Neurochemistry_lecture III (2) (1) (1) (2)

AQP4 is significant for water transport between the brain and systemic circulation at three locations:

1. Perivascular end-feet
2. Subpial glial limitans
3. Ependyma

It provides the interface for this transport, facilitating the movement of water across these barriers.

AQP4 interacts with the α1-catalytic subunit of Na,K-ATPase and the mGluR5 receptor, forming an integrated oligomolecular complex in synaptic units, but does not interact with the astrocyte glutamate transporter GLT-1.

High extracellular K+ depolarizes neurons, and if K+ is not rapidly removed from the extracellular fluid, it can lower the membrane potential of adjacent neurons. During normal activity, [K+]e can elevate by 1-3mmol/l, but during epileptogenesis, it can be three to four times higher.

Glial processes regulate brain water and extracellular potassium concentration ([K+]e). They have higher K+ permeability via Kir and NKCC channels and water permeability via AQP4 compared to most neurons, and they are interconnected by gap junctions, allowing for spatial buffering of K+ efflux from neurons.

Astrocytes facilitate the spatial buffering of K+ by allowing its diffusion toward the vascular bed, which is dependent on the flow of water through AQP4 channels and K+ through Kir4.1/5.1 channels. This process occurs in the CNS parenchyma and subpial glial limitans, with Kir4.1 channels located at perisynaptic membranes and heteromeric 4.1/5.1 channels found at astrocytic endfeet.

GLUT-1 facilitates the diffusion of glucose across the blood-brain barrier and is predominantly expressed in brain endothelial cells and astrocytic endfeet, essential for glucose delivery from blood into the brain.

The SLC2 gene superfamily consists of 12 glucose transporters (GLUT1–12) and one H+-myoinositol cotransporter (GLUT13), all having 12 transmembrane segments with N- and C-termini on the cytoplasmic side and specific N-linked oligosaccharide side chains on extracellular loops.

Astrocytes metabolize glucose to lactate, which can be transferred into neurons via monocarboxylate transporters MCT1 and MCT2, supporting neuronal energy needs.

A hereditable mutation in the GLUT1 gene leads to a limited glucose supply to the brain, resulting in a syndrome characterized by infantile epilepsy, acquired microcephaly, and hypoglycorrhachia.

The brain relies on facilitators for glucose supply rather than active transport, meaning that glucose supply is largely regulated by local blood flow.

GLUT3 occurs mainly in the plasmalemma of neurons and to a lesser extent in nonsynaptic intracellular vesicles.

GLUT2 and glucokinase are part of a glucose sensor system that modulates feeding behavior and regulates glucose uptake by peripheral tissues, with their mRNAs expressed in the hypothalamus.

GLUT8 is expressed in regions such as the hippocampus, amygdala, olfactory cortex, hypothalamus, brainstem, and posterior pituitary nerve endings. It is mainly found in intracellular vesicles and may be translocated to the plasmalemma by unknown regulatory stimuli, indicating an endocytic recycling model of regulation.

Insulin activates the rapid translocation of GLUT4 from an intracellular vesicle compartment to the cell surface, resulting in increased cellular uptake of glucose.

HMIT is an H-coupled myoinositol symporter with high expression in neurons and glia of the hippocampus, hypothalamus, cerebellum, and brainstem. It is involved in the regulation of inositol metabolism, which affects various mood and behavior patterns.

Integral membrane proteins mediate various signal-transduction and active-transport pathways. They can interact with intracellular proteins, extracellular components, and form specific junctions with other cells.

Glycosylated proteins and lipids contribute to the stabilization of interactions at the extracellular surface through hydrogen bonding among glycosyl residues, enhancing cell communication and adhesion.

A monotopic integral membrane protein has a single alpha-helix polypeptide structure that does not fully span the membrane. It features a positively charged amino group (NH3+) extending above the membrane and a negatively charged carboxyl group (COO-) extending below the membrane.

Plasma membranes are distinguished by the presence of glycolipids and glycoproteins on their outer surfaces, as well as the attachment of cytoskeletal proteins to their cytoplasmic surfaces.

