Active Transport and Factors Affecting Facilitated Diffusion

PART 1: ACTIVE TRANSPORT

A. Primary Active Transport

• Na⁺-K⁺ ATPase
• H⁺ ATPase
• H⁺-K⁺ ATPase
• Ca²⁺ ATPase

• A larger alpha (α) subunit (~100,000 molecular weight) - contains 3 Na⁺ binding sites on the intracellular side, 2 K⁺ binding sites on the extracellular side, and ATPase activity
• A smaller beta (β) subunit (~55,000 molecular weight) - anchors the complex in the lipid membrane

1. Three Na⁺ ions bind to the intracellular sites, and two K⁺ ions bind to the extracellular sites
2. ATPase is activated → cleaves one ATP molecule → releases high-energy phosphate
3. This energy causes a conformational change in the protein
4. 3 Na⁺ are extruded outward; 2 K⁺ are transported inward
5. Net result: low intracellular Na⁺, high intracellular K⁺, and a resting membrane potential of about -70 mV

Fig. 4.12 - The Na⁺-K⁺ pump: 3 Na⁺ out, 2 K⁺ in, powered by ATP hydrolysis (Guyton & Hall)

• Maintains Na⁺/K⁺ concentration gradients across cell membranes
• Establishes the negative intracellular electrical potential
• Controls cell volume - prevents osmotic swelling and lysis
• Powers nerve signal transmission (60-70% of energy in nerve cells goes to this pump)

B. Secondary Active Transport

1. Co-transport (Symport)

• High extracellular Na⁺ (maintained by the Na⁺-K⁺ pump) provides the driving force
• A carrier protein has two binding sites on the exterior: one for Na⁺, one for glucose
• When both bind simultaneously, a conformational change occurs transporting both into the cell
• This moves glucose against its concentration gradient using Na⁺'s downhill gradient
• This is critical in the intestinal epithelium and renal tubules for glucose absorption
• A similar mechanism exists for amino acid absorption (at least 5 types of amino acid co-transporters)

2. Counter-transport (Antiport)

• Na⁺ moves inward down its gradient; Ca²⁺ is simultaneously transported outward
• Critically regulates intracellular Ca²⁺ concentration in cardiac and smooth muscle cells

• Na⁺ moves in; H⁺ is extruded outward
• Important in acid-base regulation

Key distinction: In secondary active transport, energy comes indirectly from the Na⁺ gradient established by the primary Na⁺-K⁺ pump. The process is therefore still energy-dependent, just one step removed.

PART 2: FACTORS AFFECTING FACILITATED DIFFUSION

Fig. 4.7 - Facilitated diffusion approaches a maximum rate (V_max) while simple diffusion increases linearly (Guyton & Hall)

Factors That Affect the Rate of Facilitated Diffusion

• The rate of facilitated diffusion is proportional to the concentration difference (Co - Ci) between outside and inside the cell
• Net diffusion ∝ (Co - Ci)
• A larger gradient → faster net transport rate
• However, unlike simple diffusion, this relationship is not linear - it approaches a maximum (V_max) as the carrier becomes saturated

• Each carrier protein can only flip conformation at a finite rate
• When all carriers are occupied by substrate molecules, the maximum transport rate (V_max) is reached - this is called saturation or the transport maximum (Tm)
• Increasing the number of carrier proteins in the membrane increases V_max
• Example: Insulin stimulates the translocation of GLUT4 glucose transporters from intracellular vesicles to the plasma membrane, increasing membrane carrier density and raising V_max for glucose uptake in skeletal muscle, adipose tissue, and heart by 10- to 20-fold

• Each carrier is specific for a particular molecule or group of structurally similar molecules
• For example, GLUT transporters handle glucose, galactose, and fructose (structurally similar monosaccharides) but not other sugars
• Competitive inhibition can occur when two similar substances compete for the same carrier

• Higher temperature increases the kinetic (thermal) energy of molecules
• This increases both the rate of molecular collisions with the carrier and the speed of conformational changes in the carrier protein
• Within physiological range, increased temperature generally increases the rate of facilitated diffusion

• For charged particles, the electrochemical gradient (not just concentration) determines net movement
• An electrical potential difference can either enhance or oppose diffusion of ions through their carriers
• At the Nernst potential, electrical and concentration forces exactly balance, producing no net diffusion

• Increased hydrostatic pressure on one side of the membrane increases the force with which molecules strike the membrane
• This slightly increases the rate of diffusion (including facilitated diffusion) toward the low-pressure side

Summary Table

IMMUNITY: CELL-MEDIATED AND HUMORAL IMMUNITY

PART 1: CELL-MEDIATED IMMUNITY (CMI)

Definition

Origin and Maturation of T Cells

• T cells originate from pluripotent stem cells in the bone marrow
• They migrate to the thymus for maturation and education
• In the thymus, T cells undergo:
  • Positive selection - T cells that can recognise self-MHC are kept
  • Negative selection (clonal deletion) - autoreactive T cells (those that react to self-antigens) are eliminated → this creates self-tolerance and prevents autoimmunity
• Mature T cells leave the thymus and circulate through blood and lymphoid tissues

Antigen Recognition - Role of MHC

• MHC Class I proteins (on all nucleated cells): present endogenous antigens (e.g., viral peptides, tumor antigens) → recognized by CD8+ T cells
• MHC Class II proteins (on APCs only): present exogenous antigens (e.g., extracellular bacteria) → recognized by CD4+ T cells

Fig. 35.8 (Guyton & Hall) - T-helper cells coordinate the entire immune response via lymphokines

Types of T Cells and Their Functions

1. T-Helper Cells (CD4+ / Th cells) - Most Numerous (~75% of T cells)

• IL-2 (Interleukin-2): amplifies the T-helper response (positive feedback); stimulates proliferation of cytotoxic T cells and regulatory T cells
• IFN-γ: activates macrophages to become more efficient killers
• Lymphokines stimulate B-cell growth and differentiation into plasma cells and antibodies
• Lymphokines slow macrophage migration (concentrating them at the site of infection)

HIV/AIDS destroys CD4+ T-helper cells, leaving the immune system almost completely paralyzed - this is why AIDS patients suffer from opportunistic infections.

