Transcript Second Messenger Activated Ion Channels: TRP Channels

Second Messenger Activated Ion
Channels: TRP Channels
Lindsey Biggs, Greg Loney
Oct, 2011
Transient Receptor Potential (TRP) family of
cation channels
Drosophila trp mutation – resulted in
transient response to light
Typically flux Na+ and/or Ca++
Single AA substitution alters cation selectivity
Often referred to as store operated channels
or ‘SOCs’
Expressed in both excitable and nonexcitable
cells
Ubiquitous channel family with various
methods of activation:
• Ligand (chemosensation)
• Thermal (temperature)
• Mechanoreceptor (somatosensation)
Group 1
TRPC
TRPV
TRPM
Total number of channels expressed by
exemplary species:
Worms – 17
Flies – 13
Mice – 28
Humans – 27
Group 2
TRPA
TRPN
Legend:
Green = Ankyrin repeats
Pink = Coiled coil domain
Blue = TRP domain
“P” = Pore domain
Group 1
TRPC
TRPV
TRPM
Group 2
TRPA
TRPN
Ankyrin – Present on NTD of TRP channels, typically 33 AA residues per
repeat. Generally labeled as protein-protein interaction motifs and
anchoring. Bind ATP, PLC, etc and may be important for channel sensing and
gating properties
TRP box – Located on CTD of S6, potentially important for channel
closing/inactivation (?). Includes the invariant EWKFAR sequence that is
conserved in all Trp proteins (?).
TRP channels are activated by signal transduction pathways.
For a cell at rest, extracellular to intracellular ratios of Ca++ are ~20,000:1. It is
likely that Ca++ modulates all TRP channels in some way, either directly or
indirectly.
TRP channels (e.g. TRPV1) are inhibited by PIP2 until it is hydrolyzed by PLC. PIP2
interacts with positively charged areas of channel (TRP box).
Some channels (TRPM5) may be SOCs.
Three theories of SOCs:
1. Direct link between ER and channel
2. Putative second messenger
3. Fusion of vesicles containing SOC TRP
channels
In vivo identification and
manipulation of the Ca2+ selectivity
filter in the drosophila transient
receptor potential family
Liu, Wang, Postma, Obukhov,
Montell, Hardie
2007
Methods
• Trp null mutants demonstrate a transient LIC and sever retinal
degeneration.
• Generated mutant flies so that TRP was the only light sensitive
channel present. TRPL mutants demonstrate highly selective Ca2+
current.
• Asp621 and Asp626 are the only acidic residues unique to TRP
sequence and are substituted in trpl mutants
– Mutant flies were back-crossed to generate three different point
mutated flies at Asp621 and one at Asp626
• Whole cell recordings were conducted from dissocated ommatidia
Figure 1:
a. Sequence homology
between WT (trp) and
trpl mutants
b. Protein expression
amongst WT and
mutants
c. Immunolocalization
of TRP channel –
targeted to
rhabdomeres
Figure 2:
Reversal potentials obtained
in control and biionic
conditions (cation:Cs+) for
flies expressing WT channels
and point-mutated
channels. Brief flashes were
presented to ommatidia
held at 10mv holding
potential steps
Figure 3:
Similar reversal potentials
obtained under biionic
conditions (cation:Cs+) for
flies expressing WT channels
and point-mutated channels
in which the charge of the
putative pore residue was
titrated. Heteromultimers
demonstrated intermediate
Erev . Residues are plotted in
order of decreasing charge
Figure 4:
Reversal potentials obtained
under biionic conditions for
various mutants as a
condition of cation size (A)
and the inferred
permeability ratios for these
same cations
Figure 5:
a.
Top trace is control noise
present before channel
activation. Bottom trace is
steady-state noise after
activation
b.
Using both the control noise
(top trace) and measured
reversal potentials, data were
fit to an equation in order to
estimate the amount of
single channel conductance
c.
Quantum bumps (single
absorbed photons)
Figure 6:
a.
Inhibition of LIC as a function of Mg2+ in a Ca2+ free bath
b.
