• Stages of Cellular Metabolism :

  • Stage 1 = large macromolecules are hydrolyzed into their building blocks.

  • Stage 2 = building blocks are further degraded into a few common metabolites.

  • Stage 3 = small molecular weight metabolites like acetyl-CoA are degraded yielding ATP.

• ATP = High energy carrier

• NADH = electron / proton carrier

• In fermentation / anaerobic environments :

  • ATP is produced by regenerating NAD+ for glycolysis ( lactate dehydrogenase )

• Fibromyalgia = decreased lactate dehydrogenase = increased pyruvate and lactate

• Warburg Effect = tumors shift from aerobic to anaerobic metabolism

• Regulated Glucose Entry :

  • insulin binds to receptor , triggers translocation of GLUT4 into the cell membrane

Regulation of Glycolysis :

• The citric acid cycle indirectly regulates glycolysis through the levels of ATP , NADH , and citrate

• High levels of these molecules signal that the cell has enough energy ,

  • leading to the inhibition of phosphofructokinase ( PFK )

• Cataplerotic TCA Reactions = decrease efficiency

• Anaplerotic TCA Reactions = help beta cells in pancreas sustain TCA cycle

  • also by-products of these reactions help trigger exocytosis of insulin granules

Glucose-stimulated-insulin-release ( two phases ) :

• Acute Phase of Insulin Release = pre-stored insulin granules released

  • glycolysis causes high ATP levels from glycolysis

    • ATP blocks potassium channels

      • cell depolarizes

        • calcium enters via voltage-gated calcium channels

          • causes insulin release from pre-stored insulin granules

          • lasts a few minutes

• Latent Phase of Insulin Release = glucose enters beta cells of pancreas

  • by-products of anapleorotic pathways trigger exocytosis of insulin-containing granules

  • produces delayed but amplified insulin release

Bi-Functional Enzyme ( 6PFK2/FBPase2 ) :

• Fasting :

  • Glucagon ➡️ PKA ➡️ Phosphorylates Bi-Functional Enzyme ➡️ Less Kinase Activity ➡️ Less F-2,6-BP ➡️ Less PFK1 Activation ➡️ Less Glycolysis

• Fed :

  • Insulin ➡️ PI 3-Kinase ➡️ De-phosphorylates Bi-Functional Enzyme ➡️ More Kinase Activity ➡️ More F-2,6-BP ➡️ More PFK1 Activation ➡️ More Glycolysis

• ETC makes 15 times more ATP than glycolysis , but glycolysis is way faster

• Electron transport moves electrons from high-energy carriers ( NADH and FADH₂ ) through the electron transport chain , releasing free energy as they move to lower energy states towards oxygen.

• This free energy is used by protein complexes to actively pump H⁺ ions from the mitochondrial matrix into the intermembrane space, establishing a chemiosmotic gradient.

• The proton gradient stores potential energy , which drives protons back into the matrix through ATP synthase , converting the free energy into ATP synthesis.

   

• NADH: Nicotinamide adenine dinucleotide ( reduced form )

• NAD⁺: Nicotinamide adenine dinucleotide ( oxidized form )

• CoQ: Coenzyme Q ( ubiquinone )

• CoQH₂: Ubiquinol ( reduced form of CoQ )

   

Five Electron Carriers Used :

1. Flavoproteins ( FMN in Complex I , FAD in Complex II ) :

• Accept and donate 2 electrons ( FMN/FMNH₂ , FAD/FADH₂ )

2. Iron-Sulfur ( Fe-S ) Proteins ( in Complexes I, II, and III ) :

• Transfer one electron at a time using iron atoms in Fe-S clusters

3. Ubiquinone ( CoQ ) ( mobile between Complexes I , II , and III ) :

• Transfers both electrons and protons , shuttling electrons between complexes

4. Cytochromes ( in Complexes III , IV , and mobile cytochrome c ) :

• Heme proteins that carry one electron at a time , utilizing iron ( Fe²⁺ / Fe³⁺ ) in heme groups

5. Copper Centers ( CuA and CuB in Complex IV ) :

• Transfer one electron at a time , with copper ions ( Cu⁺ / Cu²⁺ ) facilitating electron transfer and oxygen reduction

