Basic Receptor Functions

Definition of Receptor

The term "Receptor" is used across various scientific fields:

• Sensory Physiology: An end organ or group of end organs of sensory or afferent neurons, specialized for sensitivity to stimulation like touch or heat.
• Cell Biology: Specific protein molecules in cell surface membranes or organelles that bind complementary molecules like hormones, neurotransmitters, antigens, or antibodies. These interactions initiate a cascade of events leading to a biological response.
• Pharmacology: Cellular macromolecules specifically and directly involved in chemical signaling between and within cells. Receptors mediate the effects of drugs by binding to them and initiating a response.
• Chemistry: A molecule or polymeric structure within or on a cell that specifically recognizes and binds a molecular messenger. The binding is highly specific, like a lock and key.
• Medicine: A molecule on the cell surface or within a cell (usually the nucleus) that recognizes and binds specific molecules, producing a cellular effect. These effects can range from changes in gene expression to alterations in cell metabolism.

Basic Concepts of Receptor Function and Signaling

• Humoral Signaling Steps:

1. Ligand (agonist) binds to receptor.
2. Ligand activates the receptor.
3. Each receptor activates multiple transducers (complex signaling: amplification, divergence, convergence, cross talk). This allows for a coordinated and nuanced cellular response.
4. Active signaling molecules modulate cell functions.
5. Negative feedback reduces sensitivity. This prevents overstimulation and maintains homeostasis.

• Classification of Signaling:
  • Intracrine: A signaling molecule acts inside the cell that produces it.
  • Autocrine: A cell secretes a hormone or chemical messenger that binds to autocrine receptors on the same cell, leading to changes in the cell.
  • Paracrine: A cell produces a signal to induce changes in nearby cells, altering the behavior or differentiation of those cells.
  • Juxtacrine: Signaling occurs through direct physical contact between cells.
  • Endocrine: Hormones or chemical messengers are released into the bloodstream and act on target cells at a distance.

Functional Classification of Receptors

• Membrane Bound:
  • Metabotropic: G-protein coupled receptors that use second messengers like cAMP, IP3, and DAG to initiate a cellular response. These receptors are involved in a wide range of physiological processes.
  • Ionotropic: Ligand-gated ion channels selective for ions like Na^+, Ca^{2+}. The binding of a ligand to these channels causes them to open, allowing ions to flow across the cell membrane.
• Nuclear/Cytoplasmic:
  • Intracellular receptors: Steroid hormone receptors that, upon ligand binding, translocate to the nucleus to influence gene transcription. These receptors play a critical role in regulating gene expression.

Illustration: Shows different receptors with different signaling molecules.

Major Receptor Types

• G-protein coupled receptors: Activation of G proteins. These receptors are the largest and most diverse family of membrane receptors in eukaryotes.
• Ion-channel receptors: Ions flow through ion channels. These receptors are involved in rapid signal transmission.
• Receptors with intrinsic enzyme activity: Tyrosine kinase receptors phosphorylate proteins. These receptors are involved in cell growth, differentiation, and survival.
• Intracellular receptors: Hormone-receptor complex affects DNA transcription. These receptors are located in the cytoplasm or nucleus.

Different Signalization of Receptors

• Agonist-Receptor Interactions:

1. Activation of conductance
2. G-Protein Activation and Generation of Second Messenger
3. Phosphorylation of Tyrosines on Key Signaling Molecules
4. Transport to the Nucleus and Activation of Transcription and Translation
5. Activation of Cell Signaling

G-protein Coupled Receptors Modulate Multiple Second Messenger Systems

• Stimulatory (M1, M3, M5):
  • Coupled to PLC (Phospholipase C). Produces Ins(1,4,5)P3 (IP_3), leading to Ca^{2+} release. This pathway is involved in smooth muscle contraction and glandular secretion.
• Inhibitory (M2, M4):
  • Coupled to AC (Adenylyl cyclase). Decreases cAMP production. This pathway is involved in reducing heart rate and inhibiting neurotransmitter release.
• Receptor, G-protein, Effector protein and Target action listed in figure with neurotransmitters.