I-V relationship of LIC. Asp626 mutants demonstrated a rectification
current similar to WTs. The slope of the conductance between -10
and 40mv was lower in WT, presumably due to voltage dependent
Mg2+ block (Ringer’s solution)
c.
Demonstration of channel blockade by La3+ (left) and ruthenium red
(right) - both polyvalent ions. La3+ presumably utilizes a distinct
residue
Figure 7:
A-D. Traces from WT and various mutants demonstrating the waveforms
of flash induced LIC in both baths that contain Ca2+ and those that do not.
E. Time-to-peak and time-to-decay. Note AspD621G mutants’ kinetics were
not at all Ca2+ dependent. Often referred to as the fastest known Gprotein coupled signaling cascade.
Figure 8:
A-D. Responses to maintained
illumination in WT and mutants. Asp621
mutants’ responses quickly decayed to
baseline thus mimicking the original trp
null mutants.
E. Kir2.1 responses in WT and mutants.
Kir2.1 is inwardly-rectifying K+ channel
that is PIP2 dependent.
Summary
•First demonstration, in vivo, that TRP is a pore
forming channel that is selectively permeable to
Ca2+ .
•Altering a single amino acid residue in the
putative selectively filter abolished Ca2+
selectivity and resulted in a phenotype that was
indistinguishable from the original trp null
mutant.
•All that work summed in two points….thats why
its in J Neuro…
Requirement of calcium-activated
chloride channels in the activation
of mouse vomeronasal neurons
SangSeong Kim, Limei Ma, C. Ron Yu,
2011.
Review of TRPC2 in the accessory
olfactory system
• TRPC2 is found in all vomeronasal sensory neurons, but
not in the main olfactory bulb or brain.
– Located at the dendritic tip of the VSNs.
• Field potential and extracellular recordings showed
that responses in TRPC2 -/- mice to urine components
were absent or diminished.
• TRPC2 -/-mice show behavioral abnormalities:
– Deficit s in male-male aggression and express subordinate
behavior. (Also, seen in females).
– Abnormalities in sexual behavior; increased male-male
mounting, but normal mating with females.
F. Zufall, 2005. Review.
See for references.
TRPC2 is gated by DAG
http://www.springerimages.com/Images/LifeSciences/1-10.1007_s00424-005-1432-4-9
Conditions used to study Cl- current
Control:
Figure 1
Control/mimicking natural conditions:
Intracellular solution: contained ClExtracellular solution: also contained ClExpected effect upon channel opening:
Cl- flow out of cell, resulting in an
inward current flow (expulsion of
negative current).
Conditions used to study Cl- current
Control:
Figure 1
MSF-:
Methanesulfonate (MSF-):
Non-permeable anion subsituted
for Cl- in intracellular solution.
Intracellular: very low Cl- levels
Extracellular: High/normal ClExpected effect upon channel
opening: very little to no Cl=
mediated inward current.
Conditions used to study Cl- current
Control:
MSF-:
Low e.c.
chloride:
Figure 1
Gluconate substitution:
Substitue non-permeable gluconate in
place of chloride in extracellular solution.
Intracellular: High/control Cl- levels
Extracellular: Low Cl- levels
Expected effect upon channel opening:
large outflow of Cl- anions, resulting in a
large inward current.
Urine activated chloride conductance
in VNO neurons
Figure 1
• As expected, altering intracellular (I.C.) chloride reduced the inward
current.
• Niflumic acid:
– Cl- channel blocker.
• Treatment reduced the induced inward current by 70%, similar to
MSF- condition.
VNO neuron sensitivity to small
currents
• Amplitude of inward current was
very low, ranging from 2-12 pA.
This is 10-100 times lower than for
olfactory neurons, but this is
supported by data from field
recordings, showing 10-100 fold
differences between MOE and
VNO epithilium.
Figure 1
Urine-activated Cl- current requires
extracellular Ca2+
• E.C. calcium levels
were then altered to
determine the role of
Ca2+ in these Clcurrents.