NADH ( Complex I entry ) :

• Complex I pumps 4 H⁺

• Complex III pumps 4 H⁺

• Complex IV pumps 2 H⁺

• Total: 10 H⁺ pumped per electron pair from NADH

• Total ATP = $\frac{10}{4}=2.5$$\frac{10}{4} = 2.5$

FADH₂ ( Complex II entry ) :

• Complex II does not pump H⁺ but passes electrons to CoQ

• Complex III pumps 4 H⁺

• Complex IV pumps 2 H⁺

• Total: 6 H⁺ pumped per electron pair from FADH₂

• Total ATP = $\frac{6}{4}=1.5$$\frac{6}{4} = 1.5$

H⁺ Required to Make ATP :

• It is estimated that about 4 H⁺ ions are required to synthesize 1 ATP molecule. This includes:

  • 3 H⁺ ions for ATP synthesis through ATP synthase.

  • 1 H⁺ ion to transport ADP and phosphate ( Pi ) into the mitochondrial matrix for ATP synthesis.

Each full cycle of the ETC allows the production of roughly 2.5 ATP per NADH and 1.5 ATP per FADH₂ due to the difference in proton pumping.

Effect on pH :

• The proton pumping into the intermembrane space causes :

  • a decrease in pH ( more acidic ) in the intermembrane space

  • and a relative increase in pH ( more basic ) in the mitochondrial matrix

• This pH difference , along with the charge difference ( more positive in the intermembrane space ) , creates the proton motive force

Cytochrome C reduction by Cytochrome C Reductase ( complex III )

• involves a unique mechanism known as the Q cycle

  • the Q cycle moves 4 H⁺ ions into the intermembrane space and transfers 2 electrons to reduce 2 cytochrome c molecules , while regenerating ubiquinol.

  1. Ubiquinol (QH₂) Oxidation: One QH₂ molecule donates two electrons. One electron reduces cytochrome c, while the other is transferred to cytochrome b, where it reduces ubiquinone (Q) to semiquinone (Q•⁻).

  2. Proton Release: During QH₂ oxidation, 2 H⁺ ions are released into the intermembrane space.

  3. Second Ubiquinol Reaction: A second QH₂ molecule repeats the process, with one electron reducing another cytochrome c and the other fully reducing semiquinone to regenerate QH₂ at the Qi site.

  4. Proton Release Again: Another 2 H⁺ ions are released, contributing to the proton gradient.

O Reduction to H₂O by Cytochrome C Oxidase (Complex IV) :

1. Electron Transfer from Cytochrome c:

   • Two cytochrome c molecules donate one electron each to Complex IV via CuA and heme a.

   • These electrons are transferred to the heme a₃-CuB center, where atomic oxygen (O) binds.

2. O Binding:

   • One atomic oxygen (O) binds to the heme a₃-CuB catalytic site in Complex IV.

3. Electron and Proton Transfers:

   • The two electrons from cytochrome c begin reducing the atomic oxygen (O).

   • Two protons from the mitochondrial matrix are used to form hydroxyl intermediates (OH⁻) at the active site.

4. Water Formation:

   • To fully reduce the hydroxyl intermediates (OH⁻) to water (H₂O), two additional protons from the mitochondrial matrix are used.

   • As a result, one water molecule (H₂O) is formed and released into the mitochondrial matrix.

5. Proton Pumping:

   • During the reduction of one oxygen atom, two protons from the mitochondrial matrix are pumped into the intermembrane space, contributing to the proton gradient.

• Total ATP from "substrate level phosphorylation" in glycolysis :

  $$\mathrm{Glucose}+2\mathrm{ATP}⟶2\mathrm{Pyruvate}+2\mathrm{NADH}+4\mathrm{ATP}$$
  $$\text{Total}=\text{4 ATP}-\text{2 ATP}=\text{2 ATP}$$

• Total ATP from "substrate level phosphorylation" in TCA Cycle :

  $$\text{2 Pyruvate}\ast \left(\mathrm{Acetyl}-\mathrm{CoA}⟶3\mathrm{NADH}+\mathrm{GTP}+\mathrm{FADH}{\phantom{A}}_2+2\mathrm{CO}{\phantom{A}}_2\right)$$
  $$\text{Total}=\text{2 GTP (ATP)}$$

