whether the P-subunit triggers the transporter transition or merely assists in substrate transfer

Whats the difference between direct interactions and allosteric interactions?

• Direct interactions: Proton/substrate binding at the same site.

• Allosteric interactions: Ion binding alters electrostatic networks that facilitate substrate binding and occlusion

How does salt bridges, ion-binding residues, and protonation effect structural reorganization/transport efficiency?

• Salt bridge formation and breakage regulate substrate affinity.

• Ion-binding residues control transporter gating.

• Protonation events trigger conformational transitions

What does high affinity binding ensure?

outward-facing state ensures directional transport

What does leakage prevention mechanisms ensure?

substrates are transported only when coupled with their respective ions

In symporters, when is the transport cycle completed?

• when the transporter resets to its outward-facing state after releasing its ions and substrate.

• This resetting occurs spontaneously, allowing the transporter to be ready for another cycle of substrate and ion uptake

How is antiporters different then symporters? What does this ensure

• antiporters must bind a counter-transported ion or substrate to reset to their outward-facing state.

• This requirement ensures strict coupling, preventing uncoupled transport and maintaining ion homeostasis

Describe the mechanisms of Na+/H+ antiporters

• The ions bind at the same sites, typically coordinated by a conserved aspartate residue.

• The transport follows a competitive equilibrium model, often described as a ‘ping-pong’ mechanism

Describe the mechanisms for elevator-type antiporters

a conserved aspartate in the transport domain faces the hydrophobic scaffold domain, preventing conformational changes unless an ion (H⁺ or Na⁺) binds

Give an example of H+-coupled multidrug exporters and how it works

E. coli MdfA, where:

• Protonation of TM1 aspartate (D34) drives the outward-to-inward transition via rearrangement of flexible helices.

• Drug binding occurs at the deprotonated D34 site, facilitating H⁺ release and transport.

• A similar mechanism is observed in EmrE, another H⁺-drug antiporter

How has some Na+-coupled symporters evolved?

require K⁺ counter-transport for resetting, enhancing transport efficiency

How do human EAATs (Excitatory Amino Acid Transporters) work? How does archaeal homologues differ?

• K⁺ binding is required for resetting the transporter to the outward-facing state.

• EAATs symport three Na⁺ ions, one proton (H⁺), and counter-transport one K⁺ ion.

• The K⁺ ion binds in the glutamate binding site, using shared residues that also coordinate Na⁺ and the substrate.

• K⁺ binding closes the inner gate and facilitates elevator transitions of the transporter

• By contrast, archaeal homologues do not require K⁺ for resetting, as their gates can spontaneously shut

In systems like the serotonin transporter (SERT), dopamine transporter (DAT), and the bacterial LeuT, K⁺ counter-transport:

• Reduces Na⁺ and substrate affinity competitively, characteristic of a ping-pong antiport mechanism.

• Increases transport rate and enhances concentration gradients

REMINDERS

• Symporters reset spontaneously, completing the transport cycle after ion and substrate release.

• Antiporters require counter-transported ions or substrates to reset, ensuring tight coupling.

• K⁺ counter-transport enhances transport capacity in Na⁺-coupled symporters.

• Some antiporters deviate from the ping-pong mechanism, such as CLC H⁺–2Cl⁻ exchangers, which use simultaneous ion binding for transpor

How do plasma membranes very among mammals?

• Lipid headgroups

• Acyl chain length

• Degree of unsaturation between the inner and outer leaflets

what maintains membrane asymmetry and what disrupts it?

• Maintaining this asymmetry requires energy-dependent transport systems

• Lipid scramblases can disrupt this asymmetry(blood coagulation, immune cell activation, phagocytosis, apoptosis

how do membrane porteins differ in their interactions with lipids?

• Bilayer bulk properties (e.g., membrane thickness)

• Annular lipids (forming a belt around proteins)

• Specific lipid-protein interactions

How do some proteins adapt to thickness of resident membranes?

• Endoplasmic reticulum (ER) and Golgi proteins have shorter transmembrane helices, suited to thinner organellar membranes.

• Plasma membrane proteins have longer transmembrane helices to match thicker membranes

How do some membrane proteins exhibit bell-shaped activity dependence on membrane thickness?

MelB transporter has optimal melibiose uptake when the bilayer thickness matches its transmembrane helix length.

