IUBio

Nigericin help

Dan Bond drb13 at cornell.edu
Thu Jul 11 13:22:29 EST 1996


In article <4s34a5$1t9 at park.interport.net>, jmarti at interport.net (JAM)
wrote:

> I have read conflicting information regarding the effects of nigericin
> on live cells in normal physiologic media: (1) Dissipates _membrane
> potential_ only, while not disrupting active transport or other
> physiologic activities OR (2) Dissipates _pH gradient_ in addition to
> potential and affects all cellular processes.



If you had a membrane impermeable to ions (no other channels or
exchangers) nigericin would catalyze the electroneutral exchange of protons
for potassium (or Na to a lesser extent).  It should actually interconvert
delta pH to delta psi. Assuming the exterior of the cell has a lower pH
than the inside,  nigericin alllows H+ in at the expense of K+ or Na+, and
the net charge difference (delta p) accross the cell membrane is the same. 
Along the same lines,  valinomycin should, in theory,  interconvert some or
all of the delta psi to delta pH.

However, living cells often have large concentrations gradients of
potassium (high inside) and sodium (low inside) accross the cell membrane,
which nigericin should also dissapate.  The cell may try to regain these
gradients, using either active (~P-linked) or secondary (ion-linked)
transport and the futile cycling of protons and ions can deenergize the
cell. Thus I've always had to make measurements of the various gradients to
see if my cells behave like other cells.

If you want to measure transport in the absence of all chemiosmotic forces,
 use a combination of valinomycin and nigericin (or monensin).  This will
knock out all ion, charge, and pH gradients quite quickly.  Classic
uncouplers like CCCP (only works under aerobic conditions) or TCS will also
do this, although I like the combination better.  Then,  *make sure* you
give your cells an energy source that they can use to generate ATP from
substrate-level phosphorylation. Intracellular ATP measurements are a nice
check to make sure the ionophores haven't denergized them completely.

The simplest way to determine if delta pH or delta psi is the driving force
is to make an artificial one.  E.g,  equilibrate concentrated cell
suspension in a pH 7 buffer and dilute into 5 (delta pH only).  Or, 
equilibrate cells in a high potassium buffer and dilute into a potassium
free buffer (delta psi only).  The beauty of this approach is that you can
control the magnitude of the driving force, and can leave out the
ionophores.

Sorry if this is all stuff you've heard before.

Dan Bond



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