ION CHANNEL : A SITE OF DRUG ACTIONS

1. ION CHANNEL :A SITE OF DRUG ACTIONS Heny Ekowati Pharmacy Department Medicine and Health Sciences Faculty Unsoed, 2013

2. Ion Channels The plasma membrane is 6-8nm thick, and consists of a mosaic of lipids and proteins. The lipid is hydrophobic, and will not allow ions through. Ions are surrounded by waters of hydration. To move through the hydrophobic lipid bilayer, water molecules would need to be stripped off the ion. This takes too much energy.

3. Ion Channels The solution is to provide ions with specialized pathways such as ion channels that will permit ions to cross with most or all of their water molecules. Ion channel pores therefore provide ions with a polar environment.

4. Structure of Ion Channels Ion channels are large assemblies of proteins, which make up subunits, which combine to form functional channels.

5. Important features: • Gating (opening and closing) • What are the features that cause the channel to open and close? • Ion selectivity • Why does a K+ channel not allow Na+ through? • Molecular features related to their function. • What are the structural features that determine the function of the channel?

6. Gating • What are the features that cause the channel to open and close?

7. Gating: Ligands Phosphorylation Controlled by different types of stimuli: Voltage Stretch

8. 3 modes of gating: Gating • Involves conformational changes in the ion channel protein. • Each channel protein has two or more conformational states (e.g., open & closed) that are relatively stable. • Each stable conformation represents a different functional state.

9. Ion Selectivity: 0.095 nm • Why does a K+ channel not allow Na+ through? 0.133 nm

10. Why does a K+ channel not allow Na+ through? Ion Selectivity: Concept of waters of hydration: Increase the effective diameter of the Na+ ion. Thus: Pore Size is 1 mechanism for selectivity.

11. Ion Selectivity: So why does an Na channel exclude K? Channels have a specialized region that acts as a molecular sieve  The SELECTIVITY FILTER. This is where an ion sheds its waters of hydration & forms a weak chemical bond with charged or polar amino acid residues that line the walls of the channel.

12. Klasifikasi • Kanal ion terdapat pada hampir di setiap sel, fungsinya untuk transport ion, pengaturan potensial listrik melintasi membran sel, dan signaling sel. • Klasifikasi : • - Kanal ion teraktivasi voltage (voltage-gated channel) • - Kanal ion teraktivasi ligan (ligand-gated channel) • - Kanal ion teraktivasi nukleotida siklik atau kalsium • (cyclic nucleotide-gated channel atau calcium-activated • channel) • - Kanal ion teraktivasi oleh kekuatan mekanik (stretch • activated channel) • - Kanal ion terhubung protein G (G protein-gated channel)

13. Voltage-Gated Ion Channels CLOSED OPEN Depolarisasi Repolarisasi

14. Voltage-Gated Ion Channels • A class of ion channels gated (opened and closed) by the trans-membrane potential difference (voltage). • There are many, many, types. Among these are: • Na+ Channels • K+ Channels • Ca2+ Channels • Cl- Channels. There are actually many types of Na, K, Cl, and Ca Channels, classified according to pharmacology, physiology, and more recently- molecular structure.

15. Voltage-gated ion channels • Involved in: • Initiation and propagation of action potentials • Control of synaptic transmission • Intracellular ion homeostasis • Other aspects of intracellular function • Acting as activators of intracellular enzymes • Coordinating signals between cell membrane and internal organelles (e.g., mitochondria).

16. Ligand-Gated Ion Channels • Typically, these are ion channels located on the postsynaptic (receiving) side of the neuron • Some act in response to a secreted (external) ligand- typically a neurotransmitter such as • Acetylcholine (Ach) • GABA • Glycine • Glutamate • Some act in response to internal ligands such as cGMP and cAMP, and are also regulated by internal metabolites such as phosphoinositides, arachidonic acid, calcium.

17. Ligand-Gated Ion Channels Among the first ligand-gated channels to be thoroughly characterized and cloned is the Ach channel. • 5 subunits, each made of 4 membrane-spanning components (M1-M4) • 2 Ach molecules need to bind in order to open the channel pore. • Fluxes Na and K.

