Action Potential Issue #16 - ALA Scientific Instruments

“Proudly Celebrating Our 20 th Anniversary”
“Furthering Life Science through Innovative Instrumentation”
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“Providing electrophysiology
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Issue #16 Winter/Spring 2006 ALA Scientific Instruments
“Furthering Life Science through Innovative Instrumentation”
ALA News Release
In Memoriam
1100 Shames Drive,
Suite 110
Westbury, NY 11590
To:
Special Symposium at Biophysical Society Meeting
Drug Discovery for Ion Channels VI
Date: Friday, February 17, 2006
Time: 8:00 AM - 5:00 PM
Location: ballroom J, Salt Palace Convention Ctr
Salt Lake City, UT
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516-997-5780
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E-Mail:
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Internet:
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In ALA’s continuing effort to bring only the finest instruments to our
clients, we have formed an agreement with Scientifica Ltd. to supply
the new Patch Star micromanipulator, along with the rest of their
product line. "We're very excited about working with Scientifica
because of their vast knowledge and experience in our field,” said
ALA President Alan Kriegstein. ALA staff is available for installations
and demonstrations in the NY metro area, and other locations with
advanced notice. Let us show you how Scientifica products can be
combined with our other fine instruments to build a perfect rig.
Product Update
The NEW Scientifica PatchStar is a high precision, ultra-stable
micromanipulator, which can move not only in XYZ and but also in
a virtual approach axis.
Key Features...
*Ultra stable - Drifts less than 1µm over 2
hours
*Electrically Silent - even single channel
recordings are routine
*Nanoresolution - 20ηm movement provides
piezo like feel
*20mm working travel - speed of motion for
all applications
*Revolutionary "Smart Sensor" - detects the angle of your headstage
for automatic smooth and straight virtual approach.
*Wide choice of user control device - Control Cube, Joystick or
Patchpad
*Sliding bracket and rotation stages - included at no additional cost
*Designed by and built for electrophysiologists
*Free 2 year warranty for added peace of mind
Scientifica’s Movable Top Plate is an ultra stable, smooth moving
mounting platform designed to combine electrophysiology with confocal
and multiphoton microscopy. It offers absolute stability and
precise alignment without disruption to sensitive preparations. The
MTP allows you to search your preparation without moving your
scope while keeping delicate patches in place. Exceptionally
smooth movement, coupled
with a rugged single structure
ensures that there is no movement
once a position has been
reached.
Key Features...
*Extremely stable
*Compatible with ALL major upright fixed stage microscopes.
*Compatible and designed for use with ALL major manipulators
*Can be used to combine electrophysiology with confocal
microscopy.
*Crossed roller bearings for super smooth movement
*Attachable anywhere on a metric or English threaded table
*Height adjustable for different samples.
Jonathan J. Adams: 1927-2006
ALA Scientific Instruments mourns the
passing of our founder Jonathan (John)
Adams. John was a loving husband,
caring father, Holocaust survivor, and
an exceptional human being. John was
a kind gentle soul. Despite living
through the worst that humanity had to
offer he rebuilt his life and lived with dignity
and style and never lost hope that
someday people would live with compassion
and understanding for each
other. He was a mentor, a teacher and
a good friend. He had a positive impact
on all the lives he touched.
He will be missed!
In 1960 John formed Medical Systems Corporation (MSC). MSC
was the very first and only exhibitor at the first Society for
Neuroscience meeting. Each of the following people, now active
in the field of instrumentation, worked at Medical Systems at some
point and has been influenced by John in a positive manner:
Milan Kessler (1940-2001) founder of Instrutech Corp., Andrew
Pomerantz, Vice President of ALA Scientific Instruments, Alan
Kriegstein, President of ALA Scientific Instruments, Harry
Benedict, former President of MSC., and George Kai, President of
Microdata Instruments.
After John retired from Medical Systems Corp. his love of science
would lead him to Jurgen List and the famous List patch clamp
amplifiers. In 1986, they combined to form Adams and List
Associates Ltd. to import the new developments in patch clamping
that came out of the Max Planck Institute. That company, now in
its 20th year, is ALA Scientific Instruments. John Adams retired
from ALA in 1993 and moved to Florida with is wife Dorothy, who
passed in 2002. He is survived by his daughter Lucy, her husband
Bob, and their two daughters.
