Overview of ion channel proteins. What do ion channels do? Three important points:

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1 Overview of ion channel proteins Protein Structure Membrane proteins & channels Specific channels Several hundred distinct types Organization Evolution We need to consider 1. Structure 2. Functions 3. Requirements for synthesis What do ion channels do? Three important points: 1. No channel does everything we will talk about today (some functions are mutually exclusive!), however all channels have many of these functions. 2. I am oversimplifying and generalizing to make sense of a very complex field. 3. All channel functions are due to protein structures formed by amino acids found in the channel molecule. Morales; Physiology 405/505 1

2 What do ion channels do: Selectivity Selectivity for ions: Na +, K +, Ca 2+ Anions vs cations Some are very non-selective (conexins) This can be complex, take Na + vs K + What do ion channels do: Gating Gating is the channel s response to external stimuli that either permits or stops the flow of ions It involves opening & closing Gating can involve many parts of the channel, and depends on the mechanism Gating requires a stimuli that is specific Channels are usually classified by their gating stimuli Gating can be fast 10 to 100 times that of the fastest enzymes Morales; Physiology 405/505 2

3 Why is speed important? Cell signaling (Phosphorylation, G proteins, camp) Action potentials, muscle contraction <sec Ion channels min hours >hours Transcription Translation, turnover, processing (3.6 ms) Time in hours There are many kinds of gating stimuli Voltage-gated channels require depolarization (rarely hyperpolarization) Ligand-gated channels require small molecule binding Outside the channel: Neurotransmitters Inside the channel: Ca 2+ Cyclic nucleotides (camp) Other gating stimuli include cold, heat, mechanical stretch Morales; Physiology 405/505 3

4 What do channels do: Inactivation Inactivation is a process that closes a channel. It is distinct from closing Not all channels need to be closed to inactivate What do channels do: Inactivation One channel may have multiple inactivation mechanisms Once a channel has inactivated, it is possible that the channel s gate could open or close without ions passing through the pore This covers biophysics: What else? Morales; Physiology 405/505 4

5 What do ion channels do: Modulation Modulation: Any external factor which changes the frequency of rate of any channel function Examples: Phosphorylation or any other protein modification Other signaling cascades (G-proteins) All Biosynthesis (transcription/translation)? All mechanisms Neurotransmitters, NO of protein???? Ca 2+, camp modulation Environmental factors Drugs, EtOH, toxins Additional subunits Calmodulin What ion channels do: Assemble Most channels are multi-subunit complexes built around the central pore All channels must have at least one protein subunit that participates in pore formation Most channels probably have subunits not involved in pore formation: Ancillary subunits P Morales; Physiology 405/505 5

6 Apical Epithelial Cells What do ion channels do: Localization K + HCO 3 - Neurons Na + Cl - K + Na + Basolateral Cl - Others? Yes, turns out to be very common Ion channel protein diversity That s a lot of functions. All organisms have ion channels. They are located in all tissues & organs. They are involved in many biological processes. They are central to the most complex processes, like thought & muscle movement. So that means there are only a couple of types we have to know about, that way they ll be easier to learn, right? Morales; Physiology 405/505 6

7 There are organizing principles Morales; Physiology 405/505 7

8 Organizing principle Channels are members of multiprotein families They have relationships that can be used to understand function Some Definitions Homology: The property of having a common ancestor in evolution 2 proteins with similar or nearly identical sequences are usually homologous. There are other ways to establish homology than through primary sequence 1. You will often see sequence similarity & homology confused in the literature. 2. Sequences can be identical, almost or nearly identical; they can be different. 3. Genes, proteins, RNA, & organisms can be homologous, not sequences. 4. There is no such thing as 50% homologous. Morales; Physiology 405/505 8

9 Some more definitions Family: An arbitrarily defined group of genes or gene products (proteins) that are homologous, and often have related biological properties Super-Family: The largest group of genes or gene products that are homologous. Properties within a super-family can often vary widely, and there may be little apparent connection Because of the modular nature of many proteins, they can belong to more than one super-family Membership in a super-family may not be obvious from inspection of two members A final definition Phylogeny: Understanding homologous relationships between gene products This type of diagram does not suggest evolutionary relationships! It is merely a graphic representation of sequence similarity Morales; Physiology 405/505 9

