Cellular Transport Mechanisms

• Active transport involves ABC proteins and Solute Carrier proteins (SLCs.)
• Active transport goes against the electrochemical gradient and requires energy.
• Passive transport goes along the electrochemical gradient, like simple diffusion through a channel.
• Key active transport protein categories include: THey are all Priamary tránporters
  • P-type ATPases are a class of ATP-powered transport proteins that are characterized by their ability to undergo phosphorylation (transfer PO43- and conformational change) during the transport process. These transporters play a crucial role in maintaining ion gradients across cellular membranes, as they actively transport ions such as sodium, potassium, calcium, and hydrogen against their respective concentration gradients. P-type ATPases are vital for various physiological processes, including muscle contraction, nerve impulse transmission, and the regulation of cellular osmolarity. The function of P-type ATPases is driven by the hydrolysis of ATP, which provides the necessary energy for the transport mechanism. Their importance in cellular homeostasis and signal transduction highlights their essential role in both plant and animal cells.
  • V-type ATPases: These proton pumps are integral to various cellular processes, particularly in acidifying organelles such as endosomes and lysosomes, and they help maintain pH homeostasis in cells.
  • F-type ATPase: More commonly referred to as ATP synthase, these remarkable enzymes are integral in facilitating energy production through essential biological processes such as cellular respiration and photosynthesis. They are responsible for synthesizing adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi). This synthesis is driven by a proton gradient, which is established across membranes during these metabolic processes. ATP synthase operates through a rotary mechanism, enabling the conversion of the energy stored in the proton motive force into chemical energy, vital for various cellular functions.
  • ABC transporters: This diverse group of membrane proteins, known as ATP-binding cassette transporters, plays a crucial role in the transport of a wide variety of molecules across cellular membranes. They energetically utilize the hydrolysis of adenosine triphosphate (ATP) to facilitate the movement of substrates against their concentration gradients, highlighting their importance in maintaining cellular homeostasis. ABC transporters are vital in various physiological processes, including the absorption of essential nutrients, the elimination of toxins, and the regulation of cellular drug resistance, which can significantly impact the effectiveness of therapeutic interventions in medical treatments.

Channel Proteins

• Channel proteins are integral membrane proteins that allow specific ions or molecules to pass through the cellular membrane. They operate based on concentration gradients, ensuring efficient transport while reducing the need for metabolic energy, thus playing a crucial role in cellular homeostasis.
• Includes:
  • Ion channels
  • Gap junctions

Carrier/Transporter Proteins

• Can facilitate primary or secondary active transport, or passive transport.

P-type ATPases

• Primary active transporters
• Become transiently phosphorylated during their duty cycle
• Examples:
  • Na+/K+-pump
  • Ca2+-ATPase

V-type (vacuolar) ATPases

• Primary active transporters
• DO NOT get transiently phosphorylated
• Responsible for acidification of lysosomes
• Work against H+ gradient.

F-type ATPase: F0F1 ATPases

• Primary active transporters
• DO NOT get transiently phosphorylated.
• Structurally related to V-type ATPases
• Generate ATP in mitochondria
• Use H+ gradient
• Can work in reverse under physiological conditions (but this process can be reversed).

ABC Transporters

• Present in species across all kingdoms, from prokaryotes to animals
• The basic structure includes:
  • 2 ATP-binding domains (NBDs (Nucleotide-binding domains )) for binding and hydrolysis of 2 ATP molecules.
  • 2 transmembrane domains (TMDs) that collectively form the substrate binding sites.
• The ATP binding sites are built from evolutionary conserved amino acid sequences.
• The ATP molecules are collectively bound by the two ATP binding domains.
• There are two categories of ABC transporters, based on the direction of transport:
  • Exporters
  • Importers (permeases)

Exporters

Transport toxic substances (xenobiotics), metabolic products, antibiotics, drugs, and cytostatics out of cells.

• Are founded in both Prokaryotic and Eukaryotic cells
• Plasmodium falciparum uses an ABC transporter (Pfmdr) to cause chloroquine resistance in malaria.

Importers (Permeases)

• Mediate the uptake of amino acids, iron, mono- or polysaccharides, and peptides into bacterial cells.
• Found only in bacteria
• e.g., periplasmic permeases in gram-negative bacteria

Plant ABC Transporters

• Transport hydrophobic coating materials (waxes) and anthocyanins.