When does hydrophobic mismatch occur?

• Hydrophobic mismatch occurs when a protein’s transmembrane helix length and the membrane thickness differ.

What are some possible effect hydrophobic mismatch?

• Membrane deformation

• Increased energetic costs

• Changes in protein folding and oligomerization

Why do you want to minimize the costs of hydrophobic mismatch?

helps stabilize membrane proteins and shape the energy landscape of their transport cycle

What are annular lipids

surround membrane proteins, influence their actions through transient interactions

What are nonannular lipids?

Can form specific interactions by forming tight interactions with proteins, further regulate proteins

What proved that protein-proximal lipids differ from bulk lipids?

stryene maleic acid (SMA)

REMINDER

• Membrane lipid composition varies widely, influencing protein structure and function.

• Membrane asymmetry is actively maintained and plays a crucial role in cellular signaling.

• Protein adaptation to membrane thickness is essential for function, with mismatches leading to energetic penalties.

• Lipid-protein interactions shape membrane protein behavior, with annular and non-annular lipids playing key roles

How long are typical scaffold domain helices in elevator transporters? Give an example

• short or highly tilted

• In Na⁺/H⁺ exchangers, scaffold transmembrane helices 2 and 9 are only 12–14 residues long

What is the effect of the hydrophobic mismatch energetic cost?

promotes oligomerization to reduce the protein-lipid interface

Describe the membrane characteristics around the scaffold domain

membrane bilayer thinning around the scaffold domain of NapA (Na⁺/H⁺ exchanger) from Thermus thermophilus

Give an example of how lipid binding can stabilize elevator oligomers

• NhaA (Na⁺/H⁺ antiporter) forms dimers stabilized by cardiolipin, which bridges cationic amino acids at the dimer interface

• Cardiolipin synthesis increases under salt stress in E. coli, which enhances NhaA dimer assembly to help regulate ion homeostasis

Give three examples of how oligermization is lipid-indpedent

• NapA has a large (~1,800 Ų) dimerization interface, supported by an additional N-terminal helix, making it less reliant on lipids for dimerization.

• NHA2 (mammalian Na⁺/H⁺ exchanger) has one more N-terminal helix than NapA, which contributes to most oligomerization contacts.

• Delipidated NHA2 protomers exhibit a ~20 Å gap, which closes in the presence of phosphatidylinositol lipids, suggesting lipid-dependent dimer stabilization

What kind of domain does NHE9 (late endosomal Na⁺/H⁺ exchanger) have and what are the characteristics

β-hairpin lipid domain that:

• Contributes to oligomerization

• Binds phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P₂), which is specific to late endosomes

Why is lipi coordination crucial for NHE9 (late endosomal Na⁺/H⁺ exchanger) function?

• Mutation of lipid-coordinating lysines causes NHE9 to monomerize.

• PtdIns(3,5)P₂ (but not plasma membrane PtdIns(4,5)P₂) increases the Na⁺ affinity of NHE9, demonstrating lipid-specific regulation.

What are some examples of liipid dependent oligomerization?

• Purine transporter UapA requires phosphatidylinositol and phosphatidylethanolamine for dimerization and activity.

• Mutating lipid-coordinating residues results in an inactive monomeric UapA

What makes rocker-switch and rocking-bundle transporters different. Give an example

Rocker-switch and rocking-bundle transporters:

• Do not have anomalously short transmembrane helices but still form lipid-dependent oligomers.

• Example: LeuT, which functions as a monomer but can form cardiolipin-mediated dimers

Describe the structure of Mammalian neurotransmitter sodium symporter (NSS) family

Forms dimers and multimeric species stabilized by phosphatidylinositol bisphosphate lipids

Describe the structure of APC superfamily cation-chloride cotransporters

• Form obligate dimers through:

  • Extensive cytosolic domain interactions

  • A lipid-mediated transmembrane interface

• Their two protomers tilt relative to each other, distorting the bilayer, suggesting membrane modulation of transporter assembly or activity

REMINDER

• Hydrophobic mismatch in elevator transporters promotes oligomerization to reduce energetic costs.

• Specific lipids (e.g., cardiolipin, phosphatidylinositols) stabilize transporter oligomers, regulating their function.

• Organelle-specific lipids modulate transporter stability and ion affinity, fine-tuning their activity in different environments.