• With less Ca2+ in the
extracellular fluid,
there were decreases
in the urine-evoked
inward currents, thus
Ca2+ is necessary for
urine-evoked
currents.
Figure 2
Urine-activated Cl- current requires
intracellular Ca2+
• EGTA and BAPTA (Ca2+
chelating agents) were
used in I.C. solutions to
decrease I.C. Ca2+.
• With low I.C. Ca2+,
there was a reduction in
the effect of E.C. Cl- ion
substitution.
Figure 2
Activation of Cl- current in TRPC2 -/Mice
• With low I.C. Cl-, there is no
inward current, thus, all
current seen in TRPC2 -/- mice
is via Cl- channels.
• In TRPC2 -/- mice, 80% of
urine-evoked current was
conserved, therefore, a TRPC2
independent mechanism is
responsible for activation of
CACC.
Black: Cl- current
Gray: non-Cl- current
Figure 3
Intracellular Ca2+ release activates
CACC
• Since TRPC2 was the only TRP
channel found in VNO neurons,
they hypothesized that CACC was
being activated by release of Ca2+
from intracellular stores.
• In the presence of Ruthenium Red
(RR, blocks IP3 mediated Ca2+
release) and Thapsigargin (TG,
depletes I.C. Ca2+ stores), urineinduced currents were reduced.
• In the MSF- condition where Cl- currents are not
present, the effect of RR and TG are eliminated.
• In TRPC2 -/- mice, RR and TG completely block
urine-evoked response of VNO neuron.
• TRPC2 KO doesn’t alter
resting potential.
• Extracellular recordings
so as not to disturb I.C.
Ca2+.
• Urine application
increased spiking rate in
WT and KO (less firing in
KO overall).
• Block Cl-(NA or
SITS)=blocked urineinduced spike increase.
• TRPC2 channels alone
are not sufficient to
excite VNO neurons.
WT= square
KO= circle
Conclusion: CACC are required
for responses to urine.
Discussion/Conclusions
• Cl- current contributes significantly to urine-evoked VNO responses.
• This current is sufficient to drive changes in spiking rate in WT and
TRPC2 -/-.
• CACC currents were able to mediate pheromone activation
independently of TRPC2 (Need to think about this conclusion).
• Unlike the MOS, in which KO of CNG channel eliminates Cl- current,
thus the animal is anosmic, only a portion of the inward current is
carried by TRPC2, and TRPC2 influx of Ca2+.
• Ca2+ influx via activation of TRPC2, and Ca2+ release from I.C.
stores act synergistically to activate the Cl- conductance, but VNO
neurons can be excited without TRPC2.
• TRPC2 -/- behavioral phenotype may be explained by reduced VNO
function and selective loss of the basal layer of VNO neurons (Go
pathway).
Essential Role for TRPC5 in Amygdala
Function and Fear-Related Behavior
Antonio Riccio, et. al., 2009
Background: Fear circuitry
• Lateral amygdala (LA) is the input center for
auditory, somatosensory, visual, olfactory and
taste systems.
• Central amygdala(CeA) is the output center for
fear conditioning circuitry.
Background: Methods- measures of
anxiety
• Elevated Plus Maze: Time spent and number of
entries into open and closed entries.
• More time/entries into open arm= less anxiety
• Open Field test: Measure number of entries and
time spent in center of the open field.
• More time/more entries into center= less
anxiety.
• Social interaction: place animal in center
cage and record time spent in area with
novel and familiar conspecifics. Also,
record nose contacts.
http://btc.bol.ucla.edu/plus.htm
Background: Methods- fear
conditioning
http://www.scholarpedia.org/article/Emotional_memory
http://www.scholarpedia.org/article/Emotional_memory
TRPC5 expression in the mouse brain
• TRP channels, although
commonly believed to be
involved in stimulus
perception, can also be
found in neuronal tissue,
e.g. TRPC5.
• TRPC5 is localized to
pyramidal neurons in
hippocampus, lateral (LA)
and central (CeA)
amygdala.
