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Second Messenger-gated Ion channels

Second Messenger-gated Ion channels. Dr. Debra Ann Fadool 18 February 2005. CNG Ion Channels: Matulef and Zagotta. 2003. Cyclic nucleotide-gated Ion channels. Annu. Rev. Cell Dev. Biol. 19: 23-44 Zagotta Laboratory = Stoichiometry and Assembly Krammer Laboratory = Modulation

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Second Messenger-gated Ion channels

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  1. Second Messenger-gated Ion channels Dr. Debra Ann Fadool 18 February 2005 • CNG Ion Channels: • Matulef and Zagotta. 2003. Cyclic nucleotide-gated • Ion channels. Annu. Rev. Cell Dev. Biol. 19: 23-44 • Zagotta Laboratory = Stoichiometry and Assembly • Krammer Laboratory = Modulation • Martens Laboratory = Lipid Raft Localization

  2. Classic Phototransduction Cascade – Main Player is the CNG ion Channel What events occur to produce the “dark current” and subsequent depolarization? What events change to create light adaptation and hyperpolarization?

  3. Family of CNG Channels Olfactory = CNGA2, CNGA4, CNGB1b (2, 4, b) Rod = CNGA1, CNGB1 Cone = CNGA3, CNGB3 Fly = CNGL, CNG-PA C. Elegans = TAX-2, -4 Six Family Members

  4. Structural Features of CNG ion channels: • Pore • CNBD • C-linker • N-terminal • Domain • Post CNBD • Region • Modulation

  5. Pore • Has the TVGYG K channel signature sequence. • Modeled after KscA, is thought to have water-filled • vestibule intracellular to the selectivity filter. • Cations would be stabilized between the pore helices and • water molecules that hydrate the cation in the vestibule. • Putative inner helix is thought to be S6 that exhibits • conformational changes to open the pore. It is thought to • widen during nucleotide binding but is not the physical gate • that would control permeation. • Evidence for a intersubunit disulfide bond that would • form spontaneously for rapid closure of the channel in the • absence of sufficient ligand concentration. • Unlike other channels we have studied, has a specific blocker • that has a higher affinity for the closed channel state: • Tetracaine.

  6. Secondary Structure motifs Similar in CNBD and CAP Yellow = Identical Green = Conserved Blue = others Red = the cAMP molecule • B. CNBD: • Several different types of nucleotide binding proteins; one of • which is used as a model for nucleotides binding to this domain. • a. PKA • b. PKC • c. CAP – catabolite gene activator protein in bacteria • Channel Activation – VIA “Concerted Allosteric Opening Transition” • First described by Monod Model to show that independent binding • of the nucleotides (2) stabilizes a concerted opening. Energetics may • vary according to the number of ligands bound. • Distinct Order of Specificity: Structures differ by side groups • on the purine ring: • cGMP >>>>>>> cIMP>>>> cAMP

  7. Activity of Nucleotides : cGMP, cIMP, and cAMP - O C Amplitude Histogram Area o / Area o + Area c = Propen Plot the pA for a number of Vc to derive the IV relation; slope of the relation is the slope conductance in pS. All three nucleotides can bind to the CNBD but the allosteric opening transitions vary as a reflection in different Propen Propen increases with increased # of ligands bound; but the # bound of course is dictated by affinity.

  8. The Molecular Basis for Ligand Specificity….? • T560 is conserved in CNGA1: mutation affects cGMP affinity but • not cAMP. • But can’t be only source for specificity because oCNG have = • affinity for cAMP = cGMP. • D604M mutation made the selectivity reverse order; • cAMP>>>>cIMP>>>>cGMP. • Model again is taken from CAP crystal structure: Think that the • C-helices move toward the B roll of each subunit, allowing the D604 • residue to interact with the purine rings of the bound cyclic • nucleotide.