• Total NADH from 1 Glucose :

  • 2 from Glycolysis directly

  • 2 from converting the 2 Pyruvate into Acetyl-CoA

  • 6 from running the 2 Acetyl-CoA through TCA

  • Total = 10

• Total FADH2 from 1 Glucose :

  • 2 from running the 2 Acetyl-CoA through TCA

  • Total = 2

• Total ATP from NADH and FADH2 generated by previous glycolysis and TCA :

• Combined Total ATP from 1 Glucose :

Coupling :

• Glycolysis , TCA , and ETC are all synchronized by the demand for ATP

• If ATP demand is high , glycolysis produces more NADH , which feeds electrons into the ETC to maintain the proton gradient and ATP production.

• Conversely , if ATP demand is low , the ETC slows down , which reduces the oxidation of NADH , and in turn , slows glycolysis and other metabolic pathways.

• Glycolysis produces NADH by oxidizing glucose to pyruvate.

  • this NADH must be reoxidized back to NAD⁺ for glycolysis to continue producing ATP.

• The ETC is responsible for reoxidizing NADH ( and FADH₂ )

  • this reoxidation of NADH to NAD⁺ allows glycolysis to continue operating.

ATP Synthase Dimerization :

• ATP synthase can dimerize on the inner mitochondrial membrane

• These dimers help to stabilize the curvature of the cristae ( the folds of the inner membrane ) , enhancing the efficiency of proton flow through the enzyme.

• This structural organization can lead to the formation of proton "funnels" that allow more effective use of the proton motive force for ATP production.

• Dimerized ATP synthases can also increase the capacity for ATP production under conditions of high demand, such as in highly active cells.

• By organizing into dimers , ATP synthases enhance the overall efficiency of chemiosmotic coupling, allowing for more ATP to be generated from the proton gradient created by the electron transport chain.

Inner Mitochrondrial Membrane Folds / Cristae :

• Increase the Surface Area

• concentrate the proton gradient

• efficient spacial arrangement of enzymes

  • everything is right there in a pocket

Reactive Oxygen Species ( ROS ) :

• Mitochondrial ROS, particularly superoxide (O₂⁻•), are produced by electron leakage from the electron transport chain (ETC), mainly at Complex I and Complex III.

• Mode 1: High NADH/NAD⁺ ratio with low ATP demand causes superoxide production in Complex I. NADH accumulates, and fully reduced FMN reacts with oxygen, leading to superoxide formation.

• Mode 2: High proton gradient and reduced CoQ pool (CoQH₂) can trigger reverse electron transport (RET), generating large amounts of superoxide. Common in ischemia/reperfusion injury.

• ROS, particularly superoxide, can be converted to hydrogen peroxide (H₂O₂) by superoxide dismutase (SOD).

• Hydrogen peroxide can react via the Fenton reaction to form highly reactive hydroxyl radicals (•OH), which can cause cellular damage.

• Ubisemiquinone (CoQ•⁻), an unstable intermediate in Complex III, can contribute to ROS generation when it interacts with oxygen, forming superoxide.

• ROS production increases in conditions of high mitochondrial membrane potential or low ATP consumption, slowing electron flow and promoting electron leakage.

• Mitochondrial defenses, such as SOD, catalase, and glutathione peroxidase, neutralize superoxide and hydrogen peroxide to prevent excessive ROS buildup and oxidative stress.

• Ubiquinol (CoQH₂) also has antioxidant properties, helping neutralize ROS under normal physiological conditions.

Metabolic Inhibitors :

• Rotenone: Inhibits electron flow at Complex I, increasing ROS production and halting ATP synthesis.

• Cyanide and Carbon Monoxide: Inhibit Complex IV, preventing oxygen reduction and halting ATP production.

• Oligomycin: Inhibits ATP synthase, blocking ATP production by preventing proton flow through the enzyme.

