Sensing mechanical stresses, including touch, stretch, compression, and gravity, is crucial for growth and development in plants. A good mechanosensor candidate is the Ca 2؉ -permeable mechanosensitive (MS) channel, the pore of which opens to permeate Ca 2؉ in response to mechanical stresses. However, the structure-function relationships of plant MS channels are poorly understood. Arabidopsis MCA1 and MCA2 form a homotetramer and exhibit Ca 2؉ -permeable MS channel activity; however, their structures have only been partially elucidated. The transmembrane topologies of these ion channels need to be determined in more detail to elucidate the underlying regulatory mechanisms. We herein determined the topologies of MCA1 and MCA2 using two independent methods, the Suc2C reporter and split-ubiquitin yeast two-hybrid methods, and found that both proteins are single-pass type I integral membrane proteins with extracellular N termini and intracellular C termini. These results imply that an EF hand-like motif, coiled-coil motif, and plac8 motif are all present in the cytoplasm. Thus, the activities of both channels can be regulated by intracellular Ca 2؉ and protein interactions.Mechanical stimuli affect plant growth, development, and resistance to herbivores (1-6). Natural stimuli, such as wind and gravity, modify the height and shape of grasses and trees.Touch retards the elongation of inflorescence and increases resistance to herbivores in Arabidopsis thaliana. Thus, stimulus perception is fundamental to plants, and the elucidation of mechano-sensors is important for understanding the molecular basis of plant mechanics and morphogenesis.A Ca 2ϩ -permeable mechanosensitive (MS) 8 channel has been suggested as a component of mechano-sensors (7-10). Using Nicotiana plumbaginifolia seedlings carrying the Ca 2ϩ -dependent photoprotein, aequorin, as an intracellular Ca 2ϩ indicator, a previous study reported that touch elicits an immediate increase in the cytosolic concentration of Ca 2ϩ ([Ca 2ϩ ] cyt ), which may act as a Ca 2ϩ signal (11). We recently identified novel Ca 2ϩ -permeable MS channels in Arabidopsis (A. thaliana), named MCA1 and MCA2 (mid1-complementing activity 1 and 2) (12-15). Both proteins share 74% identity in their amino acid sequences, form a homotetramer, have no homology to any known ion channels or transporters, and mediate Ca 2ϩ influx upon mechanical stimulation, such as hypo-osmotic shock and membrane stretch. Genes coding for MCA orthologs are found exclusively in the plant kingdom (9, 12). Rice and tobacco MCA proteins are also shown to mediate an increase in [Ca 2ϩ ] cyt upon hypo-osmotic shock (16,17).An in silico study suggested that MCA1 and MCA2 have several motifs, such as an EF hand-like motif, coiled-coil motif, and plac8 (DUF614) motif as well as a few predicted putative transmembrane segments (12, 13). To understand the molecular and physiological functions of both proteins and their motifs, their transmembrane topologies need to be elucidated in more detail. In the present study we...

Intracellular calcium homeostasis is essential for proper cell function. We investigated the effects of heat shock on the development of and the intracellular Ca2+ levels in bovine preimplantation embryos in vitro and the effects of calcitonin (CT), a receptor-mediated Ca2+ regulator, on heat shock-induced events. Heat shock (40.5 C for 10 h between 20 and 30 h postinsemination) of in vitro-produced bovine embryos did not affect the cleavage rate; however, it significantly decreased the rates of development to the 5- to 8-cell and blastocyst stages as compared with those of the control cultured for the entire period at 38.5 C (P < 0.05). The relative intracellular Ca2+ levels at the 1-cell stage (5 h after the start of heat shock), as assessed by Fluo-8 AM, a fluorescent probe for Ca2+, indicated that heat shock significantly lowered the Ca2+ level as compared with the control level. Semiquantitative reverse transcription PCR and western blot analyses revealed the expression of CT receptor in bovine preimplantation embryos. The addition of CT (10 nM) to the culture medium ameliorated the heat shock-induced impairment of embryonic development beyond the 5- to 8-cell stage. The Ca2+ level in the heat-shocked embryos cultured with CT was similar to that of the control embryos, suggesting that heat shock lowers the Ca2+ level in fertilized embryos in vitro and that a lower Ca2+ level is implicated in heat shock-induced impairment of embryonic development. Intracellular Ca2+-mobilizing agents, e.g., CT, may effectively circumvent the detrimental effects of heat shock on early embryonic development.

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