10 Major ion channel super families Largest super family includes voltage-gated channels: 4 protein subunits around one central pore Many gating mechanisms Among the oldest proteins known Neurotransmitter-gated channels 5 subunits/pore Chloride channels ClC-type--2 subunits/2 pores CFTR--? Aquaporin (water) 4 subunits/4 pores Conexins (lots of things) 6 subunits/one pore IP 3 -R & Ryanodine Ca 2+ release channel 4 subunits, one pore S5-S6 Super Family Four subunits around a pore Very old: the genes are seen in all organisms Eukaryotes (plants, animals, fungi) Bacteria (E. coli, most common bacteria) Archia (Thermophiles, Halophiles, Methanobacterium) Many members Voltage-gated K +, Na +, and Ca 2+ channels Ca 2+ -K + channels Inward rectifier and leak K + channels Cyclic-nucleotide-gated cation channels Stretch channels? Hyperpolarization-activated cation channels TRP sensory stimulus signaling channels Morales; Physiology 405/505 10

11 The S5/S6 Super Family starts with channels with two membrane spanning segments M1 S5 M2 S6 K + selectivity How many subunits? Gating? Six Membrane Spanning Segments Voltage-gated K + channels add the S4 voltage sensor Extracellular Intracellular S1 S2 S3 S4 + S S6 This is an α helix. How would you describe it? AILRIIRLVRVFRIFKLSRHSKGL Morales; Physiology 405/505 11

12 Cyclic nucleotide-gated & Ca 2+ gated channels add ligand binding X X X camp Ca 2+ C terminus Ligand binding opens channel They have S4 Segments--weak voltage-gating One member is activated by hyperpolarization The TRP channels add amazing functionality TRP: Transient receptor potential Cation or Ca 2+ channels, jury still out Their sequences contains many regions implicated in signaling processes & protein binding Activated by Inositol-lipids Light Heat or hot foods Cold or MeOH Stretch Structure: 6 transmembrane sequences, no S4 charge. Present in many nonneuronal tissues heat sensitive TRP in keratinocytes. Involved in Mechanosensory transduction (touch) Olfaction Osmolarity Pain Defective in polycystic kidney disease, skin tumors, mucolipidosis type IV Morales; Physiology 405/505 12

13 Voltage-Gated Na + & Ca 2+ : One peptide, same plan VG Na + and Ca 2+ channels string four 6 TM channel subunits into one polypeptide: Advantage is lack of symmetry: Each subunit can have unique functions P P Proposed Evolutionary Pathways Primordial Channel? +Pore NP-gated ENaCh XC-ATP Duplication TWIK GluR GluR +Glu BP Ligandgated MthK +Ligand Binding KscA Kir +Ligand Binding +S4 KvAP VG-K + Na + & Ca 2+ Duplication Morales; Physiology 405/505 13

14 There are many other designs: Neurotransmitter-gated channels Commonly referred to as ligand-gated (which is wrong) neurotransmitter-gated channels, which is better, but there are channels not related that are also neurotransmitter-gated Five-fold symmetry The neurotransmitters include: Acetyl choline γ amino butyric acid (GABA) Serotonin Neurotransmitter-gated channels They are often described as receptors (eg ACh receptor) because this was how they were discovered. Super-family not as extensive as the 4 subunit group, but were: The first to be cloned The first to have structural info The first to have single channel currents measured Morales; Physiology 405/505 14

15 The Prototype: The Nicotinic Ach receptor Binds to nicotine There is also a muscarinic ACh receptor, but it is not a channel Cation selective, but no preference for Na + over K + Each channel has 5 related subunits: α, α, β, γ, δ Exact arrangement unknown, most of the protein is outside the cell ACh channels have 4-membrane spanning segments The N & C Termini are extracellular Each subunit has 4 membrane spanning sequences Morales; Physiology 405/505 15

16 ACh receptor composition No subunit binds ACh alone, so 5 identical subunits can t make a channel The subunits are 50% identical The best characterized receptor is from muscle, but there are many ACh receptor subunits expressed in brain; their function is unknown The GABA & Glycine receptors are related to the ACh receptor BIG difference, they conduct chloride ions, not cations They are also made up of different types of subunit GABA: α 2, β, γ, δ subunits (binding similar to ACh) Gly: α 3, β 2 subunits (only α required for binding) GABA receptors are very important drug targets Alcohol Benzodiazapines Barbituates Morales; Physiology 405/505 16

17 The Big Finish While there are many kinds of channels, understanding one, helps understand the others. BUT, do not mistake this for redundancy. Each of the channels & channel subunits probably have unique functions, although currently much of this is very mysterious. Morales; Physiology 405/505 17