1.) Fatty Acyl-CoA Synthesis ( activation ) :

• investment / activation cost = 2 ATP equivalents

2.) Carnitine - Transport into Mitochondria :

1. Conjugate with Carnitine

   $$\mathrm{Fatty}\mathrm{Acyl}-\mathrm{CoA}+\mathrm{Carnitine}\stackrel{\mathrm{Carnitine}\mathrm{Palmitoyl}\mathrm{Transferase}1(\mathrm{CPT}1)}{\leftrightarrow }\mathrm{Acyl}-\mathrm{Carnitine}$$

   • CPT1 is an integral membrane protein found in the outer membrane of mitochondria

   • CPT1 is the rate-limiting enzyme of β-oxidation and is inhibited by malonyl-CoA

2. Acyl-Carnitine enters mitochondrial matrix using carnitine-acylcarnitine translocase ( CACT )

3. Acyl-Carnitine is converted back into Fatty Acyl-CoA

   $$\mathrm{Acyl}-\mathrm{Carnitine}\stackrel{\mathrm{Carnitine}\mathrm{Palmitoyl}\mathrm{Transferase}2(\mathrm{CPT}2)}{\to }\mathrm{Fatty}\mathrm{Acyl}-\mathrm{CoA}+\mathrm{Carnitine}$$

4. Carnitine recycles back to cytosol

3.) β-Oxidation :

1. Oxidation – Formation of a double bond in acyl-CoA dehydrogenase step.

2. Hydration – Addition of water across the double bond in enoyl-CoA hydratase step.

   $$\mathrm{Trans}-\mathrm{\Delta }{\phantom{A}}^2-\mathrm{Enoyl}-\mathrm{CoA}+\mathrm{H}{\phantom{A}}_2\mathrm{O}\stackrel{\mathrm{Enoyl}-\mathrm{CoA}\mathrm{Hydratase}}{\to }\beta \text{-}\mathrm{Hydroxylacyl}-\mathrm{CoA}$$

3. Oxidation – Conversion of hydroxyl to a keto group in L-3-hydroxyacyl-CoA dehydrogenase step.

4. Thiolysis – Cleavage of the carbon-carbon bond by β-ketothiolase, producing acetyl-CoA and a shortened acyl-CoA.

Net :

Total Acytl-CoA for Even Numbered Fatty Acid :

Total Rounds of β-Oxidation for Even Numbered Fatty Acid :

For Unsaturated Fatty Acids :

• once it reaches the double bond , it has to be isomerized

  • this costs $1{\text{FADH}}_2$$1\ \text{FADH}_2$ molecule

  • therefore , unsaturated fatty acids yield less ATP than saturated

3 Fates of Fatty Acids in Enterocytes :

1. Reformed into TAGS , packaged into chylomicrons , shipped out

2. Act as transcription factors

3. Beta Oxidation

Lipid Transfer in Muscle Cells :

• Absorption Option 1 :

  1. Chylomicrons in the bloodstream are broken down by lipoprotein lipase ( LPL ) into FFAs

  2. FFAs enter muscle cells directly via CD36 receptor

• Absorption Option 2 :

  1. VLDL in the bloodstream are broken down by LPL into FFAs

  2. FFAs enter muscle cells directly via CD36 receptor

• Once Inside :

  1. FFAs enter nucleus and bind to PPARδ

     1.1 PPARδ dimerizes with RXR

     1.2 PPARδ genes are transcribed ( example = PDK4 )

  2. Pyruvate dehydrogenase kinase 4 ( PDK4 ) phosphorylates pyruvate dehydrogenase

     • decreases glycolysis

     • increases fatty acid oxidation

Triglyceride Synthesis :

• Prep :

  1. Glucose enters adipocytes via GLUT-4 ( insulin-dependent )

  2. Dihydroxyacetone phosphate is converted into glycerol-3-phosphate via glycerol-3-phosphate dehydrogenase

• TAG Generation :

  1. $\mathrm{Glycerol}\text{-}3\text{-}\mathrm{phosphate}+\mathrm{FA}-\mathrm{CoA}\stackrel{\mathrm{GPAT}}{\to }1\text{-}\mathrm{acylglycerol}\text{-}3\text{-}\mathrm{phosphate}(\mathrm{lysophosphatidic}\mathrm{acid})$$\ce{Glycerol-3-phosphate + FA-CoA ->[GPAT] 1-acylglycerol-3-phosphate ( lysophosphatidic acid )}$