A bitopic type I integral membrane protein crosses the membrane once with its positively charged amino group (NH3+) extending below the membrane and a negatively charged carboxyl group (COO-) extending above. In contrast, a bitopic type II protein also crosses the membrane once, but its negatively charged carboxyl group (COO-) extends above the membrane while the positively charged amino group (NH3+) extends below.

A polytopic integral membrane protein is characterized by having multiple alpha-helix polypeptide structures that cross the membrane multiple times, weaving in and out of the lipid bilayer.

The types of proteins associated with the membrane include:

1. Protein associated via protein interaction: A globular protein attached to the membrane surface through interactions with other proteins.
2. Acyl-anchored protein: A protein attached to the membrane via a fatty acyl group, extending into the lipid bilayer.
3. Phospholipid-associated protein: A protein anchored to the membrane via a glycosylphosphatidylinositol (GPI) anchor, linked by a glycosyl residue.

Neural cell adhesion molecules (NCAMs) are cell-surface glycoproteins that can activate fibroblast growth factor receptors when clustered. This activation sequesters a complex of NCAM, BI spectrin, and protein kinase C ẞ2 (PKCẞ2) into rafts, which is implicated in neurite outgrowth.

Spectrin binds to phosphatidylinositol-4,5-bisphosphate through a pleckstrin-homology (PH) domain and also interacts with phosphatidylserine, facilitating membrane associations.

Ca2+ influx initiates protein and membrane associations through various mechanisms, including allosteric regulation of protein binding surfaces. A notable example is the Ca2+-dependent binding of calmodulin to other proteins, and annexins exhibit Ca²+-dependent associations with cell membranes by interacting with phospholipids.

Primary active transporters transduce free energy from ATP hydrolysis into electrochemical energy, which is stored in the transmembrane concentration gradients of ions such as Na+, K+, Ca2+, and protons. This energy is utilized by membrane channel proteins for signaling and by secondary transporters to concentrate other ions and molecules.

Secondary active transporters rely on an ion gradient to transport specific ligands uphill across membranes. They are crucial for various neural functions, including packaging neurotransmitters into vesicles, terminating signals at synapses, and transporting metabolites.

Facilitators are membrane proteins that allow specific molecules to diffuse across membranes, often under regulatory control. Unlike active transporters, facilitators cannot perform 'uphill' transport, meaning they do not move substances against their concentration gradient.

The main types of membrane transport proteins include:

P-type transporters create cation gradients, which are essential for neuronal conductance, electrical signaling, and driving secondary transporters.

P-type transporters generate Ca2+ gradients that are primarily used for regulating intracellular signaling.

The similarities in structures and reaction mechanisms of Na+/K+ and Ca2+ pumps have provided insights into the functioning of the entire class of P-type transporters.

Cu-ATPases transport Cu2+ into liver cells and bile; mutations in these transporters can lead to certain neurological diseases.

VoV₁ pumps are H+ pumps located in Golgi-derived vesicles.

ABC cassettes function in the transport of various large molecules and certain pharmacologic substances.

ATP binds and 2 K+ are released.

The transition occurs when three Na+ bind and the enzyme is reversibly phosphorylated, leading to a conformational change known as the 'power stroke'.

Three Na+ bind to the pump, are phosphorylated, and then released to the extracellular side during the transition from E2-P back to E1.

Two K+ bind to the pump, and the enzyme acylphosphate is hydrolyzed, leading to the occlusion of the K+ ions.

The 'power stroke' is the conformational transition that allows the ionophoric sites to become accessible to the extracellular side, decreasing their affinity for Na+ and facilitating its release.

The active Na,K-ATPase is a heterodimer consisting of:

1. α subunit: Catalytic component containing Na+, K+, and nucleotide-binding sites, aspartyl phosphorylation site, and ouabain-binding site.
2. β subunit: Accessory component that assists in the proper functioning and localization of the α subunit.

Mutations in the human α2 and α3 isoforms of Na,K-ATPase are linked to:

1. Familial hemiplegic migraine type 2 for α2 isoform.
2. Rapid onset dystonia with Parkinsonism for α3 isoform.

Agrin acts as an endogenous antagonist of Na,K-ATPase α3 by inhibiting its function. This inhibition is significant as it plays important roles in:

• Ca2+ homeostasis
• Neuronal activity

Endogenous CTS, such as ouabain and marinobufagenin, when binding at sub-nanomolar ranges, may not significantly inhibit Na,K-ATPase pumping activity but can provoke multiple signaling events, including:

• ERK cascades
• PLC/PKC pathways
• PI3K/Akt signaling
• Mitochondrial production of reactive oxygen species (ROS)

They work together to maintain a low concentration of free cytosolic Ca2+.