Crosstalk Between Signaling Pathways

• Convergence: different receptors (Rx, Ry) activate same signaling molecule (Sy1).
• Branching: one receptor (Lx) activates multiple signaling molecules (Sx1, Sx2, Sx3).
• Cross Talk: signaling pathways influence each other (e.g., phosphorylation of FoxO by AKT).

Diagram showing how multiple components lead to Protein Synthesis and Protein Degradation.

Agonist - Antagonist

• Agonist: Enhances Cellular Activity
• Antagonist: Blocks Cellular Activity
• Partial and mixed agonists and antagonists also exist.

Desensitization as a Physiologic Phenomenon

• Initial Response: Agonist leads to biological effect.
• Repeated Response: Desensitization occurs with continued agonist exposure.
• Homologous Desensitization: Desensitization occurs only to the agonist that caused it.
• Heterologous Desensitization: Desensitization occurs to multiple agonists that signal through same pathway.

Mechanism of Desensitization

• Timescale of Desensitization:
  • <10s: Receptor phosphorylation, Arrestin binding
  • <100s: internalization
  • <1000s: Endosomal degradation

1000s: Inhibition of receptor translation

Biased Ligands

• Biased Receptors
• Balanced Signal: Ligand activates multiple signaling pathways equally.
• Biased Ligand: Ligand preferentially activates one signaling pathway over others.
• Biased Receptor: Receptor signals through one pathway more effectively than another.

One Ligand, Many Proteins: Receptor Subtypes

• Acetylcholine: Can activate both muscarinic and nicotinic receptors.
• Muscarinic: M1, M3, M5 (stimulatory) and M2, M4 (inhibitory).
  • Key effectors (examples)
    ↑ PLC
    ↑ [Ca2+]i
    ↑ MAPK
  • Key effectors (examples)
    ↑ AC
    ↑ MAPK
    ↑ GIRK ch.
• Nicotinic: α7 and α4β2 (ionotropic).
  • Key effectors (examples)
    ↑ [Ca2+]i
    ↑ VDCC
    ↑ PKC

Illustration comparing subtypes, binding sites, effectors, and signaling molecules.

One Ligand, Many Proteins: Further Examples

• Different receptors activated by the same ligand are:
  • Grouped in receptor families
  • Constitute the base for receptor classification / nomenclature
• Ligands and Types of Receptors:
  • Acetylcholine:
  • Nicotinic (Ionotropic Receptor)
  • Muscarinic (Metabotropic Receptor)
  • M1…M5
  • Glutamate:
  • GluR, NMDAR (Ionotropic Receptor)
  • mGluR (Metabotropic Receptor)
  • GABA:
  • GABAA (Ionotropic Receptor)
  • GABAB (Metabotropic Receptor)
  • Serotonin:
  • 5-HT3 (Ionotropic Receptor)
  • 5-HT1,2,4-7 (Metabotropic Receptor)
  • ATP (a purine):
  • P2Y (Metabotropic Receptor)
  • P2X (Ionotropic Receptor)

Quantitative Aspects of Receptor Function

• Ligand-Receptor Interaction & Biological Response
  L + R \rightleftharpoons LR \rightleftharpoons LR^*
  \text{Occupation: Governed by affinity}
  \text{Activation: Governed by efficacy } (\alpha, \beta)
  \text{Biological Response: Governed by activity of signaling enzymes/pathways}

Quantitative Characterization of Ligand-Receptor Interaction

• Saturability:
  • Finite number of receptor (specific ligand binding) sites [i.e., M/g tissue].
• Affinity:
  • Result of non-covalent binding.
  • K_d is the concentration of a ligand [M/L] required for binding to 50% of the receptor sites (equilibrium dissociation constant).
  • Ka is the association constant ( = 1/Kd).
• Activity (intrinsic activity or efficacy):
  • Measures a ligand's ability to induce a response by the receptors (0..100%, or -100..+100% when the receptor displays intrinsic activity).
  • Defined as the maximum response to the test agonist relative to the maximum response to a full agonist acting on the same receptor.
• Specificity:
  • The defining characteristic of receptors.
  • Distinguishes between ligand molecules with only minor structural modifications.

