Second-Messenger Gated Ion Channels

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Second-Messenger Gated Ion Channels

• Tom Mast
• Membrane Biophysics
• 10/5/07

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What is a Second Messenger?

• 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)

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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/BIO254DarkNoise
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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

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Which Channels?

• Cyclic gated nucleotide Channel A 2
• Transient Receptor Potential C 2
• Inositol 1,4,5 Triphosphate 1

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Common Subunit Structure
IP3 R1
Bosanac et al., BBA vol.1742 December 2004,
89-102
http//www.ukbf.fu-berlin.de/pharma/agschaefermult
i.html
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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
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TRPC2 and IP3R1 Signaling
C. Badland
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The TRP Channels
Notice weak voltage sensor and TRP box
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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 3040(3)551-61.

Primary Question Are DAG-induced currents
present in VNO neurons?
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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
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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
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Figure 3
Ligand specifity of the SAG-induced current
Important data IP3 does not gate current
neither do all fatty acids

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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
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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

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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
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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

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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 26278(52)52881-9.

Primary Question How do the allosteric factors
Ca and IP3 effect conformational changes in
the channel?
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Simple example of Allostery
http//biology.fullerton.edu/biol302/regulation.ht
ml
Binding of a factor at one site alters other
sites could be enzymatic activity, affinity,
conformation
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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

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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
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Figure 4
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Figure 6

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

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Figure 7 8
w/o Ca
w/ Ca
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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

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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 15569(Pt 1)91-102

Primary Question What is the allosteric model
for cGMP binding to CNGA2?
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Summary of the Canonical Cilia Cascade
CNCGs consist of three subunits A2A4ß1 in a
211 ratio A2 is required to detect most odors.
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Calculating Cooperative Binding

• An allosteric relationship
• Binding of the first ligand
• changes the affinity for future ligands at other
• to other sites
• h gt 1 positive
• h lt 1 negative
• H cannot be greater than the of binding sites

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Figure 1

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

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Figure 2

• Example of current
• used for calculations

B-D cXMP-response curves with
calculated parameters
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Figure 3

1. Activation time-courses
2. Plot of time-constants
3. Plot of activation ratios
4. Voltage-effect

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Figure 4
Activation w/ cAMP is slower At a rate
consistant W/ binding Indicates changes in
activation over cGMP is intrinsic
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Figure 5
Channels in native Ratios (211) have Similar
activation when Compared to CNGA2 Indicates
kinetics are intrinsic
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Figure 6
CNGA2 channels open spontaneously has
implications as to type of allosteric model
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Figure 8
Activation kinetics are best fit by model with 3
binding steps and with both negative and positive
cooperativity
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Conclusions

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