Human ABC Proteins

• There are 48 proteins total
• These proteins are divided into 8 subfamilies from ABCA to ABCG
• Include channel-type ABC proteins, channel regulators, and active transporters

Channel-type ABC Proteins

• Cystic fibrosis transmembrane conductance regulator CFTR = ABCC7

Channel regulators

• SUR1=ABCC8 (CFTR = ABCC7 can also have this function)

Active transporters

• Multidrug transporters: Pgp=ABCB1, ABCG2=BCRP, MRP1=ABCC1
• Oligopeptide transporter in the ER: TAP1/TAP2=ABCB2/ABCB3

Structure of CFTR Cl- ion channel

• Channel opening is induced by ATP binding to the NBDs and Protein kinase A (PKA) is an enzyme that, upon activation by cyclic AMP (cAMP), catalyzes the transfer of a phosphate group to specific serine or threonine residues on target proteins, including the regulatory (R) domain of certain proteins. This phosphorylation event causes conformational changes that modulate the activity and function of the protein, often leading to important signaling outcomes in various cellular processes.
• Channel closing caused by ATP hydrolysis
• ATP binding drives and hydrolysis drives channel gating.

CFTR roles and disease

• CFTR is expressed in epithelial cells lining the airways, intestines, and sweat glands
• CFTR involved in the production of airway surface liquid (ASL), digestive fluids, and sweat
• Mutations inactivating the CFTR protein cause cystic fibrosis (CF)
• CF is a recessive genetic disorder
• CF carriers (heterozygotes) are more likely to survive a cholera infection
• CF affects airway function by causing thick, sticky mucus buildup, bacterial infection, and widened airways.

Regulation of Airway Surface Liquid (ASL)

• ASL consists of a lower periciliary layer + upper mucus layer.
• ASL is necessary for efficient mucociliary clearance.
• Many ion-channels and transporters are involved, but CFTR is the key regulator

Types of CFTR Mutations

• Different mutations affect CFTR protein expression/function differently.
• Common mutations include G542X, F508del, and G551D.
• Some mutations affect synthesis, processing, regulation, or conductance

Pharmacological Modulation of CFTR Defects

• Potentiators, correctors, and stabilizers can modulate CFTR mutations.
• Correctors address folding defects
• Potentiators address defective channels
• Stabilizers reduce the speed at which the channel fails

KATP-channel complex

• SUR1, also known as ABCC8, and Kir6.2 are pivotal components that combine to form the KATP-channel complex, which is a hetero-octameric structure. This complex plays a crucial role in regulating insulin secretion from pancreatic beta cells in response to metabolic signals, particularly the levels of glucose. The precise functioning of these channels is essential for maintaining glucose homeostasis within the body, as they help determine when insulin should be released based on energy demands.
• Additionally, several anti-diabetic medications, such as Glibenclamide, Meglitinide, and Tolbutamide, specifically target these KATP channels to enhance insulin release, making them vital tools in the management of type 2 diabetes mellitus.

Regulation of Insulin Secretion

• Increased glucose level in the blood leads to elevated glucose uptake into beta cells
• Elevated glucose uptake increases intracellular ATP/ADP ratio, promoting ATP binding to SUR1/Kir6.2 complex.
• ATP binding causes a conformational change, closing Kir6.2 K+ channels and causing depolarization.
• Depolarization opens voltage-gated Ca2+ channels, increasing intracellular Ca2+ levels and triggering insulin secretion

Mutations in KATP-channel

• Mutations in the KATP-channel complex can lead to metabolic diseases.
• Gain-of-function mutations cause neonatal diabetes.
• Loss-of-function mutations cause (hyperinsulinemic) hypoglycemia

P-glycoprotein

• Also known as (=Pgp, MDR1, ABCB1)
• Is expressed in small intestine, the blood-brain barrier, blood-testis barrier, placenta, tumor cells, and stem cells
• Transports toxic compounds, amphiphilic and lipophilic xenobiotics, and chemotherapeutic drugs
• Resistance against chemically and functionally unrelated drugs happens because cytotoxic drugs do not reach the cell killing dose in tumor cells.
• Resistance is caused by the expression of ABC transporters in the plasma membrane of cancer cells, like: P-glycoprotein (Pgp=MDR1=ABCB1), ABCG2 (=BCRP), and multidrug resistance protein 1 (MRP1=ABCC1)

