PNNL-13501 - Pacific Northwest National Laboratory

Analysis of Receptor-Modulated Ion Channel Signaling by Confocal Optical/Patch-
Clamp Microscopy
Study Control Number: PN00010/1417
H. Peter Lu, Brian D. Thrall, Steven D. Colson
Regulated ion flux across cell membranes plays a fundamental and crucial role in live cells. Ion channels control and
regulate the ion current flux and therefore play a key role in regulating how cells respond to their changing environment.
The purpose of this research project is to develop a unique technology, by combining our extensive imaging capabilities,
a confocal scanning linear/nonlinear optical microscope, with state-of-the-art patch-clamp technologies. Application of
this unique instrumentation will present an unprecedented opportunity of seeking mechanistic and dynamic understanding
of ion channel functions and structures in living cells.
Project Description
This research project will develop a unique technology,
by combining a confocal scanning linear/nonlinear optical
microscope (see Figure 1) with state-of -the-art patchclamp
technologies, which will significantly enhance the
diagnostic and investigative capabilities of both methods
by combining real-time fluorescence measurements with
real-time single-channel current measurements in a living
cell. To illustrate the power of this approach in cell
signaling research, this technology will be used to gain an
understanding of how ion channel activities are regulated
by conformational change dynamics and channel
formation mechanisms using the gramicidin ion channels.
We designed, developed, and assembled an instrument
combining patch-clamp measurements with a real-time
scanning confocal microscope. We achieved recording
single-ion channel (gramicidin dimers) current (on-off)
trajectories with high sensitivity and established
computational data analysis of single-ion channel
stochastic time trajectories (Figures 2 and 3). In
combining single-molecule spectroscopy and optical
imaging, we have moved significantly forward in
obtaining fluorescence images of single-dansyl
gramicidin molecules and their dimers in lipid bilayers.
By combining the patch-clamp technique and confocal
fluorescence microscopy, it will become feasible to study
ligand binding and dissociation, ligand binding
cooperativity, ion channel activation and desensitization,
and ion channel protein conformational changes in an
intact cell in real-time. The measurements of ion channel
protein conformational changes correlated with ion
channel current trajectories have never been achieved
before, and they are essential for the understanding of
Ligand-dye
Lipid Bilayer Ion Channel Protein
Microscope
Objective
PatchClamp Tip (1-5 µm)
M+
Laser light with
diffraction
limited focal point.
Figure 1. We combine laser scanning confocal optical
microscopy with patch-clamp technique to study the ion
channel conformational dynamics and mechanism of the
single-ion channels. Single ligand-gated channel protein
conformational change dynamics can be studied by
monitoring fluorescence spectral fluctuations. Ligand
binding/unbinding motion and ion-channel conformational
changes are probed by two-photon confocal microscope and
ion channel open/close states are monitored by patch-clamp
ion channel current measurement. Dye-labeled ligands in
nanomolar concentrations can be introduced to a receptor
from the patch-clamp micropipette.
ion channel molecular dynamics and ion channel
mechanisms. Understanding the correlated
conformational dynamics of a single ion channel will be a
significant step in deciphering the molecular mechanisms
of ion channel functions.
Biosciences and Biotechnology 31