  9. The C Linker: • The residues of the linker are modulated by metals. • Three residues of the linker can affect gating – • R460, I465, and N466. • D. N-Terminal Domain: • Stabilizes the open state by decreasing the free energy (delta G) • of gating: Called the “autoexcitatory effect on gating”. • Ca/Cam binding to the N-terminal of CNGA2 causes a decrease • in Propen. • Olfactory adaptation: negative feedback of Ca (permeant ion) • for Ca/Cam that inhibits the N-terminal domain of the channel. • N and C terminus interact directly: Ca/Cam prevents this • interaction required for gating, and causes decrease • cAMP/cGMP from binding.

  10. Post-CNBD Region: • Also mediates the Ca/Cam modulation/inhibition. • Important for trafficking and heteromeric assembly • RP = truncated mutation in this region. • Types of Modulation: • Ca/Cam • Metals • PKC • DAG • Na/Ca K exchangers in protein-protein interatactions • Role of circadian rhythms

  11. Paper 1: Zheng and Zagotta. 2004. Stoichiometry and Assembly of Olfactory Cyclic Nucleotide-gated Channels. Neuron 42: 411-421. Key Finding: CNG channels in olfactory neurons are tetramers of a fixed, non-random (precise) stoichiometry: 2:1:1 (CNGA2, CNGA4, and CNGB1b). Secondary Finding: CNGA4 and CNGB1b have a higher affinity for CNGA2 than for self assembly Conclusion: Extramembranous intersubunit interactions promote assembly from the N-C or C-linker interactions.

  12. Background: • K channel assembly of subunits is random, whereas that for • ligand-gated ion channel (AChR) was known to be fixed to promote • ligand affinity. • Knew that CNGA2 (2), CNGA4 (4), and CNGB1b (b) were expressed • in native olfactory neurons but did not know ratio. • New that 2 could form functional homomeric channels and that • 4 and b would only express if also with 2. • 4. 2 by itself did not exhibit native biophysical properties of the • CNG olfactory channel. • Properties they analyzed • Functional expression • Activation by cAMP • Block by ditiazem • Ca/Cam modulation

  13. 4 Biophysical Properties = Ligand Functional Blocker Modulator Combinations = All 2 2 + 4 2 + b 2 + 4 + b (closest to native) Red= Low cAMP Black = High cAMP Green = Plus Blocker Time course of current inhibition due to Ca/Cam modulation; fit with single exponential. Fast Modulation most like Native

  14. Fluorescence Intensity Ratio Method to Determine Stoichiometry = FIR Using Rod CNG as a control • 458 nm CFP / 488 mm YFP • Assumption that unassembled • channels would not be membrane • inserted. • C-terminal tagged constructs • where CFP = green and YFP = yellow • Since two dye molecules so • close in proximity must subtract • any FRET-induced decrease in the • intensity of the donor dye (green) • Show linear regression of actual • data (red line), FRET correction is • computed to show very little • difference (green line), and solid lines • (black) are predicted based upon • logical potential ratios. GREEN YELLOW

  15. Now are using the olfactory combinations after optimization of the protocol. A vs. B: switching green and yellow tagged constructs for 2 and 4 – demonstrates in really 3:1. C: Fit mathematical models for different subunit ratios based upon RNA ratios. green yellow

  16. Same experimental protocol: Now 2 and b instead of 2 and 4. Same 3:1 results BUT>>>>>> Ratio in the membrane does not Necessarily = The Ratio of subunits in the physical channel; therefore……. Must use FRET to determine interaction distance and channel stoichiometry.

  17. Fluorescence Resonance Energy Transfer (FRET) • Is a distance-dependent interaction between the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon. • The efficiency of FRET is dependent on the inverse sixth power of the intermolecular separation, making it useful over distances comparable with the dimensions of biological macromolecules. • Spatial resolution beyond the limits of conventional optical microscopy.

