Toward the goal of understanding the mechanisms of motility control, the genetic dissection of Paramecium behavior in our laboratory, is now focused on the molecular basis of ion-channel activities.
We aim to identify and characterize several hitherto unknown proteins which control or modulate ion channels using a unique system where channel mutants, """"""""curing"""""""" proteins, whole-cell voltage clamp and cell-free patch clamp are all available. Three soluble protein factors were found to restore missing ion currents. The small, acidic, soluble and non-calmodulin protein which replenishes the Ca current missing in the cnrC mutant will be further purified to homogeneity. A protein phosphorylation activity which correlates with cnrC-curing will be further studied to clarify the enzymology involved. Calmodulin appears to restore the Ca-dependent K current missing in the pntA mutant and a different soluble protein restores the same current missing in pntB mutant. These proteins will be purified, those from wild type and the mutants compared, and their roles in enzyme cascades examined. Second messengers, enzymes, their effectors and antibodies related to protein phosphorylation cascades will be injected into Paramecium to see whether they affect ion currents through various channels. These molecules will also be added to baths in which single-channel recording is being carried out (by the planar-lipid bilayer or the patch clamp method) to see whether they have effects in vitro. Cytoplasmic fractions will be tested similarly to find unknown elements, including the """"""""curing proteins,"""""""" which may govern channel functions directly. We will continue to generate and analyze mutants using different mutagens and screening methods. We will focus on the Ca channel, the Ca-dependent K channel and the Ca-dependent Na channel since they can now be studied by several molecular methods. We also plan to develop methods to streamline Paramecium genetics by mating them en masse. Exploiting the ability to restore Ca-channel function by mRNA injection into cnrC, we will try to clone the gene for the cnrC-curing factor using a hybrid selection procedure. It successful, this would yield material for further structural and functional studies. We will also continue to search for other curing proteins or mRNA concerning these or other ion channels to extend our knowledge on how they function and how they are controlled.
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