  2. $1\text{-}\mathrm{acylglycerol}\text{-}3\text{-}\mathrm{phosphate}+\mathrm{FA}-\mathrm{CoA}\stackrel{\mathrm{AGPAT}}{\to }1,2\text{-}\mathrm{diacylglycerol}\text{-}3\text{-}\mathrm{phosphate}(\mathrm{phosphatidic}\mathrm{acid})$$\ce{1-acylglycerol-3-phosphate + FA-CoA ->[AGPAT] 1,2-diacylglycerol-3-phosphate ( phosphatidic acid )}$

  3. $1,2\text{-}\mathrm{diacylglycerol}\text{-}3\text{-}\mathrm{phosphate}+\mathrm{FA}-\mathrm{CoA}\stackrel{\mathrm{PAP}}{\to }1,2\text{-}\mathrm{diacylglycerol}$$\ce{1,2-diacylglycerol-3-phosphate + FA-CoA ->[PAP] 1,2-diacylglycerol}$

  4. $1,2\text{-}\mathrm{diacylglycerol}+\mathrm{FA}-\mathrm{CoA}\stackrel{\mathrm{DGAT}}{\to }\mathrm{Triacylglycerol}$$\ce{1,2-diacylglycerol + FA-CoA ->[DGAT] Triacylglycerol}$

• Rate Limiting Enzyme = step 1 = conversion of glycerol-3-phosphate to 1-acylglycerol-3-phosphate

• How can intermediates impact other cell functions ?

  • 1,2-Diacylglycerol ( DAG ) is an important second messenger ( like cAMP ) that is involved in protein kinase C ( PKC ) signaling

Insulin Resistance and Impact of Plasma FFAs and Glucose on Cardiomyocytes :

• Insulin Resistance:

  • Type 1: Genetic, no insulin production.

  • Type 2: Poor diet leads to insulin desensitization and reduced insulin production by beta cells.

• Plasma Free Fatty Acids (FFAs) and Glucose in Cardiomyocytes:

  • Decreased insulin signals adipocytes to break down fats, increasing FFAs.

  • High FFAs disrupt insulin signaling, especially in muscle and heart cells.

• Mechanism 1:

  1. Increased FFAs boost beta-oxidation, raising NADH and acetyl-CoA.

  2. Excess NADH and acetyl-CoA inhibit glucose metabolism, reducing glucose uptake.

• Mechanism 2:

  1. FFAs increase fatty acid metabolites, activating PKC.

  2. PKC phosphorylates IRS-1/2, impairing insulin signaling and glucose transport.

• Diabetic Cardiomyopathy (DCM):

  • Abnormal heart function linked to insulin resistance, ROS, and metabolic disruptions.

  • FFAs and high glucose levels cause oxidative stress and alter energy metabolism in heart cells, leading to damage.

Describe the lipid bilayer and how it varies between cells and organelles:

• The lipid bilayer is a fundamental component of cellular membranes, composed of two layers of phospholipids. Each phospholipid molecule has a hydrophilic (water-attracting) head and hydrophobic (water-repelling) tails. These properties drive the bilayer to form, with hydrophobic tails facing inward and hydrophilic heads facing outward toward the aqueous environments inside and outside the cell.

• Variations in the lipid bilayer between different cells and organelles arise from differences in the types of lipids present. For example, liver cells have a high concentration of cholesterol to help maintain membrane fluidity, while red blood cells have lipids that help them withstand the mechanical stress they encounter in circulation. Organelle membranes, like the endoplasmic reticulum or mitochondria, also differ in lipid composition to suit their specific functions

What is a lipid raft and why might they not exist?:

• Lipid rafts are specialized microdomains within the plasma membrane that are rich in cholesterol and sphingolipids. These rafts are thought to serve as platforms for organizing signaling molecules, influencing membrane fluidity, and assisting in protein trafficking.

