We have used the patch-clamp electrical recording technique on giant spheroplasts of Escherchia coli and have discovered pressure-activated ion channels. The channels have the following properties: (t) activation by slight positive or negative pressure; (it) voltage dependence; (ifi) large conductance; (iv) selectivity for anions over cations; (v) dependence of activity on the species of permeant ions. We believe that these channels may be involved in bacterial osmoregulation and osmotaxis.Ion channels are gated protein pores found in biological membranes; these channels regulate many cellular interactions with the environment, including responses to hormonal, neuronal, and sensory stimuli (1). Ion channels have been studied in animals, plants, and microorganisms (2-4). In bacteria, in vivo channel activity has not been demonstrated, although the activity of isolated channel proteins has been measured in artificial membranes (5, 6).The patch-clamp technique allows recording of current through individual ion channels in the native membrane by sucking the membrane onto a recording pipette to form a tight (gigaohm) electrical seal (7). This method has been used to study single channels in vivo in many eukaryotic cells, and it has demonstrated that the large currents measured across the membranes of a whole cell are really composed ofmany small currents passing through individual channels.The lower limit to the diameter ofthe patch-pipette opening is about 1 ,um (1); this precludes measurement ofion channels in bacteria directly. Cells of Escherichia coli, however, can become giant spheroplasts when grown in the presence of chemicals such as mecillinam to prevent cell wall (peptidoglycan) synthesis, and membrane potential has been measured in such spheroplasts by conventional electrophysiology (8). Giant spheroplasts can also be formed by growth of cells in the presence of cephalexin to prevent cell division and form filamentous "snakes"; these snakes can then be treated with lysozyme and EDTA to dissolve the cell wall (the spheroplasts can revert to normal form when returned to growth medium in the absence of these chemicals) (9). We used this latter method to make spheroplasts with a diameter of -6 ,.m. We demonstrate here the application of in vivo patch-clamp recording to such giant spheroplasts. This method should be generally applicable to any bacterial species.We discovered that a low positive or negative pressure (tens of millimeters of mercury; 1 mm Hg = 133 Pa) applied to the spheroplast membrane activates ion channels. This pressure could be caused by an osmotic difference of as little as a few milliosmolar across the membrane. We believe that these channels may allow E. coli to detect and to respond to small osmotic changes in the surrounding medium. The preliminary work has been reported in abstract form (10). MATERIALS AND METHODSMaterials. Organic components of the growth medium were purchased from Difco. Tris was purchased from Boehringer Mannheim; other salts and chemicals for preparation of...

The excretory cell extends a tubular process, or canal, along the basolateral surface of the epidermis to form the nematode renal epithelium. This cell can undergo normal tubulogenesis in isolated cell culture. Mutations in 12 genes cause excretory canal cysts in Caenorhabditis elegans. Genetic interactions, and their similar phenotypes, suggest these genes may encode functionally related proteins. Depending upon genotype and individual canal, defects range from focal cysts, flanked by normal width segments, to regional cysts involving the entire tubule. Oftentimes the enlarged regions are convoluted or partially septated. In mutants with very large cysts, renal function is measurably impaired. Based on histology and ultrastructure, canal cysts likely result from defects of the apical membrane domain. These mutants provide a model of tubulocystic disease without hyperplasia or basement membrane abnormalities. Similar apical mechanisms could regulate tubular morphology of vertebrate nephrons.

SUMMARYMany unicellular tubes such as capillaries form lumens intracellularly, a process that is not well understood. Here we show that the cortical membrane organizer ERM-1 is required to expand the intracellular apical/lumenal membrane and its actin undercoat during single-cell C.elegans excretory canal morphogenesis. We characterize AQP-8, identified in an ERM-1 overexpression (ERM-1[++]) suppressor screen, as a canalicular aquaporin that interacts with ERM-1 in lumen extension in a mercury-sensitive manner, implicating water-channel activity. AQP-8 is transiently recruited to the lumen by ERM-1, co-localizing in peri-lumenal cuffs interspaced along expanding canals. An ERM-1[++]-mediated increase in the number of lumen-associated canaliculi is reversed by AQP-8 depletion. We propose that the ERM-1-AQP-8 interaction propels lumen extension by translumenal flux, suggesting a direct morphogenetic effect of water-channel-regulated fluid pressure.

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