Olfaction Ion channels in olfactory transduction

Transcription

1 Olfaction Ion channels in olfactory transduction Anna Menini Neurobiology Sector International School for Advanced Studies (SISSA) Trieste, Italy Odorant molecules vanilla camphor apple musk Olfactory bulb lemon peppermint Olfactory epithelium hyacinth raspberry jasmine amber Nasal cavity fish garlic chocolate eucalyptus Nostril pathway green pepper grapefruit Retronasal pathway marigold urine fatty skunk woody 1

2 The Olfactory Epithelium Scanning electron micrograph of the human olfactory epithelium Firestein S Nature 2001 Morrison and Costanzo J Comp Neurol 1990 Scanning electron micrograph of the human olfactory epithelium Scanning electron micrograph: cilia of a human olfactory sensory neuron 1 µm Morrison and Costanzo J Comp Neurol 1990 Morrison and Costanzo J Comp Neurol

3 An isolated olfactory sensory neuron OLFACTORY TRANSDUCTION Olfactory sensory neurons - convert an odorant stimulus into an electrical response - graded depolarizing potential - generation of action potentials Electrical response to odorant stimuli of an olfactory sensory neuron Odorant response Whole-cell recording V holding = -50 mv Anna Boccaccio 3

4 1,000 odorant receptor genes Firestein S, Picco C, Menini A 1993 J Physiol Nobel Prize in Physiology or Medicine 2004 Olfactory transduction AC camp ATP Ca Ca CNG Linda Buck Richard Axel for their discoveries of odorant receptors and the organization of the olfactory system α β γ Ca Ca Ca Ca Ca Ca Ca Na Na Ca Na Na Na Na Na inside outside 4

5 Resting electrochemical gradients at the apical end of olfactory sensory neurons Caged compounds Caged compound Uncaged: physiologically active Photorelease of camp in the cilia A Photorelease of camp E chloride = 0 mv Symmetrical 50 mv B 50 mv -50 mv -50 mv Menini 1999 Curr Opin Neurobiol Boccaccio and Menini 2007 J Neurophysiol 5

6 Photorelease of camp: Blockage by Niflumic Acid Photorelease of camp E chloride = -50 mv Gluconate in pipette A NFA B NFA 46 mv -54 mv -64 mv V holding = -50 mv Boccaccio and Menini 2007 J Neurophysiol Boccaccio and Menini 2007 J Neurophysiol The Ca-activated current can be up to 90% of the total transduction current in the presence of 1 mm extracellular Ca and symmetrical outside inside OR Activation of CNG ion channels 1 m α γ β ATP AC camp CNG channel Ca 2 Na CaM 6

7 Ionic channels directly activated by cyclic nucleotides Photoreceptors: Fesenko, Kolesnikov, Lyubarsky, 1985 Nature camp-gated currents in an excised inside-out patch 0.5 mm Olfactory sensory neurons: Nakamura and Gold, 1987 Nature 20µm Pifferi S, Boccaccio A, Menini A 2006 FEBS Letters camp-gated currents Modulation by Ca-calmodulin of camp-gated currents Hill equation K 1/2 = 2.8 µm n = 1.3 Chen and Yau 1994 Nature 7

8 Olfactory CNG channel subunits Ca concentration increases in the cilia and has a dual effect 1- inhibitory 2- excitatory Pifferi S, Boccaccio A, Menini A 2006 FEBS Letters outside Inhibition CNG channel Ca 2 Na Ca inhibitory effects: in combination with Calmodulin inside OR 1 m α γ β ATP AC camp CaM PDE CaM 1- inhibition of camp-gated channel 2- activation of Phosphodiesterase AMP 8

9 outside inside OR 1 m α γ β ATP Amplification AC camp CNG channel Ca 2 Na CaM - Ca 2 activated - channel - Ca excitatory effect: direct activation of current In situ gradient measurements (rat, mucus/knob concentrations) [-] i = 69 mm [-] o = 55 mm Ecl = 6mV (Reuter et al. 1998) outside Na/Ca exchanger CNG channel Ca 2 Na Ca 2 activated - channel Ca-activated currents in an excised inside-out patch inside OR 1 m α β γ AC CaM - - Na /Ca 2 exchanger 0.5 mm ATP camp CaM PDE AMP Pifferi S, Boccaccio A, Menini A 2006 FEBS Letters 9

10 Ca-activated current: Calcium sensitivity Ca-activated current: anion selectivity Hill equation K 1/2 = 2.6 µm n = 3.2 K 1/2 = 4.7 µm n = 2.5 Ca-activated current: blockage Molecular identity of Ca-activated channels is still unknown Pifferi S, Pascarella G, Boccaccio A, Mazzatenta A, Gustincich S, Menini A, Zucchelli S 2006 PNAS 10

11 Ca-activated channels TMEM16 (ANO) Family Wadelius cloned the gene responsible for Best macular distrophy 1998 Nathans reported that Bestrophins are chloride channels 2002 Qu, Fischmeister and Hartzell mbest2 is a Cadependent chloride channel 2004 TMEM16 (ANO) Are Ca-activated chloride channels Caputo et al 2008 Yang et al Schroeder et al 2008 Yang et al Nature Heterologous expression of Tmem16b in HEK cells EGFP/Tmem16b native Ca sensitivity Tmem16b Ca 2 20µm 20µm Pifferi S, Dibattista M, Menini A 2009 Eur J Physiol Pifferi S, Dibattista M, Menini A 2009 Eur J Physiol 11

12 SISSA Trieste, Italy Join us! PhD and Post-doc fellowships are available 12