• However, the existence of lipid rafts is debated because they are difficult to observe directly. Some argue that these rafts might not exist as stable structures but could instead be transient assemblies that form and disassemble quickly. The fluid nature of the lipid bilayer and the challenges in isolating these domains experimentally contribute to the skepticism about their existence

How can proteins insert into the lipid bilayer?:

• Proteins can insert into the lipid bilayer through several mechanisms. Some are synthesized with hydrophobic regions that anchor them into the membrane, known as integral membrane proteins. Others attach to the bilayer by interacting with lipid molecules or by covalent attachment to lipids (lipid-anchored proteins). Transmembrane proteins span the bilayer, while peripheral proteins associate loosely with the membrane surface via interactions with integral proteins or the lipid head groups

Why do cell membranes have mechanisms to restrict or allow protein movement?:

• Membranes regulate protein movement to maintain functional organization and facilitate processes such as signal transduction, cell communication, and transport. For example, restricting movement can keep proteins in specific regions of the membrane (like ion channels at synapses). Allowing movement, on the other hand, enables proteins to diffuse and participate in dynamic processes, such as signaling cascades, that require interactions with other membrane components. Mechanisms like cytoskeletal attachments and membrane microdomains (e.g., lipid rafts) play roles in these regulatory processes

• Inside of cell = high potassium

  • $V_{K^{+}}=-90mV$$V_{K^+} = -90\ mV$

• Outside of cell = high sodium

  • $V_{{\text{Na}}^{+}}=+60mV$$V_{\text{Na}^+} = +60\ mV$

• 2 Force Gradients :

  • electrical

  • chemical = mass = concentration

• Lipid bilayer = insulator , separates 2 conductors. extracellular and intracellular

• Asymmetries of ion concentration across lipid bilayer = voltage = membrane potential

Nernst Equation :

• calculates membrane potential $\left(V_m\right)$$\left(\ V_m \ \right)$ for a single ionic species

• The Nernst potential is the voltage that would exist if the membrane were only permeable to one ion

Electrochemical Equation :

• accounts for how an ion behaves under the actual membrane potential , which is influenced by all ions and the currents across the membrane.

So how to find the actual membrane potential $\left(E_m\right)$$\left(\ E_m \ \right)$ ? They use the "Parallel" or "Chord" Conductance Equation :

• its a "weighted average" of all the membrane potentials

• It uses the "permeability" of some ion channel , aka its "conductance" $\left(g\right)$$\left(\ g \ \right)$ , as the weight for each channel.

  • higher permeability = higher rate of ions traveling into or out of cell

• So total averaged membrane potential over all the different types of ion channels in the membrane

• This gives the true "resting membrane potential"

The Driving Force :

• an ion will move down its electrochemical gradient

• it wants to move , in or out of the cell , in such a way that the entire membrane potential $\left(E_m\right)$$\left(\ E_m \ \right)$ becomes closer to that particular ion's equilibrium/Nernst potential

• remember , sodium , if left to itself , balances out at $+60mV$$+60\ mV$

  • potassium , if left to itself , $-90mV$$-90\ mV$

• ok , so if we take a snapshot of the current membrane potential ,

  • and lets say the total $E_m=-50mV$$E_m = -50\ mV$ , meaning the cell is currently hyperpolarized in time ,

  • for sodium , it would love the cell to be at $+60mV$$+60\ mV$

    • so its goin to rush in , to try and bring $E_m$$E_m$ more positive , and closer to $+60mV$$+60\ mV$

  • but for potassium , it wants the cell to be at $-90mV$$-90\ mV$

    • so its going to want to leave the cell , to make the membrane more negative , and closer to $-90mV$$-90\ mV$

Liddle's Syndrome :

hypokalemic ➡️ hyperpolarized cells ➡️ reduced firing rate ➡️ arrhythmia , seizures , muscle spasms

Cholera :

Vibrio cholare produces cholera toxin ➡️ stimulates adenylate cyclase ➡️ increased cAMP ➡️ blocks sodium and chloride transporters ➡️ high concentration of osmoles outside cells , in the gut ➡️ water dumps into gut ➡️ diarrhea ➡️ dehydration

Treatment = high sodium and glucose. The sodium-glucose co-transport system is not affected by cholera toxin. Water follows back into the cells

• Small and lipophilic molecules $(O_2,C{O}_2,N_2,\text{benzene})$$\left(\ O_2\ ,\ CO_2\ ,\ N_2\ ,\ \text{benzene} \ \right)$ are really the only things that can readily cross the plasma membrane readily

• Everything else needs help

  • transporters and ion channels

• 3 Major Classes of Transmembrane Proteins :

  • receptors , ion channels , transport proteins ( ion transporters / carrier mediated transport )

S4 segment :

• a conserved transmembrane region

• consists of a series of positively charged amino acid residues that form a spiral of charges traversing the membrane.

