Types of drug receptors

1. Types of drug receptors • Practically all receptors are proteins: • Enzymes • Ion channels • Ligand-gated channels: Ion channels that open upon binding of a mediator • Voltage-gated channels: Ion channels that are not normally controlled by ligand binding but by changes in the membrane potential • ‘Metabolic’ receptors – hormone and neurotransmitter receptors that are coupled to biochemical secondary messenger / effector mechanisms

2. Physiology and pharmacology of membrane excitation • Excitable cell types: • Nerve cells • Myelinated nerve fibers (fast transmission) • Non-myelinated nerve fibers (slow transmission) • Muscle cells • Skeletal muscle • Heart muscle • Smooth muscle “striated”

3. Membrane potentials and excitability • Both excitable and non-excitable cell membranes have an electrical potential across their cytoplasmic membranes • The membrane potential chiefly depends on the asymmetric distribution of sodium and potassium ions, and with some cells calcium ions across the cell membrane • In the ‘ground state’, the orientation of the membrane potential is negative inside

4. 3 Na+ Na+ Ca++ Glucose How is the asymmetric distribution of ions across the membrane maintained? 2 K+ ADP + Pi ATP 3 Na+ K+ Cl- K+ Na+

5. Ionic basis of membrane potentials and excitability • In the resting state of excitable cells – and throughout in the non-excitable cells – the interior of the cell is electrically negative against the outside • Electrical excitation (the ‘action potential’) consists in a brief, transient reversal of the orientation of the membrane potential • Both the resting potential and the action potential are diffusion potentials

6. - - - - - - - - - - + + + + + + + + + + Diffusion potentials (1) no potential (electroneutrality)

7. - - - - - - - - - - + + + + + + + + + + Diffusion potentials (2) still no potential (electroneutrality) + -

8. - + Diffusion potentials (3) negative positive - + - + - + - + - + - + - + - + - +

9. - - + + - - + + Diffusion potentials (4) Driving force 1: Entropy (equalize concentrations on both sides) Driving force 2: Electroneutrality (equalize charges on both sides)

10. Cout  ln E = Cin E: R: T: F: z: ln: Cin, Cout The equilibrium diffusion potential Gas constant (8.31 J  K-1mol-1) Absolute temperature (K) Faraday constant (96500 Coulomb/mole) Number of charges of single ion (1 with K+ and Na+, 2 with Ca++, -1 with Cl-) Natural logarithm (base: e = 2.71828) Inside and outside concentrations of the diffusible ion species R  T z  F The Nernst equation describes the diffusion potential at equilibrium

11. Inside Outside Equilibrium potential Na+15 mM 150 mM +60 mV K+ 150 mM 6 mM -90 mV What if there are multiple diffusible ions? (1) Intra- and extracellular cation concentrations: Actual resting membrane potential: -70 mV

12. Cout  ln Nernst equation: E = Cin R  T z  F PK  [K+]out + PNa  [Na+]out R  T  ln E = PK  [K+]in + PNa  [Na+]in F What if there are multiple diffusible ions? (2) Goldman equation (special case for Na and K):

13. PK  [K+]out + PNa  [Na+]out R  T  ln E = PK  [K+]in + PNa  [Na+]in F The Goldman equation and the role of ion channels P = Permeability – this is where the ion channels come in

14. PK  [K+]out + PNa  [Na+]out R  T  ln E = PK  [K+]in + PNa  [Na+]in F The Goldman equation and the role of ion channels (2) change don’t change

15. + + - K+ - Na+ - K+ Na+ - - K+ Na+ - - K+ Na+ - - Na+ - The cellular resting potential is essentially a potassium potential negative positive K+

16. + + Voltage-gated sodium channels will open upon reversal of the resting membrane potential negative positive Na+ negative positive

17. + + + Voltage-gated sodium channels propagate the action potential negative positive - - - - Na+ Na+ outside inside Na+ K+ K+ K+ K+ - - - - positive negative spreading action potential