• Oligomerization mechanisms extend beyond elevator transporters, with lipid-dependent interactions observed in LeuT, NSS, and APC superfamily members

How does hydrophobic mismatch vary throughout the transport cycle? Give an example

• influencing membrane deformation and energy costs.

• Example: Glt_Ph and Glt_Tk transporters exhibit significant membrane deformations in their inward-facing states.

What bulk lipid properties effect state energies and transport kinetics?

Bulk lipid properties such as thickness and elasticity affect state energies and transport kinetics

What connections do lipid interactions often mediate?

catalytic and regulatory regions of transporters

What is BetP (bacterial betaine–sodium symporter)?

lipid-mediated regulation in response to osmotic stress

Describe the structure of BetP (bacterial betaine–sodium symporter)

• Anionic lipids (phosphatidylglycerol and cardiolipin) bind at the trimer interface.

• The C-terminal regulatory domain interacts with the bilayer, stabilizing a down-regulated state with high substrate affinity and reduced dynamics.

• Osmotic stress (via cytoplasmic K⁺ increase and membrane packing changes) causes partial unfolding of the C-terminal domain, disrupting the lipid and interprotomer interactions, and activating the transporter.

• Monomeric mutants of BetP remain functional but lose their osmotic stress response

What is ClC-7 (lysosomal Cl⁻/H⁺ exchanger) inhibited by?

ClC-7 (lysosomal Cl⁻/H⁺ exchanger) is inhibited by PtdIns(3,5)P₂, which likely stabilizes interactions between its regulatory CBS (cystathionine-β-synthase) domain and transmembrane transporter domain, limiting conformational flexibility

What is NHE1 (plasma membrane Na⁺/H⁺ exchanger) activaed by

activated by PtdIns(4,5)P₂, which binds to its C-terminal regulatory tail, facilitating its detachment and activation

What is SPNS2 (sphingosine-1-phosphate transporter, MFS fold) activated by?

PtdIns(4,5)P₂, which binds to an intrinsically disordered regulatory region, altering transporter dynamics

What role does cholesterol play?

• Binds and stabilizes transporters

• Modulates activity

• Ensures localization to membrane microdomains

Describe SERT and DAT (serotonin and dopamine transporters) relationship with cholesterol

share a conserved cholesterol-binding site in bundle domains.

• Cholesterol stabilizes the outward-facing state, activates transport, and modulates antagonist binding affinity

What is phosphatidylethanolamine?

a zwitterionic lipid, influences ion-coupling mechanisms:

• Competes with salt bridges, modulating conformational state energies

How does LacY (lactose permease) relate to Phosphatidylethanolamine (PE)?

• PE facilitates deprotonation of E325 after sugar release into the cytoplasm.

• PE is essential for H⁺-coupled sugar uptake but not for uncoupled counterflow (sugar exchange)

How does MeIB (melibiose transporter) relates to Phosphatidylethanolamine (PE)?

• Na⁺- and H⁺-coupled melibiose uptake is reduced in an E. coli strain lacking PE.

• In contrast, only H⁺ symport is affected in strains lacking phosphatidylglycerol and cardiolipin

What is Phosphatidylserine (PS)?

enriched in the inner leaflet of the plasma membrane, can facilitate intra-bundle salt bridge disruption:

• Regulates rocker-switch GLUT glucose transporters.

• Essential for the activity of Lyp1 (rocking-bundle H⁺-coupled lysine transporter)

What is the effect of unsaturated lipids in membranes and transporters?

Unsaturated lipids increase membrane fluidity, which also enhances the function of GLUT and Lyp1 transporters

REMINDER

• Hydrophobic mismatch influences state-dependent membrane deformation and transport cycle energetics.

• Direct lipid-mediated regulation is less studied in transporters than in ion channels but plays crucial roles in specific cases.

• Lipid interactions can stabilize regulatory regions, affecting transporter activity (e.g., BetP, ClC-7, NHE1, SPNS2).

• Cholesterol binds to and stabilizes transporters, influencing conformational states and drug binding.

• Phosphatidylethanolamine (PE) and phosphatidylserine (PS) play essential roles in ion-coupled transport and rocker-switch transporter regulation.

• Unsaturated lipids improve membrane fluidity, enhancing transporter function