• It is likely that these S4 segments serve as voltage sensors.

  • That is, the positive charges “feel” the membrane potential , and move in response to changes in the membrane potential ,

    • such that channels activate following a depolarization.

Acetylcholine Receptors ( AChR ) :

• hetropentamero

• each of the five subunits winds through the plasma membrane 4 times

• $4\ast 5=20$$4 * 5 = 20$ transmembrane spanning alpha helixes

• AChRs are kinked in the middle

  • when 2 ACTH bind between the residues , the kinks twist in opposite directions around each other

    • this forms a larger whole , opening the channel

Aquaporin:

• Aquaporins are specialized water channels with both hydrophilic and hydrophobic regions.

• Hydrophilic residues in the channel align H₂O molecules in a single file, allowing them to pass.

• Hydrophobic regions repel ions, including H₃O⁺, ensuring only water passes through.

• Asparagine residues in the central part of the channel disrupt proton transfer (Grotthuss mechanism), preventing H₃O⁺ from entering.

Potassium Channel:

• Potassium channels have both hydrophilic and hydrophobic regions that create a selective environment for K⁺ ions.

• The selectivity filter has binding sites optimized for K⁺, allowing it to interact with carbonyl groups in the filter.

• Although Na⁺ is smaller, it cannot interact properly with the filter, making its passage energetically unfavorable.

• This combination ensures only K⁺ is allowed through, maintaining ionic balance.

What are the four main receptor subtypes organized fastest to slowest?

• Ligand-Gated Ion Channels ( ionotropic ) : Fastest response time ( milliseconds ); involved in rapid synaptic signaling.

• G-Protein Coupled Receptors ( GPCRs ) ( metabotropic ) : Slower than ion channels ( seconds ); activate intracellular signaling pathways.

• Enzyme-Coupled Receptors: Slower response ( minutes ); often involve kinase activity leading to phosphorylation cascades.

• Nuclear / Intracellular Receptors: Slowest response ( minutes to hours ); typically involve changes in gene expression by interacting with DNA.

What do we mean by affinity and efficacy?

• Affinity: The ability of a drug to bind to its receptor. High-affinity drugs bind effectively at lower concentrations.

• Efficacy: The ability of a drug-receptor complex to produce a physiological response. A drug can have high affinity but low efficacy.

The dissociation constant $\left(K_d\right)$$\left(\ K_d \ \right)$ :

• determined, representing the concentration at which 50% of receptors are occupied.

• $K_d$$K_d$ is the equilibrium constant. The lower this number , the higher the affinity of a drug for the receptor

• Left shift on velocity vs concentration curve = more affinity

• Right shift on velocity vs concentration curve = less affinity

• Let some binding event / reaction of Agonist ( A ) and Receptor ( R ) :

• $K_m$$K_m$ ( Michaelis constant ) reflects both the rate of the enzymatic reaction and the substrate affinity. It is influenced by how quickly the enzyme-substrate complex forms and breaks down. Thus, it’s not just about affinity but also the catalytic efficiency of the enzyme.

• $K_d$$K_d$ ( dissociation constant ) is strictly about affinity. It only reflects how tightly a ligand binds to its receptor, without involving any reaction rate or further steps beyond binding.

Agonists: binds to a receptor = produces a response

• Example = fentanyl

Antagonists: binds to the same receptor = doesn't produce a response

• Example = narcan

Mechanisms that can inactivate a receptor :

• Receptor Sequestration: Receptors are internalized and temporarily unavailable.

• Receptor Down-Regulation: Receptors are degraded, reducing their numbers on the cell surface.

• Receptor Inactivation: Receptors are modified to become less responsive.

• Inactivation of Signaling Protein: Some third party protein inactivates a secondary messenger protein

• Inhibitory Protein Production: Signaling pathways produce proteins that inhibit receptor activity.

