Voltage-gated K+ channels have been found in tissues other than excitable cells, but their functions in non-excitable cells, including hematopoietic cells is poorly understood. The P.I. has discovered a shaker-like channel in human myeloblastic (ML-1 cells. Preliminary data using whole-cell patch clamp technique demonstrate that this channel is voltage-gated and can be activated by 12-O-tetradecanoylphorbol-13-acetate (TPA) which is known to induce differentiation in this cell line. After treatment with a differentiation-inducing dose of TPA, the kinetics of the current inactivation become radically altered within 6 hours. This channel alteration is prior to the appearance of differentiating phenotype. Based on these preliminary studies, the P.I. has heterologously the K+ channel by injection of Xenopus oocytes with mRNA from ML-1 cells. The P.I. now proposes to further characterize this K+ channel and to explore its role in cell proliferation and differentiation. This will be accomplished through the following specific aims: (i) A further understanding of the channel characteristics and regulation will be obtained using single channel patch clamp at different configurations; (ii) A further characterization of the expressed K+ channel in oocytes will be obtained by using the two-microelectrode voltage clamp and patch clamp techniques; and (iii) The full length cDNA encoding the primary nucleotide sequence of the channel gene will be obtained. This will be accomplished using both plaque hybridization to a cDNA library from ML-1 cells and expression in Xenopus oocytes. Polymerase chain reaction (PCR) will also be used to prime and to amplify probes for the hybridization experiments. Positive clones of the K+ channel will be sequenced and expressed in oocytes and mammalian cells. The long-term goal of the project is to study, at the molecular level, functioning of the K+ channel in ML-1 cells in order to better understand its role in cell proliferation and differentiation. This may have significance, not only for understanding differentiation in normal cells, but also for understanding how to induce this process to suppress malignant growth in leukemic cells, such as ML-1.
| Xu, B; Lu, L (1994) Protein kinase A-regulated Cl- channel in ML-1 human hematopoietic myeloblasts. J Membr Biol 142:65-75 |
| Lu, L; Yang, T; Markakis, D et al. (1993) Alterations in a voltage-gated K+ current during the differentiation of ML-1 human myeloblastic leukemia cells. J Membr Biol 132:267-74 |
| Lu, L; Markakis, D; Guggino, W B (1993) Identification and regulation of whole-cell Cl- and Ca(2+)-activated K+ currents in cultured medullary thick ascending limb cells. J Membr Biol 135:181-9 |
