Transcript Second-Messenger Gated Ion Channels

Second-Messenger Gated Ion Channels
Tom Mast
Membrane Biophysics
10/5/07
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)
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
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
Which Channels?
Cyclic gated nucleotide Channel A 2
Transient Receptor Potential C 2
Inositol 1,4,5 Triphosphate 1
Common Subunit Structure
IP3 R1
Bosanac et al., BBA vol.1742 December 2004, 89-102
http://www.ukbf.fu-berlin.de/pharma/agschaefermulti.html
Common Pore Region
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
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++
TRPC2 and IP3R1 Signaling
C. Badland
The TRP Channels
Notice: weak voltage sensor and ‘TRP’ box
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?
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
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
Figure 3
Ligand specifity of the SAG-induced current
Important data: IP3 does not gate current
neither do all fatty acids
Figure 4
Important Data: TRPC2 KO neurons
Lack the SAG-induced current of WT
Whole-cell currents
A. w/o SAG in WT
B. w/ SAG in WT
C. 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
Figure 5
A. Whole cell recording in Current-clamp
dilute urine activates neuron
C-D. This activation has an I-V relationship
similar to SAG-induced currents
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
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
Paper 2: IP3 R1
Hamada K, Terauchi A, Mikoshiba K.
Three-dimensional rearrangements within inositol 1,4,5trisphosphate 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?
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
Figure 1
A . Incubation of IP3R1 with a
lysine-protease results in
different fragment patterns
dependent on Ca++ concentration
B. Also dependent on C-term of
cytoplasmic domain which is
involved in tetramerization
C. Location of epitopes used in
western analysis
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
Figure 4
Figure 6
Modeling the 3-D
Shape of IP3R1
Based on
Transmission
Electron
micrographs
(fig 4)
A. w/o Ca++
B. w/ Ca++
Figure 7 + 8
w/o Ca++
w/ Ca++
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
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?
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.
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
Figure 1
A. Experimental set-up:
inside-out patches exposed
to light-sensitive cGMP
B. Light pulse
C. Example current
Figure 2
A. Example of current
used for calculations
B-D [cXMP]-response
curves with calculated
parameters
Figure 3
A. Activation time-courses
B. Plot of time-constants
C. Plot of activation ratios
D. Voltage-effect
Figure 4
Activation w/
cAMP is slower
At a rate consistant
W/ binding
Indicates changes
in activation over
[cGMP] is intrinsic
Figure 5
Channels in native
Ratios (2:1:1) have
Similar activation when
Compared to CNGA2
Indicates kinetics are intrinsic
Figure 6
CNGA2 channels open spontaneously
has implications as to type of allosteric model
Figure 8
Activation kinetics are best fit by model with 3 binding steps
and with both negative and positive cooperativity
Conclusions
CNGA2 channels have a greater affinity for
cGMP
CNGA2 channels display cooperative binding
CNGA2 and hetereomultimer channels are
affected by Vm