CCB Lenox, March 2005

1
CCB Lenox, March 2005

• Imaging and modeling calcium signals from
  individual ion channels

Ian Parker, Angelo Demuro Jian-Wei
Shuai Dept. Neurobiology Behavior, University
of California, Irvine
2

• The era of the well-mixed cell is behind
  us. Signaling is punctate, and many important
  questions in cell signaling need to be
  considered as four-dimensional processes
• Olaf S. Andersen, Editorial, J. Gen.
  Physiol. 1253-12, 2005

We are visualizing the dots (single-channel
events) that make up cellular calcium signals
3
Extracellular and intracellular calcium sources
Plasmalemmal channel
IP3
Cytosol
Pump
IP3R
Cell Membrane
ER
Pump
Ca2
4
Motivations to develop functional single-channel
Ca2 imaging
1. To study the Ca channels themselves
previously possible only by the
electrophysiological patch-clamp
technique. Patch-clamping has limitations
including - lack of spatial information regarding
channel location inability to obtain
simultaneous, independent recordings from
multiple channels need for physical access of
pipette possible disruption of channel function
during seal formation. 2. To image the
spatial and temporal distribution of
cytosolic Ca2 around an open Ca2 channel.
5
Imaging gt400 channels at once
6
Ca2 signals are large and fast near the
channelmouth, but small and slow only 1 mm
away. So, to get a faithful record of channel
gating, we need to record local, near-membrane
signal.
7
The model
z
y
50nm
x
Buffers Immobile - 300 mM Kd 2 mM Fluo-4
dextran - 40 mM Kd 3 mM D 15 mm2 s-1
Channel 0.1 pA for 10 ms
8
Distribution of free Ca2 and Ca-bound indicator
around a channel
Free Ca2
Ca-bound indicator
0.1 pA Ca current for 10 ms
9
Smaller sampling volumes provide better kinetic
resolution
Fluorescence signal (Fluo-Ca) sensed throughout
different volumes of cytosol centered around the
channel mouth
Initial rate of decay of signal following channel
closure
Some very small SI prefixes femto f
10-15 (1 fl 1 x 1 x 1 mm) atto
a 10-18 zepto z 10-21 yocto y 10-24
(2 ymol 1 molecule)
10
But, stochastic variations in number of Fluo-Ca
molecules introduce excessive noise at small
volumes
Signal ( Ca ions that enter through
channel and bind Fluo) SD of noise
(square root of TOTAL number of Fluo-Ca
molecules) Signal-to-noise ratio
Molecular shot noise will predominate over
other noise sources (photon shot noise, camera
dark noise, camera read-out noise)
11
Optimal compromise between kinetic resolution and
noise level achieved with sampling volumes 0.03
0.1 fl
How can we actually achieve this?
12
Total internal reflection microscopy
air
glass
13
Total internal reflection microscopy
air
glass
14
Total internal reflection microscopy
Evanescent wave
air
glass
15
Total Internal Reflection Fluorescence
Microscopy(TIRFM)The evanescent wave formed by
refraction at a glass/water interface extends
only a few hundred nm into the aqueous phase.
16
Model point-spread functions of confocal and TIRF
microscopes
17
Cultured cells expressing GFP-tagged membrane
protein imaged by conventional epifluorescence
18
The same cells viewed by TIRFM
19
TIRFM imaging of single-channel Ca2 signals
Ca2 entry through plasma membrane channels
expressed in Xenopus oocytes
20
(No Transcript)
21
Optical single-channel recording Single Channel
Ca2 Fluorescence Transients (SCCaFTs)
22
Imaging Ca2 flux through single nAChRsSCCaFTs
generated by ACh are blocked by co-application of
curare
23
The Channel ChipRepresenting the simultaneous
activity of hundreds of channels
24
The Channel ChipRepresenting the simultaneous
activity of hundreds of channels
25
Imaging single channel events with high time
resolution SCCaFTs recorded at 500 frames s-1
26
Comparing single channel properties of muscle
abgd and neuronal a4b2 nAChRsChannel open time
histograms generated from SCCaFT duration
measurements
Neuronal a4b2 nAChRs
Muscle abgd nAChRs
27
Ca2 influx through nicotinic channels depends on
electrochemical driving force
28
Gating of nACh channels as a function of ACh
29
Spatial width of experimental SCCaFTs matches
simulation
So We can locate channel with precision lt100 nm
30
Restricted mobility of channels in membrane
Diffusion coefficient for free motility of a
protein in membrane D 3 mm2 s-1 So mean
displacement after 60s would be 25 mm sqrt
(4 D 60)
31
Channel MappingnAChR channels are randomly
distributed in the oocyte membrane
32
Advantages of optical patch-clamp recording
Massively parallel - simultaneous and independent
recording from many hundreds ion channels with
time resolution approaching that of patch-clamp
recording
Applicable to both voltage- and ligand- gated ion
channels with partial Ca2 permeability
Allows spatial mapping of the functional ion
channels and measurement of their motility
Permits comparison of gating properties of many
ion channels in relation to their location in the
cell membrane
33
Thanks to
Angelo Demuro Single-channel imaging
Jian Wei Shuai Modeling
The NIH for support GM 48071, GM 65830
34
The End!
Badwater Ultramarathon Death Valley 135
miles, 13000ft ascent, 127F
35
(No Transcript)
36
Depletion of extracellular Ca2 is not a problem
0.1 pA, 1s
50 nm
40 mm
37
2. Intracellular Ca2 release through clusters of
IP3 receptor /channelsHow many channels in a
cluster, and how are they distributed?
38
Extracellular and intracellular Ca2 signals
Puffs from IP3R channels in the ER membrane
SCCaFTs from plasma membrane channels
39
Hierarchical Organization of Ca2 Signals
blip
puff
z
x
Two Basic Questions
How many IP3R channels open during a puff?
How large is the width of an IP3R channel cluster?
40
Puff Initiation following a Trigger Event
Space
Time
Averaged Trigger Duration 12 msec,
Spatial width at half peak 0.6 mm. Averaged
Puff Rising Time 18 msec,
Spatial width at half peak 1.6 mm,
Duration at half peak 30 msec. Peak Ratio
Puff peak / Trigger peak 6.7
/ 1.1 (DF/F0) 6.1.
41
Channel Dynamics during a Puff
Average number of open Channels
Puff Rise T18 ms
Trigger T12 ms N1
N? L?
N0
42
Puff Model
6mm
L
Immobile Buffer
L Cluster width N Total number of open
channels during a puff
Free Ca2
EGTA
6mm
Fluo4 Dextran
6mm
43
Modeling a Puff
L
L760 nm N30
L? N?
Free Ca2 (mM)
FluoCa (mM)
44
Confocal Image of a Model Puff
Spatial Profile
1.6mm
0.6
Time Trace
45
Estimating the number of open channels,and their
distribution within a Cluster
Average Peak Ratio R0 6.0 , Average Puff
Width W01600nm
R6.0, W1600
Number of open channels during a puff
Cluster Width (nm)
46
Conclusions
1. Data
Experiment
Model
Trigger
610
Width at Half Peak (nm)
600
Puff
1600 30
1600 32
Width at Half Peak (nm) Duration at Half Peak
(ms)
Ratio
6.1
5.9
Puff Peak / Trigger Peak
2. Prediction
760 (400 - 1000) 30 (20 - 45)
Cluster Width (nm) Open Channel during a Puff