Multidrug Resistance (MDR)

• Is caused by the expression of ABC transporters (with wide substrate spectrum) in the plasma membrane of cancer cells: P-glycoprotein (Pgp=MDR1=ABCB1), ABCG2 (=BCRP) and multidrug resistance protein 1 (MRP1=ABCC1)
• Chemotherapy can induce the expression of ABC transporters
• Stem cells and tumor stem cells also express ABCB1 and ABCG2

ABCG2

• It is an ABC transporter with a wide substrate spectrum
• Is expressed in tissue barriers and stem cells
• Its endogenous/physiologic substrate is uric acid.
• Decrease of function can lead to an elevation of uric acid level in the blood → increased risk of gout.
• Symptoms of gout: uric acid precipitates and forms deposits (tophi) in joints and in the surrounding tissues that can cause recurrent attacks of acute inflammatory arthritis
• Are transported by ABC transporters
• Bile components like phosphatidylcholine, cholesterol, and bile acids are transported by ABC transporters.
• Detoxification in the liver = conjugation of toxic compounds with glutathione and glucuronide

TAP1/TAP2

• TAP1/TAP2 (ABCB2/ABCB3) is an oligopeptide transporter in the ER membrane
• Degrades cellular and certain viral proteins in the proteasomes to oligopeptides and pumps these into the lumen of ER by TAP1/TAP2 transporter.
• The oligopeptides bind to MHC I molecules in the ER lumen
• The complex is transported to the plasma membrane and presented to cytotoxic T-cells.

Solute Carrier (SLC) Proteins

• Are proteins that transport water-soluble small molecules like inorganic ions, amino acids, oligopeptides, nucleotides, vitamins, hexoses, drugs, and drug metabolites
• ARe proteins with functional diversity that do so through either:
  • Secondary active transporters: symporters or antiporters (that depend on Na+, H+ or Cl- gradients.)
  • Passive transporters: uniporters (e.g., glucose uniporters (GLUT1-GLUT14))
• More than 600 SLCs have been identified
• Exhibiting large structural diversity with different numbers of transmembrane segments
• Localized to plasma membrane or membranes of intracellular organelles
• Over 80 SLC proteins have been linked to metabolic diseases

SLCs and Intestinal Transport

• SLC proteins are relevant in intestinal glucose and amino acid transport
• Directional transport of glucose and amino acids can be achieved by the segregation of transport proteins
• Intestinal cells can couple Na+-glucose symport on the apical surface with Glut2 glucose uniport to establish a transmembrane glucose gradient

Intestinal Uptake

• The majority of coupled (secondary active) transport processes depend on the Na+ gradient, which is built up by the Na+/K+- ATPase
• The Na+/K+-ATPase is responsible for ~30% of total energy consumption in the cell!
• Na+/glucose symporter drives glucose uptake from the gut lumen in the apical part of intestinal epithelial cells
• Glut2 (glucose uniport) transport glucose from the epithelial cells to the blood in the basolateral surface of intestinal epithelial cells (passive transport)
• Increased glucose level in the blood leads to to elevated glucose uptake into the beta cells increased intracellular ATP/ADP ratio →ATP binding to SUR1 conformational change → closure of Kir6.2 K+-channel → depolarization→ elevated intracellular Ca++ level → insulin secretion (typical example for regulated secretion)
• Insulin binding to the insulin receptor (expressed on the surface of muscle cells)→......→Glut4 expressing vesicles fuse with the cell membrane → elevated Glut4 expression →glucose uptake to the muscle cells →decrease of blood glucose level
• Chloride channel in the apical membrane of epithelial cells
• The function is mucus production
• (CF): recurrent airway infections, pneumonia, digestion problems, infertility* Sites of expression: airways, pancreas, intestinal epithelium, sweat glands, reproductive organs
• Treatment: in case of F508 deletion „corrector" molecules support the normal folding of the protein
• diseases where it is relevant is recurrent airway infections, pneumonia, digestion problems, infertility

Explore distinctions between cellular transport mechanisms, including exporters and importers. Investigate the roles of ABC transporters such as Pfmdr in drug resistance and plant physiology. Understand how inhibitors like ouabain affect nutrient uptake in intestinal cells.