Expression of transient receptor potential mRNA isoforms and Ca2+ influx in differentiating human stem cells and platelets

Store-regulated Ca2+ entry (SOCE) is an important mechanism of elevating cy tosolic [Ca2+](i) in platelets, though the Ca2+ influx channels involved ar e still unclear. We screened human platelets and their precursor cells (hum an stem cells and megakaryocytes) for the presence of candidate influx chan nels, i.e., isoforms of the Trp family of proteins. Primary stem cells were cultured with thrombopoietin to allow differentiation into megakaryocytes. The undifferentiated stem cells (CD34(+)) showed mRNA expression of only a spliced variant Trp1A. Immature (CD61(+)/CD42b(low)) and mature (CD61(+)/C D42b(high)) megakaryocytes as well as platelets expressed in addition unspl iced Trp1 as well as Trp4 (less abundant) and Trp6 isoforms. This unspliced isoform appeared to be specific for cells of the megakaryocyte/platelet li neage, since immature (CD14(+)/CD61(-)/CD42b(-)) and mature monocytes expre ssed only the Trp1A isoform. This conclusion was confirmed by the presence of Trp1A. 3, 4 and 6 transcripts in the immature megakaryocytic Dami cell l int, and of Trp1, 1A, 4 and 6 transcripts in the more mature CHRF-288 cell line. The up-regulation of Trp1, 4 and 6 in the lineage from primary stem c ells to mature megakaryocytes and platelets was accompanied by increased in flux of extracellular Ca2+ after pretreatment of the cells with thapsigargi n or thrombin. Expression of new Trp isoforms in the differentiated cells i s thus accompanied by increased SOCE. (C) 2001 Elsevier Science B.V. All ri ghts reserved.