The concentration is between 10^-8 and 10^-7 mol/l, which is more than 10,000-fold lower than extracellular free Ca2+.

Most intracellular Ca2+ is stored in the endoplasmic reticulum.

One Ca2+ is transported for each ATP hydrolysed.

PMCAs require activation by binding calmodulin at very low Ca2+ levels, which increases the affinity of the substrate Ca2+ site by 20- to 30-fold.

At least four PMCA genes form a multigene family, and alternative splicing leads to many isoforms that are differently expressed and regulated in various cell types. These isoforms are activated by factors such as acid phospholipids and protein kinases.

SERCA pumps Ca2+ from the cytosol into the endoplasmic reticulum (ER) for storage, helping to maintain calcium homeostasis within the cell.

SERCA-2b is the major isoform expressed in the brain, predominantly found in neurons.

The IP3 receptor (IP3R) releases Ca2+ from the ER when it binds the signal molecule IP3, facilitating calcium signaling within the cell.

The plasmalemma Na/Ca antiporter is crucial for the rapid restoration of low cytoplasmic [Ca2+] levels following plasma membrane depolarizations, working in coordination with other calcium pumps.

Na/Ca exchangers (NCXs) are crucial for rapidly lowering high pulses of cytoplasmic Ca2+, removing cytoplasmic Ca2+ up to 10 times faster than SERCA or PMCA pumps.

The NCX exchange process is electrogenic, exchanging three Na+ ions for one Ca2+ ion.

Three Na/Ca-antiporter isoforms are expressed in the brain: NCX1 and NCX3 are found in neurons, while NCX2 is predominantly found in glia.

Na,K-ATPase a subunits are coordinated with Na/Ca exchangers (NCXs) and calcium pumps, indicating a functional relationship in ion regulation.

Astrocytes, hippocampal neurons, and arterial myocytes express a1 diffusely in their plasmalemma, while a2 in astroglia and a3 in myocytes and neurons display reticular patterns that colocalize with NCX patterns in each cell type.

Cytoplasmic Ca2+ concentration is maintained at less than micromolar levels through:

• Ligand- and voltage-regulated channels that allow Ca2+ entry.
• Na+/Ca2+ antiporter in plasma membranes that exchanges Na+ for Ca2+.
• P-type Ca-ATPases in plasma membranes and endoplasmic reticulum that pump Ca2+ out of the cytoplasm.
• The inwardly directed Na+ gradient maintained by Na/K-ATPases (a2 or a3) which drives the Na+/Ca2+ antiporter.

Mitochondria can participate transiently in calcium homeostasis by taking up or releasing Ca2+ when the capacities of other systems (like the Na+/Ca2+ antiporter and Ca-ATPases) are exceeded. This helps to regulate cytoplasmic Ca2+ levels during periods of high demand.

The release of Ca2+ from the endoplasmic reticulum (ER) is triggered by second messengers such as IP3 or Ca2+ itself, which respond to various receptor systems activated by signaling pathways.

P-type copper transporters are crucial for neural function as they facilitate the transport of Cu2+ ions, which are essential for the synthesis of copper-containing proteins like ceruloplasmin. Deficiencies in these transporters can lead to neurological diseases such as Wilson's and Menke's diseases.

Wilson's disease is caused by a mutation in a gene coding for a copper transporter, primarily expressed in the liver and brain. This mutation leads to impaired Cu2+ transport, resulting in decreased synthesis of ceruloplasmin, which can cause significant neurological symptoms due to copper accumulation in the brain.

Menke's disease is caused by a mutation in a gene that codes for a copper transporter responsible for regulating intestinal Cu2+ absorption. This defect leads to inadequate copper levels in the body, which can result in various neurological issues.

The primary function of the VOV1-ATPase in neuronal cells is to generate the proton-electrochemical gradient that energizes the H+-antiporters, which concentrate neurotransmitters from the cytosol into presynaptic vesicles.

