Anion channels perform important roles in a variety of fundamental physiological processes including whole animal salt and water balance, germ cell development, and control of muscle contraction. C. elegans offers unique experimental advantages for defining the molecular basis of anion channel function and regulation. To begin exploiting C. elegans as a model system for characterizing anion channel biology, we have carried out electrophysiological studies on oocytes, intact and dissociated early embryos, and various cultured differentiated cell types. Undifferentiated, dissociated embryonic cells express a robust outwardly rectifying mechanosensitive anion current, I Cl,mec , that is activated by membrane stretch and depolarization. The characteristics of I Cl,mec include an anion-to-cation selectivity of 4:1, an Eisenman type I anion selectivity and sensitivity to inhibitors of anion and mechanosensitive channels. I Cl,mec is detected in >80% of membrane patches. The unitary conductance of the I Cl,mec channel is 6 pS at +/-100 mV. Macroscopic currents of 40-120 pA at +100 mV are typically observed in inside-out membrane patches formed using low resistance patch pipettes. Isolated membrane patches of early embryonic cells therefore contain 60-200 I Cl,mec channels. I Cl,mec was not detected in unfertilized oocytes. To ascertain when I Cl,mec is activated, we isolated staged embryos from single worms. Robust channel activity was observed in all cells from intact 1-8 cell stage embryos and in dissociated embryonic cells with diameters ranging from 5 to 25 m m. These observations indicate that I Cl,mec is activated shortly after fertilization and that channel activity persists up to at least the ~100 cell stage of development. Cells of early embryos exhibit little or no obvious morphological differentiation. To determine whether I Cl,mec is expressed in differentiated cell types, we cultured dissociated embryonic cells on glass cover slips. Culturing freshly isolated blastomeres on cover slips coated with agents that promote cell adhesion stimulates differentiation within 24 h into the major cell types that comprise the newly hatched L1 larva. Cells cultured on uncoated cover slips do not adhere to the growth substrate and remain viable for many days, but do not appear to differentiate morphologically. I Cl,mec was not detected in cells with well-defined neuronal morphology (n=4), muscle morphology (n=12) or in ASER neurons (n=10). Because I Cl,mec is regulated by membrane stretch, we also patch clamped cultured neurons expressing
mec-7 ::GFP.
mec-7 encodes a b -tubulin expressed largely in mechanosensory neurons (Hamelin et al., EMBO J . 11:2885-2893, 1992). I Cl,mec was not detected in cell-attached patches on cultured
mec-7 ::GFP-expressing neurons (n=15). We also failed to detect I Cl,mec in whole-cell current recordings from mechanosensory neurons under conditions designed to isolate anion channel currents. Robust I Cl,mec channel activity was, however, detected in morphologically undifferentiated embryonic cells maintained in culture for six days. These results indicate that I Cl,mec activity is dramatically reduced in differentiated cells. Reduction of channel activity is not due to maintenance of cells in culture, but instead appears to be due to attainment of a differentiated state. The abundance of I Cl,mec expression in early embryos, its activation shortly after fertilization, and the significant reduction of channel activity in differentiated cell types suggest that I Cl,mec may play roles in early embryonic development. We are currently using reverse genetic strategies to identify the genes that encode the I Cl,mec channel and its associated regulatory machinery.