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benzodiazepines and conductance increase of GABAa

John Mihic jmihic at bgsm.edu
Wed Sep 17 15:55:33 EST 1997

In article <5vp661$sj$1 at fremont.ohsu.edu>, jonesmat at ohsu.edu says...

Matt, now look what you did.  Here I was contentedly wasting my time preparing 
a lecture and writing grant proposals, when you have to come along and distract 
me with your analysis of the Eghbali et al. paper.

You have some very valid concerns.
>A few methodological comments:
>        In the recent paper (Eghbali, Curmi, Birnir and Gage, Nature 388,71-75),
>a rather unusual drug application method was used. Most previous studies
>have used outside-out patches. Instead, the authors usually used on-cell
>patches, and *injected* GABA and diazepam into the recording pipette
>during the experiment. It is not clear why this was done, and one might
>worry about the possible effects of changing the intrapipette pressure
>(e.g., activation of stretch sensitive chloride channels). 

They state that they controlled for injection technique by injecting GABA with 
DMSO (vehicle to dissolve diazepam, for the uninitiated).  And in Fig. 2, they 
show a gradual increase in conductance by diazepam using the inside-out patch 
technique and reversal of the diazepam effect by flumazenil. 

>        Along a similar line, I can recall no experiment in which they showed
>that the large conductance was in fact sensitive to GABA-A receptor
>antagonists (correct me if I missed it). This would seem to be a crucial

I think the flumazenil reversal (Fig. 2B) is more convincing than anything they 
could have done at the GABA-A site.

>        The effect reported was highly variable from cell to cell, and showed a
>"ceiling" effect. That is, channels that were already large were less
>potentiated. Once again, one might worry that some of the observed
>chloride channels (the larger ones) were something other than GABA-A

>        Drug washout was not shown. Because stationarity is such a great 
>in single channel studies, this also seems to be an important control.

In the abstract they say that the effect of diazepam was reversible but I didn't see 
any data presented on that.  In fairness to the authors, Nature really limits how 
much data you can present in a Letter.

>        The drug effect was "graded". That is, rather than the amplitude
>histogram shifting in weight from low amplitude to high amplitude peaks,
>the peak remained sharp but moved gradually to the right. This suggests
>that there must be a "graded" response of *each* channel (or each
>functionally coupled cluster of channels, if such things exist) to
>diazepam, rather than a single binding site with an all or none action.
>In fact, such a graded effect suggests that there must be an awful lot of
>diazepam sites (more than five, say) per channel, each of which can
>modulate conductance to some degree. Oddly, whole cell diazepam
>dose-response curves usually give pretty low Hill slopes, which might be
>interpreted as there being only one or two diazepam sites per channel. In
>fairness, though, both the multibarreled pore idea and the binding
>stoiciometry of GABA and benzos remain open questions, and are not easy
>to address experimentally.

The authors state they used low concentrations of GABA in these studies.  
Benzodiazepine potentiation is not observed when high concentrations of GABA 
are used.  However, if conductance is changed by benzos, wouldn't you expect to 
still see enhancement at saturating GABA concentrations?   

>Obviously it's good to keep an open mind about such a potentially
>important finding. But the potential importance is also a good reason to
>show a lot of stringent controls.

Ahh, the sweet smell of scepticism.  Neil indeed trained you well.   


S. John Mihic, Ph.D.
Dept. Physiology & Pharmacology
Bowman Gray School of Medicine of Wake Forest University
Winston-Salem, NC 27157

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