Молекулярные и клеточные механизмы обоняния и вкуса. Колесников С.С. Институт биофизики клетки РАН

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1 Молекулярные и клеточные механизмы обоняния и вкуса Колесников С.С. Институт биофизики клетки РАН

2 Introduction in Cell Physiology.

3 Bilayer permeability for different species

4 Mosaic structure of the plasma membrane

5 Typical intracellular and extracellular ion concentrations

6 Ion channels Физиология клетки. Введение Open Closed Selectivity: Na+, K+, H+, Ca2+, Cl-, cationic, non-selective Gating: voltage, ligand binding, ph, mechanical stimuli, temperature, light

7 Cell surface receptors G-protein coupled receptors Ionotropic receptors Receptor tyrosine kinase Receptor histidine kinase Tyrosine kinase - linked receptors Receptor tyrosine phosphatase Receptor serine/threonine kinase Receptor guanylate cyclase

8 H, Ca + 2+ G-protein coupled receptors, camp, ATP, opioids, lipids, amino acids Seven transmembrane-spanning (7TM) receptors Heptahelical receptors Stimuli: Serpentine light, odorants, receptors tastants peptides, proteins GPCRs > 1000 Effectors γ β α 16 α-subunits G-proteins: 5 β-subunits 14 γ-subunits Adenylyl cyclase Phospholipases Cβ and A2 cgmp phosphodiesterase Ca 2+ channels + K channels PI3 kinase

9 Receptor guanylyl cyclase Receptor domain GC domain GTP cgmp

10 Cyclic nucleotides 3,5 -cyclic AMP (camp) 3,5 -cyclic GMP (cgmp) AC PDE GC PDE ATP camp AMP GTP cgmp GMP

11 DAG IP3

12 camp signaling EPAC PKA Ion channels

13 PLC signaling

14 Olfaction

15 Complexity of the mammalian olfactory system Grueneberg ganglion AOB Brain VNO Septal organ MOE MOB

16 Glueneberg ganglion Main olfactory epithelium Main olfactory bulb glomeruli Accessory olfactory bulb olfactory sensory neurons lumena basal Septal organ Vomeronasal organ BV apical

17 Main olfactory epithelium (A) Olfactory sensory neuron (B) Supporting cell

18 Population of chemosensory cells in MOE Odorant receptors Trace amine receptors Receptor-GC?? G olf ACIII CNG2A PDE4 CNGA3 PDE2 TRPM5 gustducin PLCβ2 TRPM5 Microvillar OMP-negative Ciliary OMP-positive

19 Physiological era

20 Distinct cell subpopulations contribute additively to electroolfactogram Vt = ΣaiNiΨi Vt SC OSN

21 Responses of the main olfactory epithelium to different odorants

22 camp generation in olfactory cilia Boekhoff, Breer, PNAS 1992

23 Patch clamp method for recording channel activity Amplifier Recording pipette Assayed cell

24 Electrical excitation of olfactory neurons by odorants lilial Current (pa) Peak current (pa) Time (s) Lilial (µm)

25 camp,µ Μ camp-gated current (pa) ms 50

26 AC transduction cascade in olfactory cilia Receptor Odorant camp-gated channel Ca2+ CNGA2 PDE4 RK + γ β Golf Arrestin α ACIII camp PDE1 CaM + Cl - 3Na 4Na + 1Na / 2Cl-/1K + 1Ca2+ Cl-in ~ mm Cl-out ~ mm 1K + 1Ca2+

27 Adaptation

28 Adaptation machinery binding transduction response

29 Definition of cellular adaptation Control response Adapted response

30 lilial Two pulse protocol for assaying adaptation

31 Adaptation of olfactory sensory neurons is mediated by Ca 2+ A 3 mm Ca 2+ Cineol, 300 µ M 1 sec 50 pa B 3 mm Mg 2+, no Ca 2+ Cineol, 300 µ M Kurahashi & Shibuya, Brain Res., 1990

32 Ca2+-dependent elements of the olfactory transduction cascade Receptor Odorant Ca2+ PDE4 RK γ β Arrestin camp-gated channel Golf α ACIII camp PDE1 CNGA2 Cl + CaM + - 3Na 4Na + - 1Ca2+ 1K 1Ca2+ 1Na / 2Cl /1K + +

33 Olfactory CNG channel CaM site CNGA2 CNBD CNGB1b CNGA2 CNGA2 CaM site CNGB1b CNGA4 CNGA4 After Trudeau & Zagotta, J.Biol.Chem. 2003

34 1.0 Normalized open probability 0.5 Control K 1/2 = 1.4 µ M Calmodulin K 1/2 = 29 µ M camp concentration, µ M

35 Molecular era

36 Richard Axel Linda Buck The Nobel Prize in Physiology or Medicine 2004 for the discovery of odorant receptors

37 Species Number of OR genes Man 388 Chimpanzee 399 Macaque 326 Mouse 1063 Rat 1259 Dog 822 Caw 1152 Opossum 1198 Class I (fish-like) ~ 10 % Class II ~ 90% Chicken 300 Xenopus 1024 Zebra fish 155 Fugu 86 Drosophila 62 C.elegans 1278