Quantitative Characterization of Biological Response

• Dose-response Curves:
  • E_{max} (Maximal Effect): Maximum possible biological response
  • EC{50} (Potency): The effective concentration required to produce 50% of the maximum possible biological response (E{max}).

Relationship Between Receptor Number and Biological Response

Graph showing relationship between occupied receptor and hormone concentration.

• Spare receptors:
  • increase the sensitivity of the target cell to the hormone
  • serve to prolong the biological response of a cell

The Spare Receptor Theory (Receptor Reserve)

Graph showing number of receptors versus hormone concentration and biological response.

• EC_{50} gives an indication of potentcy.

Receptor names

• classifications Nomenclature of Receptors
• Receptor families (molecular biological origin)

1. Family members arose from gene duplication and divergence.
2. Family members share structurally homologous domains.
3. Downstream events are not predictable from the type of receptor or its ligand(!)

• Nomenclature may be based on

1. Receptor morphology (7TM receptors)
2. Chemical nature of ligand (Cholinergic, Adrenergic)
3. Cell type (neuronal, ganglionic)
4. Signalling/second messengers (ionotropic, metabotropic)

Individual Receptor Subtypes Show Different Affinities To Natural or Synthetic Ligands

Table of K_A values for different muscarinic acetylcholine receptor subtypes.

Adrenergic Receptor Subtypes (7TM family)

• Original Classification (Ahlquist 1948) : α and β
• Molecular Pharmacology: α1, α2, β1, β2, β3, α1A, α1B, α1D, α2B, α2C, α2A
• Signal Transduction Effectors:Gq, Gi, Gs
• Adenylate cyclase cAMP (↑), Adenylate cyclase cAMP (↓), Phospholipase C, IP3, DAG, Ca2+

Physiologic Effects of Adrenergic Receptor Activation

• α1: Vascular smooth muscle (Contraction), Pupillary dilator muscle (Contraction - dilates pupil), Pilomotor smooth muscle (Erects Hair), Platelets (Aggregation)
• α2: Adrenergic nerve terminals (Inhibition of transmitter release), Adipocyte (Inhibition of lipolysis)
• β1: Heart (Increases force and rate of contraction)
• β2: Smooth muscle (bronchial, vascular and uterine) Relaxation, Hepatocyte (Activate glycogenolysis)
• β3: Adipocyte (Activates lipolysis)

Physiologic Effects of Cholinergic Receptor Activation

• Eye:
  • Sphincter: M3 - Contraction (myosis)
  • Ciliary muscle: M3 - Contraction (Accommodation for near vision)
• Heart:
  • SA node: M2 - Reduced heart rate (negative chronotropy)
  • AV node: M2 - Reduced conduction velocity (negative dromotropy)
  • No known effects on ventricles or Purkinje cells
• Lungs:
  • Bronchioles: M3 - Contraction (bronchospasm)
  • Glands: M3 - Secretion
• GI tract:
  • Stomach: M3 - Increased motility, cramps
  • Glands: M1 - Secretion
  • Intestine: M3 - Contraction (diarrhea, involuntary defecation)
• Bladder:
  • M3 - Contraction (detrusor muscle), relaxation (trigone/sphincter), voiding, urinary incontinence
  • Sphincters:
  • M3 - Relaxation, except lower esophageal, which contracts
• Glands:
  • M3 - Secretion (sweat, salivation, lacrimation)
• Blood vessels (endothelium):
  • M3 - Dilation via NO/endothelium-derived relaxing factor

Passive and active transport

• The following slides have information about Passive and Active Transport.

Water compartments in the body

Illustrations to show water percentage in blood plasma, intracellular fluid, interstitial fluid.

• Definitions:
  • extracellular space
  • intracellular space
  • interstitial space

Types of membrane proteins

• Structure and function of the cell membrane:
  • separation + connection -> barrier + transport
  • chemical -> concentration gradient
  • osmotical -> regulation of cell volume
  • electrical -> membrane potential
  • informational -> signal transduction

Spontaneous formation of

Figures showing formation of BLM in AIR.

