TRANSMEMBRANE ION CHANNELS

&SECOND MESSANGERS

• In the 1970s, pharmacology entered a new phase when receptors, which had until then been theoretical entities, began to emerge as biochemical realities following the development of receptor-labelling techniques , which made it possible to extract and purify the receptor material.

• Receptors have also been cloned

RECEPTOR-EFFECTOR LINKAGE

Functional Family

Physiological Ligands

Effectors and Transducers

Example Drug

β adrenergic receptors

NE, E, DA Gs; AC Dobutamine, propranolol

GPCR Muscarinic α2 adrenergic amines, Ach(M), opioids,serotonin

Gi, Gq, AC, ion channels, PLC

Atropine

Ion Channels Ligand-gated Ach (M2) Na+, Ca++, K+, Cl-

Nicotine, Gabapentin

Voltage-gated None(activated by membrane depolarization)

Na+, Ca++, K+

Lidocaine, verapamil

Transmembrane enzymes

Receptor tyrosine kinases

Insulin, PDGF, VEGF, growth factors

SH2 domain Herceptin, imatinib

Transmembrane non-enzymes

Cytokine receptors

Il-nterleukşns and other cytokines

Jak/STAT, soluble tyrosine kinases

Nuclear receptors

Steroid receptors

Estrogen, testosterone

Estrogens, androgens, cortisol

Thyroid hormone receptors

Thyroid hormone

Thyroid hormone

PPARγ Thiazolidinediones

Intracellular enzymes

Soluble GC NO, Ca++ Cyclic GMP Nitrovasodilators

Receptor Signaling Pathways

Second Messengers:• Ions (Ca2+, Na+, K+, Cl-)• cAMP, cGMP, IP3, Diacylglycerol• DNA binding – Transcriptional regulation.• Phosphorylated proteins and enzymes

via tyrosine kinase receptors.

Third Messengers:• Enzymes (PKC, PKA)• Ions (Ca2+, K+)

Receptor Signaling Pathways

EFFECTORS

Adenylate Cyclase (AC)Guadenylate Cyclase (GC)Phospholipase C (PLC)Phospholipase A (PLA2)Nitric oxide SynthaseIons

SECOND MESSENGER

cAMP

cGMP

DAG and IP3

Arachidonic acid

NO and CO

Na+, Ca2+

, K+

, Cl-

Adenylate Cyclase (AC)

Guanylate Cyclase (GC)

Phospholipase C (PLC)

Phospholipase A (PLA2)

Nitric oxide Synthase

Ions

MOLECULAR STRUCTURE OF LIGAND-GATED ION CHANNELS

Nicotinic ACh receptor

The 5 receptor subunits cluster around a central transmembrane pore that contain negatively charged aa’s that makes the pore ............. Selective.

There are 2 ACh binding sites at the extracellular portion of the receptor.

When ACh binds, the kinked α helices straighten out or swing out of the way and the channel opens.

G-protein and second messenger control of cellular effector systems

Gs stimulates AC

Β adrenoceptors, glucagon receptors, thyrotropin receptors, subtypes of DA and serotonin recptors

NE interacts with its membrane receptor only a few mseconds→Gs is generated, GTP- bound Gs may remain active for 10 seconds→original signal enormously amplified

Sutherland discovered cAMP as an intracellular mediator. It is synthesized from ATP by the action of membrane bound AC. Produced and degraded (PDEs)continuously.

cAMP regulates energy metabolism, cell division and cell defferentiation, ion transport, ion channels and the contractile proteins in smooth muscle.

cAMP activate protein kinases→ pk control protein phosphorylation.