18. Potensial sel • Depolarisasi • Repolarisasi • Hiperpolarisasi • Resting potensial

21. Na+ Channel

22. Sodium Channels - Structure • Composed of α, β-1 and β-2 subunits, but the large α-subunits carries most of the functional properties • 4 repeated motifs, each with 6 transmembrane domains • All linked together • Contain a voltage “sensor”/ligand binding domain (method of activation) • The hydrophobic S4 segment (voltage “sensor”) is found in all voltage gated Na+ channels and is absent in ligand gated Na+ channels • Selectivity filter (shell of hydration) • Inactivation gate

23. Cartoon representation of the “typical” voltage-activated sodium channel

24. Types Of Na+ Channels • Voltage gated – Changes in membrane polarity open the channel • Ligand gated (nicotinic acetylcholine receptor) – Ligand binding alters channel/receptor conformation and opens the pore • Mechanically gated (stretch receptor) – Physical torsion or deformation opens the channel pore

25. Sodium Channels - Function • Play a central role in the transmission of action potentials along a nerve • Can be in different functional states (3) -A resting state when it can respond to a depolarizing voltage changes -Activated, when it allows flow of Na+ ions through the channel -Inactivated, when subjected to a “suprathreshold” potential, the channel will not open

26. The theory is that the inactivation gate “swings” shut, turning off the channel

27. Na+ Channel Modulation • Phosphorylation • sodium channel function is modulated by serine/threonine and tyrosine kinases as well as tyrosine phosphatases (Yu et al, Science 1997) • Mutation – altered amino acid sequence/structure can change the biophysical properties of the Na+ channel • Pharmacology – block Na+ channel to reduce the conductance • Proteolysis- (cleavage) Proteases may cleave specific residues or sequences that inactivate a channel, or significantly alter the biophysical properties

28. Why Na+ Channels/Modulation Are Important • Neuronal depolarization, Action Potential • Neuronal Excitability • Cardiac Excitability • Muscle Excitability • The basis of neuronal/cardiac/muscular function relies on the propagation of action potentials, down axons, sarcolemma, myocardium, as well as requiring synaptic transmission. • Differential excitability alters the electrical conduction/transmission properties of the “circuit”

30. Na + Channel Blockers/Pharmacological Agents • Tetrodotoxin (TTX) • Amiodarone • Lidocaine • Procainamide • Mexilitine • Ketamine • Many, many others

31. Some Na+ Channels Outside The Nervous System • Naf – “Funny Current” in pacemaker cells of the heart (SA node/ectopic pacemakers) • Nav in the myocardium, sarcolemma, and T-tubules and motor endplate

33. K+ Channels

34. K+ Channels:

35. Shaker K+Channel: • Each Channel is made of 4 Subunits. • Each Subunit is made up of a large protein having 6 trans-membrane segments (S1-S6). • Between S5 and S6 there is a loop (red) that, along with the S6 segment, lines the conduction pore.

36. Shaker K+ Channel:

37. What they look like:

38. What they look like:

39. Voltage-gated K+ channels • mediate outward K+ currents during nerve action potentials. • Important advances in understanding have come from: • physiological studies, including the use of patch clamping • mutational studies of the Drosophila voltage-gated K+ channel protein, product of the Shaker gene • crystallographic analysis of the structure of bacterial K+ channels. • molecular dynamics modeling of permeation dynamics. • 4identical copies of the K+ channel protein, arranged as a ring, form the channel walls.

40. Hydropathy analysis & topology studies predicted the presence of 6 transmembranea-helices in the voltage-gated K+ channel protein. The core of the channel consists of helices 5 & 6 & the intervening H5 segment of each of the 4 copies of the protein.

41. Helices 1-4 function as a voltage-sensing domain, with helix #4 having a special role in voltage sensing. This domain is absent in K+ channels that are not voltage-sensitive.

42. TheN-terminus of the Shaker channel (or part of a separate subunit in some voltage-activated channels) is essential for inactivation. Mutants that lack this domain do not inactivate. Adding back a peptide equivalent to this domain restores the ability to inactivate.

43. A "ball & chain" mechanism of inactivation has been postulated, in which the N-terminus of one of the 4 copies of the channel protein enters the channel from the cytosolic side of the membrane to inhibit ion flow. In some voltage-gated K+ channels, entrance of the N-terminus into the channel is followed by a conformational change in the selectivity filter that contributes to the process of inactivation.

46. Ca2+ Channel