Worm Breeder's Gazette 14(1): 45 (October 1, 1995)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Department of Molecular Biology and Biochemistry, Rutgers University, New Brunswick, NJ 08855
mec-4 encodes a protein related to mec-10, DEG-1 and subunits of the mammalian amiloride-sensitive epithelial Na+ channel. mec-4 and mec-10 are co-expressed in the six touch receptor neurons and are postulated to encode subunits of a mechanosensory ion channel. This working hypothesis is exciting because, unlike voltage-gated and ligand-gated channels, mechanically-gated channels (those opened by membrane stretch or physical deflection) have not yet been characterized at the molecular level. mec-4 and related family members have a second interesting feature--specific dominant gain-of-function alleles induce swelling and degeneration of the neurons in which they are expressed. We are interested in understanding the normal and inappropriate activity of this newly identified channel class. Biophysical studies support that tension applied to mechanosensitive ion channels is required for their regulated opening and Chalfie and colleagues have identified extracellular matrix and intracellular proteins that may interact with the touch receptor channel (see wm95 p73). Thus, the determination of channel subunit topology is important for defining target regions for interaction with the cytoskeleton or extracellular matrix, contacts likely to be essential for mechanotransduction. MEC-4 has two hydrophobic domains that are theoretically capable of spanning the cell membrane. The predicted coding sequence does not include a hydrophobic N-terminal signal sequence. As a consequence, MEC-4 is expected to have an intracellular N-terminal domain, a large extracellular domain that includes two Cys-rich regions and a short intracellular C- terminal domain. We have performed biochemical experiments that support this overall transmembrane topology. We raised antibodies against 4 MEC-4 regions: the N-terminal domain, two stretches in the Cys-rich region, and the C-terminal domain. When we translate mec-4 in vitro in the presence of microsomal membranes we detect a glycoslyated product, which when treated with endoglycosidase H, yields a protein of the size predicted for MEC-4. In microsomal ER membranes, domains that would normally project into the extracellular space project into the ER lumen, where they are accessible to modifying enzymes such as those that add sugar moieties and domains that normally project into the cytoplasm project outside the lumen and are accessible to enzymes added to the reaction, such as proteases. When we translate mec-4 in vitro in the presence of microsomes and then add proteinase K to the reaction mix, the size of the reaction product is decreased by approximately 20 kD, a value consistent with proteolytic removal of the predicted 15 kD N-terminal and 3.4 kD C-terminal domains. The protease-resistant fragment is glycosylated and can be recognized by antibodies directed against the Cys- rich region but is not recognized by anti-N-and C-terminal antibodies. These results support that the N- and C-termini are normally intracellular and that no other domain of MEC-4 projects into the intracellular space. Results of gene fusion experiments support this topology--fusion of lacZ to the N-and C-termini of MEC-4 are active for b-galactosidase activity wereas fusions to the extracellular domain are not. The facts that mec-4 and mec-10 are co-expressed in the touch receptor neurons, that mec-10(d)-induced degeneration depends on mec-4, and that the mammalian channel includes multiple homologous subunits support that mec-10 and mec-4 are part of the same channel complex. In preliminary experiments on channel assembly, we have demonstrated that MEC-4 and MEC-10 can be co-immunoprecipitated when expressed in the presence of microsomes, providing the first physical evidence that these proteins associate in the endoplasmic reticulum.