The structure of VOV1-ATPase is similar to that of F0F1-ATP synthases, as both utilize a rotor and stator mechanism to pump protons. However, VOV1 is specifically expressed in Golgi-derived organelles, unlike F0F1, which is found only in mitochondria.

The V-ATPase is composed of two main domains:

1. V1 Domain (cytoplasmic) - Contains eight different subunits (A-H), including three copies of ATP binding subunits A and B.
2. Vo Domain (membrane) - A hexameric ring composed of proteolipid C subunits (5 C and 1 C') and single copies of subunits a and d.

The rotor consists of the hexameric proteolipid C ring and a stalk made of subunits D and F, while the stator consists of the remaining subunits fixed to the membrane via subunit a.

Different isoforms of subunit-a target the V-ATPase to different membranes, allowing for specific localization of the pump within various Golgi-derived organelles, thus influencing its functional role in those locations.

V-ATPase translocates protons from the cytoplasmic interface of the proteolipid hexamer into the lumen of synaptic or Golgi-derived vesicles through the rotation of the hexameric C ring. This rotation is induced by ATP binding and hydrolysis by the three subunits A, which act on subunit D to induce rotation via asymmetric conformational transitions, similar to the mechanism described for FoF₁ ATP synthase.

Aldolase facilitates the formation of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate from fructose 1,6 bisphosphate during glycolysis. It interacts with the a, B, and E subunits of the V-ATPase, and this interaction increases dramatically in the presence of glucose. Disruption of the interaction between aldolase and the B subunit leads to disassembly and loss of V-ATPase function, suggesting that aldolase may act as a 'glucose sensor' signaling the V-ATPase to dissociate, although its activity is not required for this effect.

In the presence of glucose, the V-ATPase complex undergoes reversible dissociation, indicating that glucose affects the organization of the protein complex. The diagram illustrates that the presence of glucose leads to a separation of the protein complex, while its absence promotes the assembly of the complex.

ABCA1 translocates cholesterol and phospholipids outward across the plasma membrane, delivering them to the inner plasmalemma leaflet via vesicular pathways, primarily in astrocytes and developing neurons.

Extracellular apoE binds and interacts with ABCA1 to promote cholesterol and phospholipid efflux from cultured astrocytes, resulting in apoE-stabilized HDL-like particles. ApoE also protects ABCA1 from proteolysis by calpain and increases its expression level.

In the absence of apolipoproteins, ABCA1 is rapidly proteolyzed by calpain, leading to a decrease in its expression and function.

ABCA2 is found in oligodendrocytes and is markedly upregulated during myelinization, indicating its role in the development of myelin sheaths in the nervous system.

MDR proteins can 'flip' amphipathic molecules, including membrane phospholipids and sphingolipids, from the inner to the outer leaflet of plasma membranes and are essential in blood-brain barrier function.

MDR1, or P-glycoprotein, pumps drugs out of cells, and its elevated expression during chemotherapy can decrease the chemosensitivity of cancer cells.

Defects in these genes can lead to conditions such as Adrenoleukodystrophy and Zellweger syndrome.

Secondary transporters utilize energy stored in ion gradients to transport other ions and molecules uphill. They often function as symporters or antiporters linked to Na+ or proton gradients.

SGLT1 (SLC5A1) is expressed in the luminal membranes of brain capillary endothelial cells, suggesting its role in glucose transport from blood into capillary endothelia.

SGLT1 and GLUT1 are both involved in glucose transport from blood into capillary endothelia, while glucose efflux from the endothelia into astrocytes and neurons primarily depends on GLUT1.

The low affinity of GLUT1 for intracellular glucose (Km ~25mmol/l) may necessitate the presence of SGLT1 on the luminal membrane to accumulate sufficiently high endothelial intracellular glucose, ensuring an adequate supply to the astrocytic endfeet.

Sodium symporters mediate neurotransmitter reuptake from synaptic clefts into neurons and glia, utilizing energy from the Na+ gradient across the plasmalemma.

Proton-dependent antiporters concentrate neurotransmitters from neuronal cytoplasm into presynaptic vesicles, utilizing energy from the proton gradient across the vesicle membrane.