38 Receptors operative in MOE Receptors Ligands Origin Proposed functions odorant GPCRs N = general odors MHC class I peptides food environment urine body secretions odor recognition, discrimination, attraction, repulsion social recognition of other strains trace amine GPCR N = 3-20 volatile amines urine stress response gender recognition GC-D N = 1 CO 2 uroguanyline guanylin atmosphere urine avoidance behavior Detection of cues related to Hunger, satiety, or thirst

39 GC-cGMP transduction cascade in olfactory cilia Guanylyl cyclase-d cgmp-gated channel CNGA3 cgmp PDE2 uroguanylin guanylin 0.04% OSN F/F=10% Leinders-Zufall et al., PNAS s Hu et al., Science 2007

40 Encoding

41 Tree types of cone photoreceptors

42

43 Axons of M71-espressing OSN converge into two glomeruli axons glomeruli Olfactory sensory neurons M71-LacZ transgenic mouse Vassalli et al., Cell, 2002

44 Olfactory sensory neurons OR1 OR2 OR3 OR1 OR2 OR3 Olfactory bulb Glomeruli

45 OSN responses to different odorants p-cresol m-cresol guaiacol creosol safrole trans-anethole estragole vanillin ethyl vanillin methyl salicylate cinnamic alcohol trans-cinnamic aldehyde KCl Furudono et al. Chem Senses, 2009.

46 Odorant coding odorant 1 odorant 2 mixture receptors code ORs ORs

47 Ca2+ imaging assay Light source Dichroic CCCD camera objective cell Fura-2 Fluo-4

48 Imaging of rat olfactory bulb loaded with the voltage-sensitive dye JPW3028 Johnson and Leon, J. Comp. Neurol. 2000

49 Glueneberg ganglion Main olfactory epithelium Main olfactory bulb glomeruli Accessory olfactory bulb olfactory sensory neurons lumena basal Septal organ Vomeronasal organ BV apical

50 Compound Species Effect Olfactory subsystem 3-Amino-s-triazole Mouse Attracts females? 2,5-Dimethylpyrazine Mouse Delays puberty VNO in females 2-Heptanone Mouse Extends estrus VNO 4-Ethylphenol Mouse Attracts females and VNO repels males 2-sec-Butyl-4,5-dihydrothiazole Mouse Attracts females and VNO repels males; induces 2,3-Dehydro-exobrevicomin Mouse Attracts females and VNO repels males; induces 2-Methylbut-2-enal Rabbit Attracts pups MOE to nipples Dimethyl disulfide Hamster Induces copulation VNO in males (Z)-7-Dodecenyl acetate Asian elephant Attracts males? 5-a-Androst-16-en-3-one Pig Facilitates mating MOE in females

51 Number of genes (pseudogenes) Species V1R V2R TRPC2 Man 5 (115) 0 (20) 0 (1) Chimpanzee 0 (116) 0 (17) 0 (1) Macaque? 0 (11)? Mouse 187 (121) 121 (158) 1 Rat 106 (66) 79 (142) 1 Dog 8 (33) 0 (9) 1 Caw 40 (45) 0 (16)? Opossum 98 (30) 86 (79)?

52 Key signaling molecules identified in VNO neurons basal Apical neurons: V1R, Gi2, TRPC2 Basal neurons: V2R, Go, TRPC2 apical

53

54 Transduction cascade in VNO neurons V1R/V2R Pheromone Ca2+ γ β PLC PIP2 DAG TRPC2 DAG DAG lipase α G i2 /G 0 IP3 CaM AA AA-gated channel

55 Taste Sweet Bitter Sour Salty Umami Fat?

56 Distribution of taste buds in the rodent oral cavity Soft palate Fungiform CV Epiglottis Foliate

57

58 Taste bud Lindemann, Nature 2001

59

60 V Detection of ionic stimuli with ion channels J = 105 Na+ ions/s Mucosal side C ~ 10 pf V ~ 10 mv Serosal side Q = C V ~ 10-11F x 10-3V = 10-14C ~ 105 e 1 pa ~ 107 e/sec Vm~10 mv

61 amiloride-sensitive Na+ channels H -sensitive cation channels

62 Molecular era. Gustducin McLaughlin et al. Nature 1992

63 Taste bihavior of gustducin-null mice Tastant Water Preference R > 0.5 Indifference R = 0.5 Aversion R < 0.5 R=δV 1 /(δv 1 +δv 2 ) - gustducin KO ο - wild type Wang et al, Nature 1997, 386,

64 Phototransduction cascade cgmp rhodopsin transducin γ β α cgmp-gated channel phosphodiesterase α PDE* Rh-kinase phosphorylated rhodopsin γ β α arrestin guanylyl cyclase GC PDE

