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Molecular Components of the Gravity-Directed Calcium Current in Ceratopteris: Characterization of a Membrane-Bound Calcium Pump. T. J. Bushart, S.C. Stout and S. J. Roux. Sect. Molecular Cell & Developmental Biology, Univ. Texas, Austin, TX.

   During the period in which gravity determines their developmental polarity, germinating spores of the fern Ceratopteris richardii exhibit a bottom-to-top calcium current. This current is capable of rapid reorientation, reversing direction in less than 42 sec when the spores are turned upside down. The current is parallel to the subsequent axis of cell development and its direction is opposite that of the primary rhizoid's emergence during germination. Blocking the current with nifedipine disrupts the ability of gravity to direct the polarity of spore development. The entry of calcium into the bottom of the cell is presumably mediated by a channel-type transporter and its exit from the top of the cell by an ATP-dependent pump. The function of the channel(s) and pump(s) that drive the current must be rapidly modulated by gravity and understanding how gravity does this will require identifying these transporters and characterizing the mechanisms that control their function. Toward this end, we have identified three cDNAs from a Ceratopteris EST library whose sequences predict that they encode Ca2+ATPase pumps. Two of them encode proteins that are most similar to calcium pumps known to be plasma membrane localized. The expression of one of these (CrACA1) appears relatively unchanged during the period when the gravity-directed current is strongest, i.e., the first 16 hours after germination is initiated by light, as judged both by microarray and quantitative RT-PCR assays.  Antibodies have been raised to a 93-mer peptide corresponding to the large loop between transmembrane regions 4 and 5 of CrACA1.  The specificity of these antibodies is being determined in anticipation of use in immunolocalization studies. These investigations are important first steps in the identification of the pump(s) that drive the gravity-directed calcium current and in the discovery of the mechanisms by which gravity controls pump function. (Supported by NASA grants NAG2-1586, NAG10-295 to S.J.R. and NGT5-50371 to S.C.S.)