Neurotransmitter reuptake depends on cerebral energy metabolism, with required H+- and Na,K-ATPase activities contributing substantially to the increased metabolic rate associated with neuronal activity.

The reuptake of neurotransmitters into the presynaptic cytosol and their storage in cytoplasmic vesicles is accomplished by the tandem actions of sodium symporters in plasmalemma and proton antiporters in the vesicle membranes.

The two distinct subfamilies of NSSs are SLC6 and SLC1.

The SLC6 subfamily includes Na+, Cl--dependent symporters for γ-amino butyric acid (GABAT), glycine (GLYT1,2), norepinephrine (NET), dopamine (DAT), and serotonin (SERT).

In SLC6 transporters, chloride ion diffusion does not supply energy to the system because it is passively distributed across the plasma membrane in most cells.

GABAT1-4 (SLC6A1-4) are secondary symporters that mediate GABA reuptake and are expressed in GABAergic neurons.

GLYT1 is found in glia and neurons, while GLYT2 is mainly expressed in glycinergic neurons.

GLYT1 is found on presynaptic glutamatergic nerve endings, where it interacts with NMDA glutamatergic receptors.

The SLC1 subfamily includes Na+-dependent glutamate symporters.

The SLC6 subfamily is characterized by a number of N-glycosylation sites and several phosphorylation sites.

Biogenic amine transporters are major research targets for psychotherapeutic medicines and addictive substances such as cocaine, methylphenidate, and amphetamine. They are inhibited by various drugs, both therapeutic and addictive.

Glutamate symporters in the brain are coded by five different but closely related genes: SLC1A1, SLC1A2, SLC1A3, SLC1A4, and SLC1A6.

Glutamate symporters can symport one Glu with three Na+ and one H+, and antiport one K+ within each cycle, but they differ in their cellular expression.

The density and distribution of isoforms of glutamate symporters are regulated at transcriptional and post-translational levels, and they have different regulatory interactions and are expressed in different cell types.

Astrocytes recover synaptically released glutamate via the transporters GLT1/EAAT2 (aka SLC1A2) and GLAST/EAAT1 (aka SLC1A3).

Glutamine synthetase in astrocytes catalyzes the conversion of glutamate to glutamine, which is then recycled to neurons via the neuronal membrane Na+-dependent amino acid transporter system.

Excitotoxicity occurs when conditions such as depolarization or anoxia deplete ATP, leading to decreased membrane potential and Na+ and K+ gradients. This causes glutamate symporters like GLAST and GLT-1 to fail or operate in reverse, resulting in a 100- to 1,000-fold increase in extracellular glutamate.

Acetylcholine action is terminated by hydrolysis rather than by transport. Cholinergic neurons recover choline via the high affinity choline transporter CHT-1.

CHT-1 is structurally most similar to the Na+-dependent glucose symporter and is classified within the SLC5 gene family.

Decreased intracellular pH activates Na/H antiporters (NHEs), leading to an efflux of protons at the expense of the Na+ gradient.

The stoichiometry of NHE transport is 1:1, meaning one sodium ion is exchanged for one proton.

An internal pH decrement results in protonation of a cytoplasmic site, which allosterically increases the affinity of the NHE for protons.

Growth factors and hormones can produce transient cytoplasmic alkalinisation, likely through protein kinase phosphorylation of the antiporter, which increases its affinity for cytoplasmic protons.

Anion antiporters in the SLC8 gene family primarily transport bicarbonate.

CNS energy production derives almost entirely from glycolysis, resulting in a rate of metabolic CO2 production nearly equal to the rate of oxygen consumption, approximately 1.5 mmol/l per minute in the adult human brain.

In erythrocytes, the Cl-/HCO3- anion antiporter mediates rapid uptake of HCO3- in exchange for Cl-. In the lungs, it functions in the reverse direction to exchange HCO3-.

The expression of the AE3 isoform in neurons suggests that significant anion exchange occurs across neuronal membranes.

AQP1 facilitates water transport through facilitated diffusion, driven by osmotic gradients. Its water permeability is about 3 billion water molecules per subunit per second and is reversibly inhibited by Hg2+. It exhibits low activation energy and does not involve ionic currents or translocation of other solutes, ions, or protons.