ALA Around the World
Biophysical Society - Salt Lake City, UT,
February 18 - 22, 2006 - Booth 1014-1016
FENS 2006 - Vienna, Austria
July 8 - 12, 2006 - Booth TBD
Safety Pharmacology - San Diego (Mission Valley), CA
Sept. 26 - 28 - Booth TBA
Society for Neuroscience - 36th Annual Meeting, Atlanta, GA
October 14 - 18 - Booth TBA
American Heart Scientific Session - Chicago, IL
Nov. 12 - 15 - Booth TBA
John Adams, founder of
Medical Systems and
ALA Scientific

Gap Junction Recordings with Two npi SEC Amplifiers
Jose F. Ek Vitorin and Janis M. Burt
Department of Physiology, University of Arizona, Tucson AZ 85724
Special Symposium: Biophysical Society Meeting
Drug Discovery for Ion Channels VI
Date: Friday, February 17, 2006
Time: 8:00 AM - 5:00 PM
Location: Ballroom J, Salt Palace Convention Ctr
We have used dual discontinuous single electrode voltage
clamp amplifiers in combination with fluorescence
microscopy to examine the selectivity of gap junctions
and their comprising channels. For the same junction
we first determine its permeability to fluorescent molecules
and then its macroscopic conductance (g j ); in
most cases the conductance properties of the comprising
channels (γ j ) is then examined. To determine junctional
permeability we introduce dye into one cell of a
pair and monitor its intercellular diffusion as a function
of time (typically 5-10 minutes). Figure 1 shows a pair
of Cx43-expressing rat insulinoma cells before (1A, differential
interference contrast image) and 60 seconds
after (1B, fluorescent image, false colors) accessing
one of them with a dye-containing patch pipette (whole
cell
1A
configuration). The junctional permeability to the dye
was quantified by calculating the rate constant (k 2 ) for
its intercellular diffusion (from donor to recipient). To
determine g j a second patch electrode is then placed in
the recipient cell and transjunctional potential differences
alternately established from either cell such that
g j and γ j could be calculated. The ratio between k 2 and
g j (an estimate of the junctional permeability to small
ions, like K and Cl) defines the selectivity for the fluorescent
probe (see Ek-Vitorin and Burt, 2005).
To better understand how junctional selectivity is determined
by the selectivity of the comprising channels, the
calculated k 2 must be related to the conductances of
the channels constituting the same junction.
Unfortunately, amplifiers suitable for measurement of g j
are typically not ideal for measurement of single channel
activity. Traditional (non-switching) voltage clamp
amplifiers are designed to provide for large current
injection and modest noise handling (suitable for wholecell
voltage clamp of frog oocytes) or for small current
delivery and high signal-to-noise ratio (suitable for voltage
clamp of membrane patches and small mammalian
cells). Neither of these amplifier types is ideal for gap
junction recordings, where large currents can be
1B
Figure 1. Transjunctional diffusion of the fluorescent
dye NBD-M-TMA (see ref. 1) For this
cell pair k 2 =0.675; calibration bar=10µM
required to establish a stable transjunctional voltage
between well-coupled, small mammalian cells.
Discontinuous single electrode voltage clamp (dSEVC)
amplifiers can be used to record in these circumstances.
They are capable of injecting considerable
amounts of current (Strickholm, 1995) when switched to
current injection mode, but still directly evaluate membrane
potential when in voltage recording mode. Thus,
the dSEVC amplifier allows for accurate assessment of
macroscopic g j (Muller et al, 1999) and, importantly,
also allows for channel activity of the same junction to
be determined. Typical examples of such electrical
recordings are shown next.
In figure 2, panel A illustrates the voltage protocol used
to determine g j - it consists of a 10mV depolarizing
pulse alternately applied (V 1 , V 2 ) to each side of the
junction, while keeping the opposite side at 0mV. Panel
B shows the membrane currents recorded from donor
(I 1 ) and recipient (I 2 ) cells for the described voltage
pulses; junctional current (I j ) is the downward deflection
of both traces. For this pair (pre-treated with a MAPK
inhibitor) g j was 10.8ηS. In panel C, the same voltage
protocol was implemented (only I 1 and I 2 are shown)
during application of halothane, an agent known to
Figure 2. Determining macroscopic junctional conductance
(g j , as I j /V j ). Voltage (A) and current (B) traces obtained
(from the Rin43 cell pair illustrated in figure 1) shortly after
documenting the rate of intercellular dye diffusion and during
halothane application (C). Traces taken from the SEC
amplifiers with output filtering set at 1kHz (V) and 13kHz (I);
currents were subsequently low-pass filtered at 100Hz. For
all traces, sampling rate was 1kHz. Calibration: x, 2s; y, 10
mV/100pA.
reversibly reduce junctional conductance. Note the fast
reduction of junctional current (I 2 trace during V 1
pulse), and after complete uncoupling the remnant
capacitive artifacts and the non-junctional membrane
current (I 2 trace during V 2 pulse).