• TRPC5 was not found in
glial cells or interneurons
in any of these areas.
Figure 1
TRPC5 and conditioned fear responses
• Freezing responses in wild type and
TRPC2 -/- mice were not significantly
different 30 minutes or 24 hours
after conditioning with foot shock
(0.7 mA, 2 s).
Figure 2D
• But with a less aversive foot shock
(0.4 mA, 0.5 s), TRPC5 -/- exhibited
a higher percent of freezing 24
hours after conditioning.
Figure S5A
TRPC5 and “innate fear”
Elevated plus
maze
• TRPC5 -/- mice showed decreases
in anxiety-like behavior.
Open Field
Figure 2
Social Interaction
Synaptic transmission and LTP in
cortical and thalamic inputs to the LA
in
TRPC5
-/mice
• Long term potentiation (LTP)- an
increase in synaptic strength
between two neurons after high
frequency stimulation. Effect is
due to increased sensitivity (more
receptors) of the post-synaptic
terminal.
• Paired pulse facilitation- an
increase in the EPSP evoked by the
second of two pulses. This is
thought to be due to an increase
in the probability of
neurotransmitter release from the
pre-synaptic terminal.
http://www.longtermpotentiation.com/long-termpotentiation-the-basics-of-nerve-impulsetransmission/
Membrane excitability and synaptic
function in 10-13 day old mice
Figure 3A
Membrane excitability and synaptic
function in 10-13 d.o. mice
• Neurons in TRPC5 -/- mice
show decreased synaptic
strength between cortical
fibers and LA, than WT
littermates.
– This was due, in part, to
decreased probability of
release, as seen in paired pulse
facilitation in C,D.
Figure S7
Membrane excitability and synaptic
function in 4-5 week old mice
TRPC5 leads to no differences
in firing properties, and basal
synaptic transmission in
afferent inputs to LA.
Figure 3
Long-term potentiation at cortico- and
thalamo-amygdala synapses
• Deletion of
TRPC5 channels
does not affect
LTP at cortico- or
thalamoamygdala
synapses.
Cortical input
Thalamic input
Figure 3
Probability of neurotransmitter release
from cortical and thalamic inputs
• MK-801- selective,
irreversible, NMDA
receptor blocker. NMDA
channels are blocked only
when they have been
opened by pre-synaptic
glutamate release.
– Therefore, Pr determines
the rate of the block.
• No differences between
WT and TRPC5-/-.
Figure 4
Quantal amplitude from cortical and
thalamic inputs
• Strontium (Sr2+), when
substituted for Ca2+, causes
fusion and release of a single
vesicle from the pre-synaptic
terminal.
• No differences were seen in
the amplitude of EPSCs in the
presences of Sr2+.
• Conclusion: Basal firing
properties of LA and synaptic
transmission from cortical and
thalamic fibers are not
affected by the loss of TRPC5
channels.
Figure 4
mGluR and CCK2 receptor mediated
currents
• Binding of glutamate
to the mGluR is
believed to open
TRPC5 channels.
• Blocking mGluR and
CCK (cholecystokinin)
has anxiolytic effect.
• I/V curves were similar
for WT and TRPC5-/-,
but response
amplitudes were
smaller in TRPC5-/mice.
Figure 5
• LA neurons in
TRPC5 -/- mice
fired fewer number
of action potentials
to presynaptic
stimulation, when
compared to WT.
Figure 6
• CCK4, a CCK receptor
agonist, in the E.C.
solution caused an
increase in inward
current in slices from
WT animals.
• TRP channel
antagonists (2-APB)
blocked the effect.
Figure 7
• The effect of CCK4 application
on inward current is absent in
TRPC5 -/- mice (d,e,f).
• The increase in
spike firing by
application of CCK4
was also absent in
TRPC5 -/-mice.
Conclusions
• Deletion of TRPC5 produced a decrease in
innate fear related behaviors and may be
involved in conditioned fear under some
conditions.
• These data suggest that this effect is due to a
lack of TRPC5 channel activation via mGluR1
and CCK receptors.