Temperature and voltage clamp
jonesmat at physiology.wisc.edu
Fri Nov 12 12:55:56 EST 2004
r norman <rsn_ at _comcast.net> wrote in message news:<vksuo09mt3f9oldt02gmp7n7nl9jsjq78n at 4ax.com>...
> On Mon, 08 Nov 2004 07:53:47 GMT, BilZ0r
> <BilZ0r at TAKETHISOUThotmail.com> wrote:
> >I was talking to a colleague today, who primary studies antiepileptics with
> >voltage clamp on voltage sensitive sodium channels expressed in X. Oocytes.
> >He told me he does all of his work, I believe, at 15ºC... Does that sound
> >Why would he use such a low temperature? So he could see any effects of
> >gating more readily? Any ideas?
> Why do you think 15 C is a "low" temperature? Remember, the work is
> done on Xenopus cells, not mammalian. Xenopus ordinarily lives at
> temperatures of 15 to 25 C. Preparations do last longer and remain in
> better conditions at cooler temperatures.
On the other hand, the sodium channels he studies are probably
mammalian, e.g., rat, mouse or human. So one could still argue that
this is a lower temp than what those channels see in their native
state (which is obviously not in a frog egg, but that's another
However there's quite a long history of people studying sodium
channels at low temp, even right down to 4 deg C. I think the main
reason for this is that sodium channels have really really fast
kinetics, which causes problems for accurately recording their
properties. For example, the temporal resolution of an experiment is
ultimately limited by the sampling rate, which is usually limited to
<100 KHz in standard acquisition systems. Then one needs to filter at
the Nyquist freq or below, which brings the resoltution down to 25-50
kHz at best. Actually that's probably not enough filtering to get rid
of enough noise, so in practice people will usually be working at 2-10
Khz. This means that components of the signal faster than about 100
microseconds will be distorted or lost. At room temp or 35 deg C, Na
currents can probably have rise times faster than 100 us, so they
would be very badly distorted. Plus, aside from acquisition, in order
to voltage clamp properly, the amp needs to be able to sense voltage
changes and inject current faster than what the channel is doing,
which is particularly problematic if the channel is trying to
regeneratively alter the voltage (i.e., fire a spike).
All of these problems are alleviated by lowering the temp, since it
makes everything the biology is doing much slower.
What i would worry about is whether all the transition rates of the
channel vary with temperature in the same manner or not. If not, then
the macroscopic behavior could actually be qualitatively different at
low vs high temp.
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