The 10T1/2-MRF4 fibroblast/myogenic cell system was used to address the following interrelated questions: whether distinct signaling pathways underlie myogenic inhibition by basic fibroblast growth factor (bFGF) and transforming growth factor (TGF)-␤; which of these pathways also up-regulates the fibroblast intermediate

The fibroblast intermediate conductance, calcium-activated potassium channel (FIK) is proposed here as a functional prototype for other IK channels which to date have undefined physiologic actions. FIK pharmacology in the 10T1/2-MRF4 myogenic fibroblast cell line was determined: to define the relationship of FIK to other IKs; to confirm a physiologic role for FIK; and, thus develop a hypothesis about IK channel family function. Whole cell patch-clamp electrophysiology was used to determine K(0.5) values for FIK block by the structurally related peptides charybdotoxin (ChTX) (7 nm) and iberiotoxin (IbTX) (536 nm), and a new unrelated FIK inhibitor, Stichodactyla toxin (StK) (85 nm). Peptide pharmacology for FIK was consistent with that of recently cloned IKs. ChTX and StK inhibited bFGF stimulated 10T1/2-MRF4 cell proliferation with dose-dependencies consistent with their FIK blocking actions. ChTX, StK, and IbTX also evoked MRF4-dependent transcription as measured by muscle acetylcholine receptor channel functional expression; but they did not evoke subsequent multinucleated fiber formation or myosin heavy chain expression, suggesting a role for FIK in early, rather than late, myogenic events. Thus despite structural differences, ChTX, IbTX, and StK have common effects on cell growth and differentiation reflecting their common FIK blocking action. We suggest that a major function of the IK channel family is to regulate cell growth.

We investigated small conductance (SK) potassium channel-mediated regulation of muscle-specific, ion channel functional expression in the C3H10T1/2-MRF4 cell model system, a stable fibroblast line ectopically overexpressing the myogenic regulatory transcription factor, MRF4. Mitogenic stimulation of C3H10T1/2-MRF4 cells with basic fibroblast growth factor negatively regulates MRF4 transcriptional activity, inhibiting myogenesis. Using patch clamp techniques we found that mitogenic stimulation of C3H10T1/2-MRF4 cells also up-regulated SK. SK is a charybdotoxin-sensitive, apamin-insensitive channel that exerts positive proliferative control in fibroblasts. Mitogen withdrawal, which removes negative regulation of MRF4 thus initiating myogenesis, also eliminated SK channel currents, coincident both with induction of acetylcholine receptor channels, and up-regulation of muscle inward rectifier potassium channels. Addition of the SK channel blocker charybdotoxin to growth factor-containing culture medium overcame basic fibroblast growth factorinduced negative regulation of MRF4, as evidenced by induction of inward rectifier potassium and acetylcholine receptor channel expression identical to that observed in mitogen-withdrawn cells. Thus, the SK channel can govern electrophysiological phenotype in C3H10T1/2-MRF4 cells, consistent with an ability of SK to affect MRF4-dependent transcriptional activity. SK appears to be a pivotal signaling component for growth factor regulation of both cell proliferation and differentiation.The regulated expression of ion channels is a key component of developmental processes in many cell types. In nonexcitable cells such as lymphocytes and fibroblasts, both potassium and voltage-independent cation channels contribute to proliferative and cell fate control mechanisms (1-6), while in excitable tissues, the expression of both voltage-dependent cation and ligand gated channels is a culminating event in cell differentiation. The latter scenario is prominent in mammalian skeletal muscle, for which cell maturation is marked by the up-regulation of voltage-dependent channels and conditional expression of different ACh 1 receptor channel subtypes (7). In turn, the progression of muscle fiber differentiation may be affected by the activity of these channels (8 -10).To understand ion channels as both causal and effector agents in cell growth and differentiation, we have looked at the regulation of channel functional expression in the C3H10T1/2-MRF4 myogenic model cell line. C3H10T1/2 (10T1/2) is a multipotent, fibroblast-like cell line that can be manipulated to express a phenotype characteristic of either chondrocytes, adipocytes, or myocytes (11). A muscle phenotype can be selectively produced in 10T1/2 cells via overexpression of the myogenic regulatory transcription factor MRF4. In the presence of bFGF or other mitogenic stimuli, MRF4 is unable to initiate muscle-specific gene expression, even though MRF4 protein levels remain unchanged. Upon bFGF withdrawal, negative regulation of MRF4 is re...