Milikan, Jacobus Martinus; (2000) Probing calmodulin in living neurones. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In this thesis I explored the role of the calcium binding protein calmodulin in neuronal signalling in detail. Nerve cells have a high calmodulin content and calmodulin is involved in neuronal processes such as the memory models Long Term Potentiation and Long Term Depression, and neurite outgrowth. Previous studies mainly relied on biochemical and pharmacological techniques and immunocytochemistry to study calmodulin function. I investigated the suitability of the potent calmodulin antagonist calmidazolium for studying the calmodulin-mediated processes in living pheochroma cytoma 12 (PC12) cells. I used two fluorescently labelled calmodulins to study calmodulin function in living neurones: FL-CaM, which does not change its fluorescence when it binds calcium, and TA-CaM, which increases its fluorescence upon calcium binding. In addition, I measured the intracellular calcium concentration using low-affinity calcium indicator dyes. I used FL-CaM as a concentration and localisation marker for fluorescent calmodulin and the fluorescence of TA-CaM as an indicator of calmodulin activation. I introduced fluorescent calmodulins into rat dorsal root ganglion neurones by micro-injection or by loading via a patch-pipette under whole-cell voltage-clamp. Fluorescence was collected using confocal microscopy. I found that fluorescently labelled calmodulins locate to the cell nucleus of resting cells. Calmodulin immunofluorescence, however, had a different subcellular distribution and would only accumulate in the nucleus after depolarisation of the cells. In response to a brief depolarisation, TA-CaM was activated to the same extent in nucleus and cytosol of dorsal root ganglion neurones. When calcium levels fell again, TA-CaM fluorescence showed a residual plateau when the calcium level had recovered back to baseline. Since calmodulin activity is retained after the calcium signal, calmodulin can serve as part of a system in which repetitive stimulation leads to longer term changes in nerve activity. I investigated this phenomon and binding of calmodulin to target proteins appeared responsible.

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