After uncoupling, single channel activity appears spontaneously
or can be stimulated by a partial washout of
the halothane. To document at maximum resolution the
detailed behavior of observed channels, we routinely
obtain current signals from the npi SEC amplifiers
(http://www.npielectronic.com/home.cgi?main=products) at high filtering
values (13kHz) for tape storage; these signals
are subsequently filtered as necessary for analysis.
Figure 3. Determining channel conductance (γ j ). A transjunctional
voltage is established (V 1 at ± 40mV, V 2 at 0mV)
while recording transjunctional current (I 2 ). Dotted line
marks zero transjunctional current in I 2 . Calibration: x, 5s; y,
5pA. Output filtering: voltage trace, 1kHz; current 50Hz.
Due to the excessive number of points at the set sampling
rate (2kHz), data were decimated (at 1/5) for illustration purposes.
Alternatively, the filtered (low-pass Bessel filter - LPF)
current signal can be digitized and stored directly. As
shown in Figure 3, it is also possible to record directly
from the amplifiers (without LPF) channels of the
expected amplitude. For this experiment, natural closure
of channels (I 2 ) occurred before the voltage pulses
(V 1 ) were terminated. To compensate for possible tip
potential imbalance, pulses of both polarities were
applied. Notice that single channel events are clearly
defined and their amplitudes can be easily determined.
References:
1. Ek-Vitorin JF, Burt JM. Quantification of Gap Junction Selectivity.
Am.J.Physiol Cell Physiol. 2005; 289:C1535-1546.
2. Strickholm, A. A single electrode voltage, current- and patchclamp
amplifier with complete stable series resistance compensation.
J. Neurosci. Methods 61: 53-66, 1995.
3. Muller, A., M. Lauven, R. Berkels, S. Dhein, H.-R. Polder, and W.
Klaus. Switched single-electrode voltage-clamp amplifiers allow
precise measurement of gap junction conductance. Am.J.Physiol.
276: C980-C987, 1999.
Opening remarks:
Cathy Smith-Maxwell,
Molecular Devices
Keynote address:
Targeting Voltage-Gated Calcium Channels: From Proofof-Concept
to Clinical Candidates.
Terrance Snutch, University of British Columbia and
Neuromed Technologies, Canada
Session I: Automated Drug Discovery with the
Oocyte Expression System
OpusXpress as a Tool for Ion Channel Drug Discovery.
Sean Donovan, Pfizer
The Roboocyte as a Tool for Automated Ion Channel Drug
Screening and Development.
Steven Petrou, University of Melbourne, Australia
Session II: Automated Drug Discovery with
Mammalian Cell Expression Systems Part 1
Development and Validation of Two Novel Automated
Methods of Patch-Clamp Recording.
Mark Bowlby, Wyeth Research
Screening Na + Channel Blockers Using Fluorescence
based and Automated Electrophysiological Assays.
George Ehring, Allergan
Assay Systems and Compound Identification for Potassium
Channels. Min Li, Johns Hopkins University
Session II: Automated Drug Discovery with Mammalian
Cell Expression Systems Part 2
Ion Channel Safety Pharmacology Using Automated
and Manual Electrophysiology.
Clemens Möller, Evotec AG, Germany
Novel Applications of Population Patch Clamp in Ion
Channel Drug Discovery.
Claire Townsend, GlaxoSmithKline
Screening New Drug Candidates for KCNQ1/KCNE1 (IKs)
Activity with PatchXpress 7000A.
Elena Trepakova, Merck Research Labs
Session III: Topics in Solution Delivery
An Automated Electrophysiological Screening System
for Small Sample Volumes.
Daniel Bertrand, University of Geneva, Switzerland
Session IV: Topics in Ion Channel Drug Discovery
Combination screening of ion channel targets to reduce
false positive rates. Chris Fanger, Hydra Biosciences
Small Molecule Libraries as Research Tools for Ion
Channel Drug Discovery: from Design to Drug Candidate.
Victor Panchenko, ChemDiv
CRAC Channel Inhibitors to Treat Acute and Chronic
Inflammation. Michael Xie, Synta Pharmaceuticals
Closing remarks:
Ian M. Herzberg,
ALA Scientific Instruments