Summary of membrane transport

• Direction: influx, efflux
• Type: electrogenic, electroneutral
• Mechanism:
  • through lipid phase, ion channel, mobile carrier
  • uniport, symport, antiport
• Energy backgrounds: passive, active primary, secondary, etc.
• Driving forces: concentration-, electrical-, osmotic gradients
• Interactions:
  • direct competition (for the same binding site)
  • coupled transportation (symport, antiport)
  • electrostatic interactions (between cations and anions)
  • osmotic interactions (between water and solutes)

Passive diffusion through the lipid phase

• hydrophobic substances, neutral gases (O2, CO2, N_2)
• driving force: concentration gradient
• -\Phi = D * A * \frac{dc}{dx}
  where: \Phi: flux of diffusion, D: diffusion coefficient (cm^2/s), A: surface area, \frac{dc}{dx}: concentration gradient, l: distance of diffusion
• for a discrete membrane: -\Phi = P * A * (c1-c2)

Passive diffusion through ion channels

• Outward current (positive): exit of + charges from cells (K^+, Na^+ efflux, but Cl^- entry)
• Inward current (negative): entry of + charges into the cells (K^+, Na^+ entry, but Cl^- efflux)
• Driving force: electrochemical potential gradient
• dECP = Vm-Vx
  where: Vm: membrane potential, Vx: equilibrium potential for ion X
• Ion current I*X = n * i*X
• IX = gX * (Vm – VX)
• g_X = \frac{dI}{dV}

Transport mediated by carrier proteins

• Michaelis-Menten kinetics
• Properties
  • saturation
  • competition
• Mechanism
  • uniport
  • symport
  • antiport
• V = \frac{V*{max} * [S]}{Km + [S]} where: V{max}: maximal rate of transport, K*m: concentration of substrate resulting in a transport rate of V_{max}/2
• Facilitated diffusion
• Active transport

Active transport

• Primary active transport:
  • carrier and ATP-ase in same entity
  • carrier mediated -> competition, saturation
  • energy source: ATP
  • Na+-K+ pump (all cells)
  • Ca2+ pump (RBC, muscle, heart, epithelium)
  • H+-K+ pump (parietal cells, renal tubules)
  • SR Ca2+ pump (skeletal & cardiac muscle)
  • Types of ATPase:
  • P type ATPase (phosphorilation)
  • V type ATPare (H^+ transport, acidic Vacoules)
  • H+ ATPase (membrane bound, non mithochondr.)
  • F type ATPase 8-13 polipeptid chains, (F0 & F1 subunits)
    ATP synthesis
    H+ ATPase (plasmamembrane : bacteria eucaryotes: inner mithochondrion)

Na+-K+ pump

• 3Na^+- 2K^+ stochiometria -> electrogenic
• Role of Na+-K+ pump in regulation of cell function
  • Asymmetrical ionic distribution (Na^+ and K^+ gradient)
  • Resting membrane potential (K^+)
  • Action potential (Na^+)
  • Energy for other transport processes (Na^+)
  • Regulation of cell volume (Na^+)
  • Heat production (ATP-ase)
  • Phosphorylated and dephosphorylated states (E1 – E2) -> change in conformation
  • Activation by: [Na^+]i and [K^+]e
  • Enhancement of pump activity: insulin, aldosterone, thyroxine, catecholamines
  • Specific blocker: ouabain -> depolarization, swelling of cells
    cardiac glycosides: positive inotropic action

Active transport 2.

• Secondary active transport
  • carrier and ATP-ase are separated
  • carrier mediated -> competition, saturation
  • energy source: transmembrane Na^+ gradient
  • Na^+-Ca^{2+} exchanger
  • Na^+-H^+ antiport
  • Na^+-K^+-2Cl^- symport
  • Na^+-glucose symport

Transmembrane transport of water

• Consequence of osmotic flow: change in cell volume with its consequences
  Placing cells in hypotonic solution makes them swell or even rupture while they shrink in hypertonic solution
  Hemolysis, Cerebral edema
  Illustration: Cells in Isotonic, Hypotonic, and Hypertonic solution.
• Driving force: osmotic pressure (\Pi)
• \Pi = R * T * C_{osm}
  where C_{osm} = osmotic concentration molar concentration of osmotically active particles
• 1 Osmol = 6 * 1023 osmotically active particles
• Concept of effective osmols

