Second-Messenger Gated Ion Channels

1. Second-Messenger Gated Ion Channels Tom Mast Membrane Biophysics 10/5/07

2. An intracellular signal produced in response to a stimulus usually when a ligand binds a receptor ex: cyclic nucleotides (cAMP or cGMP) calcium inositol 1,4,5 triphosphate (IP3) diacylglycerol (DAG) What is a Second Messenger?

3. Classic Physiological Role of Second Messenger: cAMP in Rods Things to notice: Amplification steps Modulation Change in ion flow Calcium Feedback Ligand binding http://openwetware.org/wiki/BIO254:DarkNoise

4. Second Messenger Channel Topics Ion species flux- specific or not, calcium Structure- subunit architecture pore and selectivity filter conformational changes tetramerization Ligand-binding- what is the ligand conformation/ shape changes kinetics

5. Which Channels? Cyclic gated nucleotide Channel A 2 Transient Receptor Potential C 2 Inositol 1,4,5 Triphosphate 1

6. Common Subunit Structure IP3 R1 Bosanac et al., BBA vol.1742 December 2004, 89-102 http://www.ukbf.fu-berlin.de/pharma/agschaefermulti.html

7. Channel Pore and Selectivity Sequence Bovine Rod CNGC RKYVYSLYWSTLTLTTIG..ETPPPV Catfish Olfactory CGNC FCYVYCFYWSTLTLTTIG..EMPPPV Bacterial K KSA TYPRALWWSVETATTVGYGDLY.PY Shaker K SIPDAFWWAVVTMTTVGYGDMT.PV Mammalian IP3R1 LLMCIVTVLSHGLRSGGGVGDVLRK Mammalian TRPC2 FNETFQFLFWTMFGMEEHTVVDMP Common Pore Region Other regions within the channels are similar (ie S4) Original channel may have been a 1 TMD Ca++ Strong et al., Mol. Bio. Evol. 1993 (10) 221-242 Due to these relatively non-selective pore regions these channels flux cations mainly Na+ and Ca++

8. TRPC2 and IP3R1 Signaling C. Badland

9. The TRP Channels Notice: weak voltage sensor and ‘TRP’ box

10. Paper 1: TRPC2 Lucas P, Ukhanov K, Leinders-Zufall T, Zufall F A Diacylglycerol-Gated Cation Channel in Vomeronasal Neuron Dendrites Is Impaired in TRPC2 Mutant Mice Neuron. 2003 Oct 30;40(3):551-61. Primary Question: Are DAG-induced currents present in VNO neurons?

11. Figure 1 A- sensory neuron in vitro B- inside-out patch: response to DAG analogue C- F I-V relationship of SAG-induced currents note: permeable to several ion species outward current block by large cation

12. Figure 2 inside-out patch: single channel responses to SAG A Low spontaneous opening w/o SAG B. increased opening C-D Frequency histograms of opening E I-V relationship of SAG-induced currents

13. Figure 3 Ligand specifity of the SAG-induced current Important data: IP3 does not gate current neither do all fatty acids

14. Figure 4 • Whole-cell currents • w/o SAG in WT • w/ SAG in WT • W/SAG in WT • and bath application • of large cation • D-F. Same a A-C except • in TRPC2 KO • G. I-V relationship WT • H. I-V relationship KO • I. Histogram of SAG • induced currents Important Data: TRPC2 KO neurons Lack the SAG-induced current of WT

15. Figure 5 • Whole cell recording in Current-clamp • dilute urine activates neuron • C-D. This activation has an I-V relationship • similar to SAG-induced currents

16. Figures 6 + 7 Below: Phospholipase C inhibitor (U-73122) blocks the urine-induced current in voltage-clamped neurons Above: DAG kinase inhibitor induces an inward Current which is abolshed by Phospholipase C inhibitor (U-73122) in voltage-clamped neurons Important data: in neurons pharmacologically increasing or decreasing endogenous DAG produces the predicted result

17. Conclusions A urine induced current is non-selective for external cations It is dependent on PLC It is abolished by gene-targeted deletion of TRPC2 It closely resembles that of a SAG-induced current

18. Paper 2: IP3 R1 Hamada K, Terauchi A, Mikoshiba K. Three-dimensional rearrangements within inositol 1,4,5-trisphosphate receptor by calcium. J Biol Chem. 2003 Dec 26;278(52):52881-9. Primary Question: How do the allosteric factors Ca++ and IP3 effect conformational changes in the channel?

19. Simple example of Allostery http://biology.fullerton.edu/biol302/regulation.html Binding of a factor at one site alters other sites could be enzymatic activity, affinity, conformation

20. Figure 1 • A . Incubation of IP3R1 with a • lysine-protease results in • different fragment patterns • dependent on Ca++ concentration • Also dependent on C-term of • cytoplasmic domain which is • involved in tetramerization • C. Location of epitopes used in • western analysis

21. Figure 2 + 3 Left: Mg++ does not affect proteolysis while Sr++ and (maybe) Ba++ Does. IP3 does not affect proteolysis. Right: IP3R1 favors a ‘windmill’ shape in certain Ca++ concentrations

22. Figure 4

23. Figure 6 • Modeling the 3-D • Shape of IP3R1 • Based on • Transmission • Electron micrographs (fig 4) • w/o Ca++ • w/ Ca++

24. Figure 7 + 8 w/o Ca++ w/ Ca++

25. Conclusions IP3R1 is sensitive to Ca++ Channel-wide conformational changes are due to Ca++ binding and not IP3 Tetramerization may play a role in the conformational changes

26. Paper 3: CNGA2 Nache V, Schulz E, Zimmer T, Kusch J, Biskup C, Koopmann R, Hagen V, Benndorf K Activation of olfactory-type cyclic nucleotide-gated channels is highly cooperative J Physiol. 2005 Nov 15;569(Pt 1):91-102 Primary Question: What is the allosteric model for cGMP binding to CNGA2?

27. Summary of the Canonical Cilia Cascade CNCGs consist of three subunits: A2:A4:β1 in a 2:1:1 ratio A2 is required to detect most odors.

28. Calculating Cooperative Binding An allosteric relationship Binding of the first ligand changes the affinity for future ligands at other to other sites h > 1 = positive h < 1 = negative H cannot be greater than the # of binding sites

29. Figure 1 • Experimental set-up: • inside-out patches exposed • to light-sensitive cGMP • B. Light pulse • C. Example current

30. Figure 2 • Example of current • used for calculations B-D [cXMP]-response curves with calculated parameters

31. Figure 3 • Activation time-courses • Plot of time-constants • Plot of activation ratios • Voltage-effect

32. Figure 4 Activation w/ cAMP is slower At a rate consistant W/ binding Indicates changes in activation over [cGMP] is intrinsic

33. Figure 5 Channels in native Ratios (2:1:1) have Similar activation when Compared to CNGA2 Indicates kinetics are intrinsic

34. Figure 6 CNGA2 channels open spontaneously has implications as to type of allosteric model

35. Figure 8 Activation kinetics are best fit by model with 3 binding steps and with both negative and positive cooperativity

36. Conclusions CNGA2 channels have a greater affinity for cGMP CNGA2 channels display cooperative binding CNGA2 and hetereomultimer channels are affected by Vm