2. Cytotoxic T Cells (CD8+ / CTLs - Killer Cells)

• Recognize antigen presented on MHC Class I (on infected/tumor cells)
• Directly attack and kill target cells
• Killing mechanism:
  1. CTL binds tightly to the target cell via antigen-specific TCR
  2. Releases perforins - hole-forming proteins that punch channels in the target cell membrane
  3. Releases granzymes (serine proteases) that enter via perforin pores and activate apoptosis
  4. Also activates Fas-FasL pathway → programmed cell death (apoptosis)
  5. The CTL detaches and can kill multiple target cells sequentially
• Targets: Virally infected cells, tumor cells, transplanted foreign cells
• After killing, CTLs can persist in tissues for months

3. Regulatory T Cells (CD4+ Tregs)

• Suppress the activity of cytotoxic T cells and T-helper cells
• Prevent autoimmunity and excessive tissue damage
• Maintain immune tolerance to self-antigens
• Secrete inhibitory cytokines (IL-10, TGF-β)
• Also generated by the thymus to suppress any autoreactive T cells that escape negative selection
• Clinical significance: Tregs may suppress anti-tumor immunity → research into downregulating Tregs in cancer immunotherapy (checkpoint inhibitors); upregulating Tregs in autoimmune diseases

Memory T Cells

• Are preserved in lymphoid tissue throughout the body
• Respond far more rapidly and powerfully on subsequent exposure to the same antigen (secondary response)
• Can last for months to years

Examples of Cell-Mediated Immunity in Action

• Defense against intracellular pathogens (Mycobacterium tuberculosis, Listeria, viruses)
• Killing of virus-infected cells (CTLs recognize viral peptides on MHC I)
• Rejection of organ transplants (CTLs attack foreign MHC molecules)
• Tumor surveillance (CTLs destroy cancer cells)
• Delayed-type hypersensitivity (Type IV) - e.g., tuberculin skin test, contact dermatitis

PART 2: HUMORAL IMMUNITY

Definition

Origin and Maturation of B Cells

• B cells originate and mature in the bone marrow (where they undergo negative selection to eliminate autoreactive B cells)
• Mature naive B cells express surface IgM and IgD as B cell receptors (BCR)
• They circulate and home to the B cell zones (follicles) of secondary lymphoid organs (lymph nodes, spleen)

Antigen Recognition by B Cells

• Membrane immunoglobulin (binds antigen)
• Igα and Igβ signaling proteins (transmit activation signal into the cell)

• Afferent lymphatics (small soluble antigens via conduits)
• Subcapsular sinus macrophages (large antigens)
• Follicular dendritic cells (FDCs) - display antigen for prolonged periods (days to weeks) to sustain B cell activation

B Cell Activation

T-Dependent Responses (Protein Antigens - Most Common)

1. Antigen binds BCR → B cell internalizes and processes antigen → presents peptide fragments on MHC Class II to a CD4+ Th cell
2. CD40L (on T cell) binds CD40 (on B cell) - this costimulatory signal is essential for full B cell activation
3. T helper cytokines (IL-2, IL-4, IL-5, IL-6, IL-21) drive B cell proliferation and differentiation

• Activated B cells move into follicles → form germinal centers
• In germinal centers, two critical processes occur:
  • Affinity maturation: Somatic hypermutation of immunoglobulin genes → B cells with higher-affinity receptors are selected (by FDC-displayed antigen) → progressively higher-affinity antibodies are produced as the response matures
  • Heavy chain isotype (class) switching: Initial IgM response switches to IgG, IgA, or IgE depending on the cytokine environment (e.g., IL-4 → IgE; TGF-β → IgA)

1. B cells differentiate into:
   • Plasma cells - antibody-secreting factories; a single B cell generates up to 5000 plasma cells, each secreting ~2000 antibody molecules per second
   • Memory B cells - long-lived cells that mediate rapid secondary responses

T-Independent Responses (Polysaccharide/Lipopolysaccharide Antigens)

• Some antigens (e.g., bacterial polysaccharides) can activate B cells without T-helper cell help
• Produce mainly IgM antibodies (no class switching)
• Little affinity maturation or memory generated
• Important in defense against encapsulated bacteria (e.g., Streptococcus pneumoniae, Haemophilus influenzae)

Antibodies (Immunoglobulins) - Structure and Classes

• Variable region (Fab): Binds antigen (unique for each antibody specificity)
• Constant region (Fc): Determines antibody class and effector function (complement activation, Fc receptor binding)

Mechanisms by Which Antibodies Eliminate Pathogens

1. Neutralization: Antibodies bind to toxins or viral surface proteins → block their ability to bind host cells (e.g., anti-tetanus toxin IgG)
2. Opsonization: IgG coats bacteria → phagocytes (macrophages, neutrophils) recognize Fc region → enhanced phagocytosis
3. Complement Activation: IgM and IgG activate the classical complement pathway → MAC (membrane attack complex) lyses bacteria; C3b opsonizes; C5a recruits neutrophils
4. Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC): NK cells and macrophages bind Fc portion of IgG on target cells → kill the target cell
5. Agglutination / Precipitation: IgM agglutinates bacteria (clumps them) → easier phagocytosis

Primary vs. Secondary Antibody Response

Comparison: Cell-Mediated vs. Humoral Immunity