Agonist-Induced Ca2+ Signaling in HEK-293-Derived Cells Expressing a Single IP3 Receptor Isoform.

In mammals, three genes encode IP3 receptors (IP3Rs), which are involved in agonist-induced Ca2+ signaling in cells of apparently all types. Using the CRISPR/Cas9 approach for disruption of two out of three IP3R genes in HEK-293 cells, we generated three monoclonal cell lines, IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK, with the single functional isoform, IP3R1, IP3R2, and IP3R3, respectively. All engineered cells responded to ACh with Ca2+ transients in an "all-or-nothing" manner, suggesting that each IP3R isotype was capable of mediating CICR. The sensitivity of cells to ACh strongly correlated with the affinity of IP3 binding to an IP3R isoform they expressed. Based on a mathematical model of intracellular Ca2+ signals induced by thapsigargin, a SERCA inhibitor, we developed an approach for estimating relative Ca2+ permeability of Ca2+ store and showed that all three IP3R isoforms contributed to Ca2+ leakage from ER. The relative Ca2+ permeabilities of Ca2+ stores in IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK cells were evaluated as 1:1.75:0.45. Using the genetically encoded sensor R-CEPIA1er for monitoring Ca2+ signals in ER, engineered cells were ranged by resting levels of stored Ca2+ as IP3R3-HEK ≥ IP3R1-HEK > IP3R2-HEK. The developed cell lines could be helpful for further assaying activity, regulation, and pharmacology of individual IP3R isoforms.

Influence of Nitrogen Availability on Growth of Two Transgenic Birch Species Carrying the Pine GS1a Gene.

An alternative way to increase plant productivity through the use of nitrogen fertilizers is to improve the efficiency of nitrogen utilization via genetic engineering. The effects of overexpression of pine glutamine synthetase (GS) gene and nitrogen availability on growth and leaf pigment levels of two Betula species were studied. Untransformed and transgenic plants of downy birch (B. pubescens) and silver birch (B. pendula) were grown under open-air conditions at three nitrogen regimes (0, 1, or 10 mM) for one growing season. The transfer of the GS1a gene led to a significant increase in the height of only two transgenic lines of nine B. pubescens, but three of five B. pendula transgenic lines were higher than the controls. In general, nitrogen supply reduced the positive effect of the GS gene on the growth of transgenic birch plants. No differences in leaf pigment levels between control and transgenic plants were found. Nitrogen fertilization increased leaf chlorophyll content in untransformed plants but its effect on most of the transgenic lines was insignificant. The results suggest that birch plants carrying the GS gene use nitrogen more efficiently, especially when growing in nitrogen deficient soil. Transgenic lines were less responsive to nitrogen supply in comparison to wild-type plants.