65 Taste specific G-protein coupled receptors I. T1R family: T1R1, T1R2, T1R3 Hoon et al., Cell 1999 II. T2R family: T1R1 T2R37 Adler et al. Cell 2000

66 Taste receptor repertoire Species T1R T2R Man 3 25 Chimpanzee 3 24 Macaque 3 26 Mouse 3 35 Rat 3 37 Dog 3 16 Caw 3 19 Opossum 3 29

67 Ca2+ imaging assay Light source Dichroic CCCD camera objective cell Fura-2 Fluo-4

68 Responses of HEK-293 cells co-expressing T1R2 and T1R3 to sweet compounds Before After 10 mm acesulfame-k 180 µm GA mm sucrose Adler et al.cell 2000

69 T2R5 T2R4 T2R8 - receptor + receptor Adler et al. Cell 2000 Responses of HEK-293 cells transfected with T2R to bitter compounds

70 Taste bud innervation bitter

71 Taste nerve responses in T1R KO mice Umami Sweet Bitter Sour Salty Wild type T1r1-KO T1r2-KO T1r3-KO T2r5-KO Chandrashekar et al., Nature 2006

72 Family of taste G-protein coupled receptors T1R1 T1R3 amino acid receptor I. T1R family: T1R1, T1R2, T1R3 T1R2 T1R3 sweet receptor II. T2R family: T1R1 T2R36 T2R bitter receptor

73 Knock-out of PLCβ2 and TRPM5 diminishes sweet, bitter, and umami responsiveness Umami Sweet Bitter Sour Salty Plcβ2-KO Trpm5-KO Chandrashekar et al., Nature 2006

74 Taste bud cells Type I Type II Type III Basal cells Afferent nerve

75 Sapid molecules Taste receptor G-protein PLCβ2 TRPM5 3 IP Ca2+ Ca2+ store Type II cell

76 How do type II cells signal to the taste nerve? salty Type I sweet bitter umami Type II sour Type III T1R/T2R receptors Gustducin Phospholipase Cβ2 TRPM5

77 P2X2/P2X3-null mice exhibit the complete loss of behavior and neuronal responses to stimuli of all taste modalities Chorda tympani nerve responses P2X2/P2X3+/+ P2X2/P2X3-/- Finger et al., Science 2005

78 Depolarization of a taste cell results in mobilization of intracellular Ca2+ in adjacent COS-1 cells 20 mv -70 mv -70 mv

79 Classical exocytotic synapse Engelman, MacDermott, Nat.Rev.Neurosci 2004

80 ATP secretion is independent of intracellular Ca mv 10 mv Membrane voltage 5 sec 200 pa WC current Intracellular Ca 5 sec 2+ Fluo 4 Fluo 4 COS-1 Taste cell

81 The non-canonical synaptic transmission in the taste bud Sapid molecules Taste receptor Na TRPM5 + DAG IP 3 Ca 2+ G-protein Phospholipase Cβ2 Depolarization 2+ Ca store VG Na -channel Na Romanov et al., EMBO J., ATP-permeable channels ATP P2X Nerve ending

82 Taste bud cells Type III Type II Type I

83 Finger, Kinnamon F1000 Biology Reports 2011 Taste isn t just for taste buds anymore

84 Taste cells in different tissues Tissue Cell type Signaling proteins Chemical stimulus Respiratory epithelium of the nose Main olfactory epithelium Non-sensory epithelium of VNO solitary chemosensory cells olfactory sensory neurons microvillar cells solitary brush-like cells T2Rs, α-gustducin, PLCβ2, TRPM5 TRPM5 PLCβ2, TRPM5 Τ2Rs, α-gustducin, TRPM5? Tracheal epithelium solitary brush cells Τ2Rs, α-gustducin, TRPM5? Alveolar epithelium, solitary brush cells Τ2Rs, α-gustducin, TRPM5? Gastrointestinal tract epithelia: stomach colon duodenium ileum solitary brush cells solitary brush cells L-cells solitary brush cells solitary brush cells T2Rs, α-gustducin, TRPM5 T2Rs, α-gustducin, TRPM5 Τ1R2/T1R3, α-gustducin, PLCβ2 T2Rs, α-gustducin, TRPM5 α-gustducin, TRPM5 Pancreatic ducts enteroendocrine cells Τ2R2/T1R3, α-gustducin, PLCβ2, TRPM5 denatonium 2-heptanon? bitter? bitter? glucose, sucrose bitter? bitter? glucose, sucrose Testis spermatozoa α-gustducin chemoattractants?

85 Sensory transduction in the enteroendocrine cell Bertrand, Front. Neurosci, 2009

86 Спасибо за внимание!

87 PKD2L1 TRP channel is a sour receptor PKD2L1 PKD1L13 Umami Sweet Bitter Sour Salty Wild type Pkd2l1-DT

88 ASIC family of H+-gated channels

89 Heterogeneity of taste bud population NaCl Sucrose Citric acid Taste cells G E Type I Salty Sweet Bitter Umami Sour Type III Basal cells Type II