Re: How does calcium chloride affect the permeability of cell membranes?

Dear Louise;
  
  For starters I should say that this is a very good question.  The reason 
I am saying this is because it does not really have a definitive answer 
yet, and although I'm reluctant to try and answer it, I'm more reluctant to 
defer the question, so here goes.
  
  At the moment(or at least as far as my references have it) the mechanism 
behind calcium chloride affecting cell permeability is unknown - genetic 
engineers and scientists know the procedure works purely through empirical 
experimentation, (in a sense, a bunch of "what"s -- but no "how" or "why"s) 
and therefore have been using it for transfection - or the addition on DNA 
to a cell for an added effect (like drug-resistant strains of bacteria, as 
an example).  

  Calcium chloride breaks down into ions in an aqueous(water) solution, 
such as a solution the cells are kept in, and the ions create a 
transient(temporary) state of "competence", or permeability, in the cell 
membrane which allows the small particles like plasmid DNA's and bacterial 
DNA's to pass through.  The "cold CaCl2" method is used for bacteria like 
e. coli, and can be used on mammalian cells also, though Calcium phosphate 
is usually used for mammalian cells.  Addition of other cations, 
detergents, and longer exposure to ions have increased the efficiency of 
transfection, but as I said, the mechanism is unknown.

  Now, if I had to venture a *guess* as to how it affected the membrane, 
and where on the membrane it affected, I would suspect it has something to 
do with the Calcium ion pumps in the cell membrane, the electrical 
charge across the membrane itself, or both.

The calcium ion pumps - which keep the Ca(+2) levels far lower inside the 
cell than outside - could possibly be overwhelmed by such a large presence 
of CaCl2.  The CaCl2 is added to the cell/bacterial suspension at a very 
cold temperature (like in an ice bath) - the cold slowing down the chemical 
and cellular reactions at first.  The solution is then warmed and the 
plasmid DNA is added to the solution while it is being warmed.
  
  This warming helps along and speeds up the chemical reaction of the CaCl2 
breaking apart into ions (Ca(+2) and Cl-), and the heat of course helps 
these ions bounce around more.  The heat also increases the cellular 
activity of the bacteria(or mammalian cells).

  This overwhelming presence of Ca(2+) outside of the cell, far more than 
previous and now in combination with heat and thus faster chemical motion/
reactions will generate intense osmotic pressure between the inside and the 
outside of the cell.  This osmotic pressure will cause Ca(+2) to be driven  
into the cell.  This in turn will force the cell to increase the activity 
of its Calcium ion pumps to force the calcium ions back out.  It is 
possible that in this cellular "state of emergency" (since the cell does 
not want large amounts of the calcium ion inside of it - it is pretty much 
poisonous to the cell in large amounts), that the cell works so hard at 
dealing with getting the calcium ions out that other things, such as 
plasmid DNA, has a better chance of getting in - either through the pumps 
themsleves or just passing through the cell membrane through endocytosis.  

  As for voltage shifts in the cell membrane, it is possible that at this 
time (during heat and excess CaCl2) there could be a voltage change going 
on across the cell membrane because of the sudden shift in ion 
concentration.  This voltage shift could make the cell membrane more 
permeable to plasmids, and again, they are merely entering the cell through 
endocytosis. 

(Voltage shifts in the cell membrane can allow for increased or decreased 
permeability, depending on the substance trying to pass through and the 
natural state of the cell.  In the fertilization process for example, a 
voltage shift in the egg once it is fertilized is referred to as the "fast 
block" reaction - the cell membrane become far less permeable to the sperm, 
thus preventing more sperm from entering into the egg.  I believe, though I 
don't have the reference handy, that this voltage shift is brought about 
by the ion pumps.)

  And of course, as I said, it could be a combination of the two of these.  
But honestly?  No one knows for sure - as I said, at least as far as my 
references go - and all of this listed above is based on speculation.
  
  But it definitely is still a good question.  I hope I've helped.
  
    - Patti


Sambrook, Fritsch, Maniatis.  Molecular Cloning: A Laboratory Manual.  
Second Edition.  Cold Spring Harbor Laboratory Press. 1989.  ISBN: 0-87969-
309-6