Tolerance and Opioid Addiction Progression

1. Normal State:

   • Baseline: Receptor activity is regulated by endogenous ligands (e.g., endorphins). Receptor numbers are stable.

2. Euphoria (Initial drug use):

   • Increased ligand (opioid) concentration overstimulates opioid receptors, producing euphoria. No tolerance yet.

3. Tolerance:

   • With repeated opioid use, receptor downregulation occurs. More ligand (drug) is required to achieve the same effect due to fewer receptors available.

4. Dependence:

   • The body adapts to high ligand levels, becoming dependent on external opioids. Endogenous ligands no longer maintain normal function, leading to withdrawal symptoms if opioids are stopped.

Post-Abstinence Risk

1. Normal After Abstinence:

   1. After stopping the drug, receptor upregulation restores receptor levels to baseline, but endogenous ligand production is still low.

2. Withdrawal:

   • Without external ligand (opioid), receptors are understimulated, causing withdrawal symptoms due to insufficient receptor activation.

3. Recovery:

   • Over time, the body’s endogenous ligands regain normal function, and receptor activity stabilizes.

4. Overdose (High Risk):

   • If opioid use resumes at prior doses, the upregulated receptors are overstimulated by the same ligand concentration, leading to overdose.

Steroid Family 1: Includes common steroids like glucocorticoids, androgens, estrogens, progesterones, and mineralocorticoids.

• These receptors are cytosolic,

• Upon ligand binding, the receptor forms a homodimer and translocates into the nucleus, initiating gene transcription.

1. steroid crosses cell membrane

2. steroid binds to receptor / heat shock protein complex

3. heat shock proteins dissociate , receptor is activated

4. receptor moves into nucleus

5. receptro binds to DNA as a dimer

6. receptor alters gene transcription , mRNA is made

7. target protein is made , elicits biological response

Steroid Family 2: Includes T3, retinoid acid, RXR, and vitamin D receptors.

• These receptors are nuclear

• After ligand binding, they form a heterodimer (often with RXR) before binding to DNA and initiating gene transcription.

1. steroid crosses cell membrane

2. steroid moves into nucleus

3. steroid binds receptor , activates receptor ( heterodimer )

4. receptor binds to DNA as heterodimer

5. receptor alters gene transcription

6. target protein is made , elicits biological response

Steroid Receptors Contain :

• Zinc finger motifs in the DNA-binding domain.

  • These are essential for binding to hormone response elements (HREs) on the DNA.

    • HRE = nucleotide sequence unique to zinc-finger

      • regulate the rate of transcription

  • zink is used as the coordinating ion

Receptor Tyrosine Kinase Activation :

1. Binding of ligand causes dimerization

2. dimerized receptor phosphorylates tyrosine

   • either auto-phosphorylation or cross-phosphorylation

3. phosphorylated receptor recruits and activates adapter proteins

4. kinase cascade

• MAP kinases are one of the most common pathways involved in kinase signaling, especially in regulating the cell cycle.

• MAP kinases activate CDKs (Cyclin-Dependent Kinases), which in turn regulate cyclins. These cyclins phosphorylate key proteins like RB (Retinoblastoma protein), which regulates whether the cell can progress through the cell cycle. Phosphorylated RB allows progression, driving the cell from G1 into S phase.

CDKs and Cyclins Act on the Cell Cycle :

• They control cell cycle checkpoints, ensuring proper cell cycle progression.

• Misregulation (e.g., through oncogene mutations like KRAS or loss of tumor suppressors like RB) can lead to uncontrolled cell proliferation, a hallmark of cancer.

• Growth Factor ➡️ MAP Kinase ➡️ $\uparrow$$\uparrow$ Cyclin D ➡️ $\uparrow$$\uparrow$ Cyclin D-Cdk4 ➡️ $\uparrow$$\uparrow$ Rb-P ➡️ $\uparrow$$\uparrow$ DNA Replication

Rb = prevents cell cycle progression via inhibition of gene transcription :

1. blocks expression of myc and fos

2. binds to E2F ( needed to go past R-site )

• Phosphorylated Rb allows transition from G1 to S-phase