Ion Channels as Effectors for Cell Growth Regulatory Signaling

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Using model cell systems for cell growth control, the principal investigator and others have advanced the concept that ion channels are integral components of the central growth factor/proto-oncoprotein/ oncoprotein growth regulatory signaling networks. Specifically, the laboratory of the principal investigator has identified a fibroblast intermediate conductance, calcium-activated, potassium channel, FIK, that is selectively up-regulated in response to growth factor or oncoprotein stimulated mitogenic signaling. In addition, specific block of FIK function with peptidyl toxins has been shown to reversibly inhibit cell proliferation. FIK has also been shown to regulate transcriptional activity of the MRF4 muscle regulatory transcription factor in 10T1/2-MRF4 fibroblasts, a model cell system for defining the linkage between signaling events at the cell membrane and growth regulatory transcription events. A key hypothesis for how FIK regulates cell growth is that it insures consistent hyperpolarization of the cell membrane to or near the potassium equilibrium potential. The model predicts that this hyperpolarizing action will, in turn, promote mitogenic calcium entry via growth factor activated, voltage-independent calcium channels. In support of this idea, preliminary studies have identified a calcium channel in 10T1/2-MRF4 fibroblasts which is activated by bFGF, an important growth regulatory peptide for these cells. Thus, a wealth of published work and preliminary data have identified FIK and peptide growth factor activated calcium channels as key physiologic regulators of cell growth. The long term goal of this project is to define how these channels regulate cell growth, thus providing a unique insight into this aspect of cell physiology. Specific project goals are: 1. Establish a mechanistic basis for the role of FIK in cell growth regulatory signaling. Physiological and pharmacological means will be used to examine the link between FIK channel regulation and growth control in fibroblast cells, with the 10T1/2-MRF4 model system being exploited to understand how FIK modulates growth-associated transcription. 2. Homology clone the fibroblast IK channel (FIK). An homology cloning strategy will be implemented based on the sequence of recently cloned human calcium-activated potassium channels. Physiology of the heterologously expressed cloned channels is remarkably similar to the fibroblast IK which this lab has extensively characterized. Cloning FIK will ultimately allow utilization of a number of approaches (inducible expression, antisense inhibition of native expression, point mutations to perturb ionic selectivity, etc.) to determine how the channel regulates cell growth.