slide1 l.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
Second Messenger-gated Ion channels PowerPoint Presentation
Download Presentation
Second Messenger-gated Ion channels

Loading in 2 Seconds...

play fullscreen
1 / 38

Second Messenger-gated Ion channels - PowerPoint PPT Presentation


  • 251 Views
  • Uploaded on

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

loader
I am the owner, or an agent authorized to act on behalf of the owner, of the copyrighted work described.
capcha
Download Presentation

Second Messenger-gated Ion channels


An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript
slide1

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
slide2

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?

slide3

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

slide4

Structural Features of CNG ion channels:

  • Pore
  • CNBD
  • C-linker
  • N-terminal
  • Domain
  • Post CNBD
  • Region
  • Modulation
slide5

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.
slide6

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
slide7

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.

slide8

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.
slide9

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.
slide10

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
slide11

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.

slide12

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
slide13

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

slide14

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

slide15

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

slide16

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.

slide17

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.
slide18

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

slide19

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

slide20

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.

slide21

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.

slide22

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.
slide23

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

slide24

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.

slide25

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.
slide26

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)

slide27

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.
slide28

Are these repetitive

experiments?

Why or Why Not?

What do they add?

slide29

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 ?

slide30

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.
slide31

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…..?)

slide32

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.
slide33

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).

slide34

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 but what is 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
slide35

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.
slide36

Using drugs to deplete cholesterol (+CD), there is a

Reduced Buoyancy of CNGA2 in the raft.

slide37

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…….)
slide38

Cholesterol Depletion (+CD) increases the

Concentration of cAMP neededto achieve

the same I/Imax in terms of current calculated

from single channel data:

I = N po i