Temperature and voltage clamp

r norman rsn_ at _comcast.net
Fri Nov 12 13:43:12 EST 2004


On 12 Nov 2004 09:55:56 -0800, jonesmat at physiology.wisc.edu (Matt
Jones) wrote:

>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:
>> 
>> >Hi,
>> >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 
>> >right?
>> >
>> >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.
>
>
>Good point. 
>
>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
>story).
>
>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.
>

All this is very true.

On the other hand, it could simply be that the guy had a very cold lab
room!  A standard joke (no doubt, an urban legend) comparing American
with British facilities is the line "Experiments were done at room
temperature, 10 C."








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