18. - 55 mV Firing level - 70 mV - 85 mV Electrical depolarization of nerve fibers can trigger action potentials External stimuli of varying amplitude time (ms)

19. ENa (+60 mV) Repolarization: K channels open Na+ channels close Depolarization: Na channels open (PNa > PK) Hyperpolarization: Na channels closed (PK >> PNa) Resting potential: PK > PNa EK (-90 mV) The Goldman equation and the action potential

20. Set voltage externally Measure resulting current across channel Planar lipid membranes allow observation of individual channels

21. Multiple opening events of a single channel in a planar lipid bilayer Externally applied voltage Multiple, successive observations open state base line / closed averaged trace Current Time

22. Patch clamping pipette channel cell

23. Cell attached mode seal

24. Whole cell mode suction seal

25. Excised patch mode seal cell ripped apart

26. Questions: • How is the action potential initiated ? • How is the action potential terminated ?

27. Action potential: Termination (1) • The ion flux through the voltage-gated Na+ channel is countered by a voltage-gated K+ channel that responds more slowly to depolarization • Both channels spontaneously inactivate Resulting membrane potential Na+ influx K+ efflux duration: a few milliseconds

28. Depolarization Spontaneous inactivation Closed Open Inactivated Slow reactivation after membrane repolarization Action potential: Termination (2) Voltage-gated channels cycle between 3 distinguishable functional states

29. Structural model of a Kv channel Extracellular space Cytosol

30. + + + - - - + + + + + + + + + + + + + + + + + + + + - - - + + + K+ K+ The KV channel’s opening gate is located in the membrane

31. + + + + + + + + + + + + + + + + - - - - - - + + The KV channel in the resting state

32. The KV channel in the open state - - - + + + + + + + + + + + + + + +

33. - - - + + + + + + + + + + + + + + + The KV channel in the inactivated state

34. Action potential: Initiation • In a resting cell, an action potential can be initiated in a variety of ways: • By synaptic transmission. Examples: Signal conduction from one nerve cell to another, from nerve cell to muscle cell • By spontaneous, rhythmic membrane depolarization. Example: Specialized cells in heart and smooth muscle • By electrical coupling to a neighboring cell via gap junctions. Example: Heart muscle, smooth muscle

35. Muscle fibers and a branching nerve ending

36. + Na+ K+ presynaptic action potential Synaptic excitation ENa Firing level EK Presynaptic terminal synaptic cleft Postsynaptic terminal

37. - - + Na+ + Synaptic excitation (2) - Na+ Na+ In synapses, ligand-gated channels open upon binding of neurotransmitters and initiate the action potential in the post-synaptic membrane

38. + + Action potential initiation in heart pacemaker cells (negative charge left behind) Ca++ Ca++ K+ Ca++ K+ K+ In heart pacemaker cells, two types of calcium channels lead to spontaneous depolarization

39. Action potential initiation in heart pacemaker cells K+ 0 mV Ca++L -40 mV Ca++T -60 mV slow, spontaneous prepotential

40. Cell excitation by electrical couplingacross gap junctions - - - - - + + + + - - - - - + + + + Gap junction

41. negative positive - + + - - + + - - + - + Permeating anions leave behind excess positive charge Permeating cations leave behind excess negative charge negative positive R * T PK* CK,out + PNa* CNa,out + PCl, * CCl, in F * ln E = PK* CK,in + PNa* CNa,in + PCl, * CCl, out What about anions? Opposite charge affects the Goldman equation:

42. Inside cell Outside cell Equilibrium potential Na+15 mM 150 mM +60 mV K+ 150 mM 6 mM -90 mV Cl- 9 mM 125 mM -70 mV Ca++ 100 nM 1.3 mM +130 mV Intra- and extracellular ion concentrations • Opening of sodium or calcium channels will increase the membrane potential (depolarization) • Opening of potassium or chloride channels will lower the membrane potential (repolarization or hyperpolarization)

43. + Cl- Sodium and chloride in excitatory and inhibitory synapses Na+ positive negative