  18. Requirements: 1. Donor and acceptor molecules must be in close proximity (typically 10–100 Å). 2. The absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor (J). 3. Donor and acceptor transition dipole orientations must be approximately parallel. 4. The distance at which energy transfer is 50% efficient (i.e., 50% of excited donors are deactivated by FRET) is defined by the Förster radius (Ro). The magnitude of Ro is dependent on the spectral properties of the donor and acceptor dyes Donor = 458 = CFP green Acceptor = 488 = YFP yellow Ratio A = Excitation of YFP (488) by CFP (458) at 458 nm laser

  19. FRET between 4 or b in the Presence of 2 subunits. FRET between the 2 subunits in presence of 4 or b. Ratio Ao = excitation of acceptor YFP in control oocytes when only 2 YFP and no b subunits; A – Ao = FRET efficiency = greater it is means closer together in physical distance. Donor = 458 = CFP green Acceptor = 488 = YFP yellow Ratio A = Excitation of YFP (488) by CFP (458) at 458 nm laser

  20. In this example: 2 that is CFP green, a 2 that is YFP yellow, and then add in an unlabeled b. Black = the total spectrum Red = 458 green (donor) Blue = Control Background Without b. Green = difference spectra Ratio A = green/black Take Home Concept! = If High Ratio A – Ratio Ao, then occurrence of FRET indicates two or more copies of a particular subunit of the channel or an interaction across the subunits.

  21. 3:1 Stoichiometry if you add either 4 or b to 2. 2:1:1 Stoichiometry if you add both 4 and b to 2.

  22. Testing Functional Expression of Homomeric Channels • In terms of current (A) and surface expression (B) • Another demonstration that only 2 shows FRET with itself, • homomeric, whereas 4 and b do not.

  23. Intersubunit Interactions Promote Assembly in the Absence of a T1 Between 2 and either 4 or b = high affinity binding (see dark lines). Therefore decreases frequency of 4 and b binding as a dimer. Two stage: First 2-4 and 2-b dimers form Then the dimers assemble into the heteromeric channels Uses a head-tail arrangement using the N and C terminal interacting domains

  24. Paper 2: Krajewski et al. 2003. Tyrosine Phosphory- Lation of Rod Cyclic Nucleotide-gated Channels Switches off Ca/Cam Inhibition. JNS 23(31): 10100-10106. Key Finding: Y498 in the CNGA1 is the phosphorylation Site responsible for Ca/Cam Inhibition. Secondary Finding: Y Phosphorylation of CNGA1 on the C- terminus can cause an uncoupling of the N-terminus of CNGB1 so that there is no Ca/Cam modulation. Conclusion: Y Phosphorylation decreases Propen whereas Dephosphorylation increases Propen.

  25. Background: • It was known that Ca/Cam binds with high affinity to the N-terminus • of the CNGB1 subunit to weaken the intramolecular interaction • Between the N and C termini of CNGB1 and CNGA1. • Y498 is on CNGA1 and Y1097 is on CNGB1; either mutation causes • a decreased affinity for cGMP to decrease gating. • IGF causes dephosphorylaton of these sites to increase • CNG sensitivity. • What the authors wish to address…..do phosphorylation and • Ca/Cam act as separate, independent modulators of the channel or is • there a common mechanism? • 6. I-O patches of transfected oocytes, spontaneously dephosphorylate • the channels, so the Propen increases over a 5-10 minute period. • 7. During dePhos…… K1/2 decreases approximately 2x change, • reflecting an increase in cGMP affinity.

  26. Pervanadate (to keep phosphory- • lated) and non-pervanadate • conditions - demonstrates that • more cGMP is needed to get same • response when the channel is • phosphorylated. • In the absence of Pervanadate • (no phosphorylation), Ca/CaM – • need more cGMP to get same response. Note D/R curves are fit with the Hill coefficient that indicates slope = # of cGMP molecules binding remains as two (no slope change)

  27. ATP gamma S is non-hydrolyzable • therefore do not get spontaneous • dephosphorylation shift over time. • Now if try and modulate with • Ca/CaM….fails to have an affect if • retained phosphorylation.