Transepithelial transport

• I. paracellular route, between two cells, barrier is: the tight junction between cells, which can be:
  • „tight” tight junction
  • „leaky” tight junction
  • only by way of passive diffusion
• II. transcellular route:
  • barriers:
    apical membrane
    basolateral membrane
    by passive or facilitated diffusion and by primary and secondary active mechanisms

Primary active transport mechanisms in epithel cells

Schematic showing active transport in the kidney.

• Na+/K+-ATPase in almost all living cells (responsible for the maintenance of the intracellular osmolality, of the resting transmembrane potential and makes secondary active transport mechanisms possible by keeping intracellular Na+ at low concentration)
• proton-pump in the parietal cells of the stomach (gastric acid secretion)
• proton-pump in the intercalated cells in the collecting duct of the kidney (acid-base balance regulation)

Transport mechanisms of epithel cells (secondary active transport)

Figures Showing reabsorption in the kidney, ilium, and glucose in the proximal ducts and the ileum

Transport mechanisms of epithel cells (secondary active transport)

Diagram showing reabsorption in the kidney and saliva secretion

Resting membrane potential

• The following slides define components of Resting Membrane Potential.

Membrane potential

• Potential difference measured between the two sides of the cell membrane (extra- and intracellular space).

The cell as a Donnan system, Donnan potential

• The magnitude of the Donnan-potential is in the range of -10 mV, thus it cannot explain the highly negative resting potential of excitable cells !
• In case of Gibbs-Donnan equilibrium:
  • Principle of electroneutrality: [K^+]A = [Cl^-]A + [Prot^-]A , [K^+]B = [Cl^-]_B
  • Principle of ionic products: [K^+]*A * [Cl^-]A = [K^+]B * [Cl^-]_B
  • Electrochemical potential must be zero for each permeant ion species (K^+, Cl^-) (this is not true for non-permeant ions).
  • Permeant ions (K^+ and Cl^-) can trespass the membrane, while the non-permeant Prot- anion cannot.
  • This results in transmembrane potential difference (Prot- -containing side negative).
  • Microscopic violation of electroneutrality because the membrane behaves as a capacitor.

Origin of the membrane potential

Figure showing Origin of membrane potential due to K^+ ion concentration difference.
Illustration with ion distributions and flow.

Nernst equation

• EK = -\frac{RT}{zF} ln \frac{[K^+]k}{[K^+]_b} R, gas constant F, Faraday number T, temperature Z, valence of the ion
  • [K^+_k], extracellular K^+ concentration
  • E_K, equilibrium potential or reversal potential of K^+
  • ln = log_e, where e = 2,718
  • [K^+_b], intracellular K^+ concentration

Other ions?

Table provides other ion information.Na+
What maintains resting membrane potential?
Na+ /
K+ ATP-ase, or Na+ /
K+ pump
Na+
Na+
Na+
~
150 mM
~
5 mM
Na+
~
15 mM
K+
~
150 mM
+
Pi
Inhibitors: Ouabain
Digoxin, digitoxin (cardiac glicosides)
K+
K+
ATP
ADP
Ouabain
Digoxin
, digitoxin (cardiac
glicosides)
K+
~
5 mM
K+
~
15 mM
Na+
~
150 mM
ATP
ADP
+
Pi

Other ions?

Table provides other ion information.

What maintains resting membrane potential?

Figure outlining gradients and ATP usage.

Contribution of the Na+/K+ pump to the resting membrane potential

• Resting potential (mV) vs time (h) Graph displaying Ouabain inhibition effects.
• Abruptly developing small amplitude depolarization -> due to absence of the outwardly directed pump-current
• Slow but progressive depolarization -> due to collapse of the transmembrane K+ gradient
• Pump stochiometry: 3Na^+- 2K^+ -> electrogenic

Theoretical experiment

• Which direction the ions