β-adrenoceptor activation affects enzymes involved in glycogen and fat metabolism in liver, fat and muscle cells. As a result energy is made available as glucose to fuel muscle contraction.

increased energy

increased energy

The PLC/inositol system discovered in 1950s. Nasal salt secretion in seabirds is accompanied by increased turnover of membrane phospholipids known as phosphoinositides

one particular member of the PI family phosphatidylinositol (4,5) bisphosphate (PIP2), which has additional phosphate groups attached to the inositol ring, plays a key role. PIP2 is the substrate for a membrane-bound enzyme, phospholipase Cβ (PLCβ), which splits it into DAG and inositol (1,4,5) trisphosphate (IP3), both function as second messengers. The activation of PLCβ by various agonists is mediated through a G-protein (Gq,). DAG being phosphorylated to form phosphatidic acid (PA), while the IP3 is dephosphorylated and then recoupled with PA to form PIP2 once again. Li, an agent used in psychiatry blocks this recycling pathway

Structure of phosphatidylinositol bisphosphate (PIP2), showing sites of cleavage by different phospholipases to produce active mediators

Cleavage by phospholipase A2 (PLA2) yields arachidonic acid.

Cleavage by phospholipase C (PLC) yields inositol trisphosphate (I(1,4,5)P3) and diacylglycerol (DAG).

PA, phosphatidic acid; PLD, phospholipase D.

KINASE LINKED RECEPTORS

Mediate the actions of a wide variety of protein mediators, including growth factors and cytokines and hormones such as insulin and leptin

This signal transduction pathway is activated not only by various cytokines and growth factors acting on kinase-linked receptors but also by GPCR ligands. It controls many processes involved in cell division, apoptosis and tissue regeneration.

KINASE-LINKED RECEPTORS

• Receptors for various growth factors incorporate tyrosine kinase in their intracellular domain.

• Cytokine receptors have an intracellular domain that binds and activates cytosolic kinases when the receptor is occupied.

• The receptors all share a common architecture, with a large extracellular ligand-binding domain connected via a single membrane-spanning helix to the intracellular domain.

• Signal transduction generally involves dimerisation of receptors, followed by autophosphorylation of tyrosine residues. The phosphotyrosine residues act as acceptors for the SH2 domains of a variety of intracellular proteins, thereby allowing control of many cell functions.

• They are involved mainly in events controlling cell growth and differentiation, and act indirectly by regulating gene transcription.

• Two important pathways are: – - the Ras/Raf/mitogen-activated protein (MAP) kinase pathway, which is

important in cell division, growth and differentiation – - the Jak/Stat pathway activated by many cytokines, which controls the synthesis

and release of many inflammatory mediators.• A few hormone receptors (e.g. atrial natriuretic factor) have a similar

architecture and are linked to guanylyl cyclase.

The G-protein consists 3 subunits (α,β,γ) Ligand binding results in replacement of GDP and this is followed by dissociation of the α- and βγ subunits, α-GTP complex dissociates from the receptor and from the βγ complex and interacts w a target protein (can be an enzyme , AC or an ion channel, βγ may also activate a target protein leading to hydrolysis of the bound GTP to GDP.

INTRACELLULAR RECEPTORS FOR LIPID-SOLUBLE AGENTS

• Increasing understanding of receptor function in molecular terms →a number of disease states directly linked to receptor malfunction

• autoantibodies directed against receptor proteins myasthenia gravis a disease of the neuromuscular junction due to autoantibodies that inactivate nicotinic acetylcholine receptors.

• Activating antibodies have also been discovered in patients with severe hypertension (α-adrenoceptors), cardiomyopathy (β-adrenoceptors), and certain forms of epilepsy and neurodegenerative disorders (glutamate receptors).

• Mutated vasopressin and adrenocorticotrophic hormone receptors can result in resistance to these hormones

• Receptor mutations can result in activation of effector mechanisms in the absence of agonists. One of these involves the receptor for thyrotropin, producing continuous oversecretion of thyroid hormone; another involves the receptor for luteinising hormone and results in precocious puberty. Adrenoceptor polymorphisms are common in humans, and recent studies suggest that certain mutations of the β2-adrenoceptor, although they do not directly cause disease, are associated with a reduced efficacy of β-adrenoceptor agonists in treating asthma and a poor prognosis in patients with cardiac failure

• Mutations in G-proteins can also cause disease , mutations of a

particular Gα subunit cause one form of hypoparathyroidism, while mutations of a Gβ subunit result in hypertension. Many cancers are associated with mutations of the genes encoding growth factor receptors, kinases and other proteins involved in signal transduction