  28. Are these repetitive experiments? Why or Why Not? What do they add?

  29. Look at Table 1: K1/2 values for cGMP activation – what pairs of values give the greatest clues about site-directed mutation function? Now in Native rod CNG channels: When Phosphorylated, Ca/CaM has NE Why do they use Population Histograms? What do these values tell you ?

  30. Mechanism of Dual Modulation by Phosphorylation and • by binding Ca/Cam • A. Ca/CaM binding to the B1 to break interaction of N-C • B. Phosphorylation of A1 pulls C terminus away so that • there is no N-C interaction for Ca/Cam to disrupt when it • binds to the B1 N –terminus. • Phosphorylation now on B1 C-terminus, can still allow • Ca/CaM to bind B1 on N-terminus to have inhibition.

  31. Paper 3: Brady et al., 2003. Functional Role of Lipid Raft Microdomains in Cyclic Nucleotide- Gated Channel Activation. Mol. Pharm. 65: 503-511. Key Finding: Movement of CNGA2 into lipid raft domains can change function of the channel by increasing affinity of cAMP. Secondary Finding: Heterologously expressed and native CNGA2 in olfactory tissue are expressed as a fraction in the lipid raft domains. Conclusion: Lipid lowering drugs could affect olfaction via functionally altering the biophysics of the CNGA2 (but they do not have the correct stoichiometry…..?)

  32. Background on Lipid Rafts: • 1. Rich in sphingolipids and cholesterol • Act to concentrate certain membrane • proteins, signal transduction cascades, • and ion channels. • Many channelopathies are attributed • to improper trafficking to the membrane • therefore rafts are important to assemble • the correct signalling molecules in a • spatially confined manner for • efficient transduction. • 4. Lipid rafts have good resistance to • solubilization with nonionic detergents • (like Triton X-100) and therefore • proteins are retained in the pellet.

  33. Control vs. High Salt (KI): To interrupt any p-p interactions Used fractionation of CNGA2 transfected HEK 293 cells – Sucrose Density Gradient Centrifugation. Track migration of the Flag epitope tagged channel by comparison with other raft associated (caveolin, flotillin) molecules but not with one that is not generally associated (transferrin R).

  34. A: 1 = wt • 2 = Flag tagged • 3 = CFP tagged • 4 = mock transfection • B: Can treat with enzymes to digest • the suspected glycosylation (lose • the upper band at 114 kDa) • C: Why are all the CNGA2 fractions • at 114 kDa and not 81 kDa like in the • control panel of A? • Demonstrates is a fraction that • co-migrates with caveolin but also • expression that co-migrates with the • transferrin R

  35. Despite the co-migration of Caveolin • and CNGA2 using sucrose density gradient, • Protein-protein interaction is not supported: • No ability to co-immunoprecipitate • (reciprocal pull down). • Confocal does not support co-localization • at the cell membrane.

  36. Using drugs to deplete cholesterol (+CD), there is a Reduced Buoyancy of CNGA2 in the raft. Demonstrates that the integrity of the CNG rich lipid raft membrane has been disturbed

  37. Typically PGE1 stimulates CNG channel • activity unless there is CD+ pretreatment. • Ca influx into the open CNG channels • causes a decrease in delta F so they • decided to invert their spectrographic • curves to denote a positive direction = • increase in Ca influx. • Even though PGE stimulation evoked • Increase in Ca influx….. • 2 interpretations: • More cAMP (wasn’t according to their • Elisa Assay). • Different biophysical property of the • CNG (turned out to be the later with • single channel analysis…….)

  38. Cholesterol Depletion (+CD) increases the Concentration of cAMP needed to achieve the same I/Imax in terms of current calculated from single channel data: I = N po i K1/2 increased from 39 to 123 uM + CD

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