Abstract
Continuous Ca2+ influx is essential to maintain intracellular Ca2+ homeostasis and its dysregulation leads to a variety of cellular dysfunctions. In this study, we explored the functional roles of spontaneous Ca2+ influx for the proliferation and differentiation of a human erythromyeloid leukemia cell line K562. mRNA/protein expressions were assessed by the real‐time RT‐PCR, western blotting, and immunocytochemical staining. Intracellular Ca2+ concentration ([Ca2+]i) and ionic currents were measured by fluorescent imaging and patch clamping techniques, respectively. Cell counting/viability and colorimetric assays were applied to assess proliferation rate and hemoglobin synthesis, respectively. Elimination of extracellular Ca2+ decreased basal [Ca2+]i in proliferating K562 cells. Cation channel blockers such as SK&F96365, 2‐APB, Gd3+, and FTY720 dose dependently decreased basal [Ca2+]i. A spontaneously active inward current (I spont) contributive to basal [Ca2+]i was identified by the nystatin‐perforated whole‐cell recording. I spont permeated Ca2+ comparably to Na+, and was greatly eliminated by siRNA targeting TRPM7, a melastatin member of the transient receptor potential (TRP) superfamily. Consistent with these findings, TRPM7 immune reactivity was detected by western blotting, and immunofluorescence representing TRPM7 was found localized to the K562 cell membrane. Strikingly, all these procedures, that is, Ca2+ removal, TRPM7 blockers and siRNA‐mediated TRPM7 knockdown significantly retarded the growth and suppressed hemin‐induced γ‐globin and hemoglobin syntheses in K562 cells, respectively, both of which appeared associated with the inhibition of ERK activation. These results collectively suggest that spontaneous Ca2+ influx through constitutively active TRPM7 channels may critically regulate both proliferative and erythroid differentiation potentials of K562 cells.
Highlights
Ca2+ is a highly versatile intracellular signal that can regulate both acute and long-term cellular functions ranging from membrane excitation, contraction, neurotransmitter release, secretion, cell growth, differentiation to death (Berridge et al 2003)
Mediated TRPM7 knockdown significantly retarded the growth and suppressed hemin-induced c-globin and hemoglobin syntheses in K562 cells, respectively, both of which appeared associated with the inhibition of ERK activation. These results collectively suggest that spontaneous Ca2+ influx through constitutively active TRPM7 channels may critically regulate both proliferative and erythroid differentiation potentials of K562 cells
TRPM7 is a predominant transient receptor potential (TRP) isoform expressed in K562
Summary
Ca2+ is a highly versatile intracellular signal that can regulate both acute and long-term cellular functions ranging from membrane excitation, contraction, neurotransmitter release, secretion, cell growth, differentiation to death (Berridge et al 2003). Disruption of Ca2+ homeostasis and dynamics can lead to a variety of cellular pathophysiology in which numerous Ca2+-mobilizing molecules including voltage-dependent Ca channels, P2X, and NMDA inotropic cation channels, Orai and TRP channels play pivotal roles (e.g., Borowiec et al 2014; Deliot and Constantin 2015). Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.
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