Potassium channels in basolateral membrane vesicles from necturus enterocytes: stretch and ATP sensitivity.

We have previously reported that ATP-inhibitable K(+) channels, in vesicles derived from the basolateral membrane of Necturus maculosus small intestinal cells, exhibit volume regulatory responses that resemble those found in the intact tissue after exposure to anisotonic solutions. We now report that increases in K(+) channel activity can also be elicited by exposure of these vesicles to isotonic solutions containing glucose or alanine that equilibrate across these membranes. We also demonstrate that swelling after exposure to a hypotonic solution or an isotonic solution containing alanine or glucose reduces inhibition of channel activity by ATP and that this finding cannot be simply attributed to dilution of intravesicular ATP. We conclude that ATP-sensitive, stretch-activated K(+) channels may be responsible for the well-established increase in basolateral membrane K(+) conductance of Necturus small intestinal cells after the addition of sugars or amino acids to the solution perfusing the mucosal surface, and we propose that increases in cell volume, resulting in membrane stretch, decreases the sensitivity of these channels to ATP.

Volume regulatory responses of basolateral membrane vesicles from Necturus enterocytes: role of the cytoskeleton.

Previous studies from this laboratory have demonstrated that basolateral membrane vesicles isolated from Necturus maculosus small intestinal epithelial cells possess a K(+) channel that is inhibited by ATP. In the present studies, we demonstrate that these vesicles, which are essentially devoid of soluble cytoplasmic contaminants, exhibit volume regulatory responses that parallel those of intact epithelial cells. Thus, suspension of these vesicles in a solution that is hypotonic to the intravesicular solution increases channel activity whereas suspension in a solution that is hypertonic to the intravesicular solution decreases, and may abolish, channel activity. These volume regulatory responses appear to be mediated by the same K(ATP) channel and depend on an intact actin cytoskeletal network. The responses to both hypotonic and hypertonic challenge are abolished by cytochalasin D or by incubating the vesicles under conditions that are known to depolymerize actin. Phalloidin, which is known to stabilize actin filaments, partially prevents the action of cytochalasin D. Thus, the present results indicate that the K(ATP) channel activity of basolateral membrane vesicles from Necturus basolateral membranes respond to hypo- and hypertonic challenge monotonically around an isotonic "set point" and that these responses depend on an intact actin cytoskeleton.

Colocalization of glycolytic enzyme activity and KATP channels in basolateral membrane of Necturus enterocytes.

86Rb fluxes through ATP-regulated K+ (KATP) channels in membrane vesicles derived from basolateral membranes of Necturus small intestinal epithelial cells as well as the activity of single KATP channels reconstituted into planar phospholipid bilayers are inhibited by the presence of ADP plus phosphoenolpyruvate in the solution bathing the inner surface of these channels. This inhibition can be prevented by pretreatment of the membranes with 2, 3-butanedione, an irreversible inhibitor of pyruvate kinase (PK) and reversed by the addition of 2-deoxyglucose plus hexokinase. The results of additional studies indicate that PK activity appears to be tightly associated with this membrane fraction. These results, together with considerations of the possible ratio of Na+-K+ pumps to KATP channels in the basolateral membrane, raise the possibility that "cross talk" between those channels and pumps (i.e., the "pump-leak parallelism") may be mediated by local, functionally compartmentalized ATP-to-ADP ratios that differ from those in the bulk cytoplasm.

Reversal of glibenclamide and voltage block of an epithelial KATP channel.

K+ channels present in basolateral membrane vesicles isolated from Necturus maculosa small intestinal cells and reconstituted into planar phospholipid bilayers are inhibited by MgATP and sulfonylurea derivatives, such as tolbutamide and glibenclamide, when these agents are added to the solution bathing the inner mouth of the channel. In addition, these channels possess an intrinsic "voltage gate" and are blocked when the electrical potential difference across the channel is oriented so that the inner solution is electrically positive with respect to the outer solution. We now show that increasing the concentration of permeant ions such as K+ or Rb+ in the outer solution reverses channel inhibition resulting from the addition of 50 microM glibenclamide to the inner solution and also inhibits intrinsic voltage gating; these effects are not elicited by increasing the concentrations of the relatively impermeant ions, Na+ or choline, in the outer solution. Furthermore, increasing the K+ concentration in the outer solution in the absence of glibenclamide inhibits voltage gating, and, under these conditions, the subsequent addition of glibenclamide to the inner solution is ineffective. These results are consistent with a model in which the voltage gate is an open-channel blocker whose action is directly reversed by elevating the external concentration of relatively permeant cations and where the action of glibenclamide is to stabilize the inactivated state of the channel, possibly through hydrophobic interactions.

Reconstitution of a KATP channel from basolateral membranes of Necturus enterocytes.

We have previously reported that basolateral membrane vesicles isolated from Necturus maculosa small intestinal epithelial cells and incorporated into planar phospholipid bilayers display a highly selective "maxi"-conductance K+ channel whose open-time probability is affected by voltage. We now report that this channel is inhibited by MgATP in the solution bathing the intracellular face of the channel but not by Mg2+ or the Na+ or K+ salts of ATP; the effects of MgATP can be prevented or reversed by MgADP. The channel is also inhibited by the nonhydrolyzable ATP analogue magnesium adenosine 5'-O-(3-thiotriphosphate) and the sulfonylurea derivatives tolbutamide and glibenclamide; all of these agents are effective in the intracellular compartment but not when added to the extracellular compartment alone. Channel activity is stimulated by the "K+ channel opener," diazoxide, which also reverses the effect of glibenclamide but not of MgATP. The possible role of this channel as a mediator of the parallelism between basolateral membrane Na(+)-K+ pump activity and the macroscopic K+ conductance of that barrier is discussed.

Effects of a Shaker K+ channel peptide and trypsin on a K+ channel in Necturus enterocytes.

We have previously demonstrated that a synthetic peptide composed of the first 22 amino acids from the NH2-terminus of the Shaker B K+ channel protein deactivates a voltage-dependent K+ channel present in basolateral membrane of Necturus small intestinal epithelial cells reconstituted into planar lipid bilayers (Dubinsky et al. Proc. Natl. Acad. Sci. USA 89: 1770-1774, 1992). We now demonstrate that this peptide interacts with the inner surface of the Necturus channel only when it is in the open or conducting configuration and that this interaction is hindered by tetraethylammonium ion, a well-established blocker of this and other K+ channels. We conclude that this peptide is an open-pore blocker of the Necturus K+ channel as it appears to be in the case of the Shaker B K+ channel. We further demonstrate that trypsin, which abolishes the ability of this peptide to block both the Necturus and the Shaker K+ channels and inhibits spontaneous inactivation of the Shaker K+ channel, also impairs the voltage-gate of the Necturus K+ channel. These findings, and others to be reported in a companion paper, suggest structural homologies between the "inactivation peptide" of the Shaker B K+ channel and the voltage-gate of the Necturus K+ channel.

Immunoisolation of a K+ channel from basolateral membranes of Necturus enterocytes.

We have reported that a peptide composed of the NH2-terminal 22 amino acids of the Drosophila Shaker B K+ channel protein, which is responsible for the inactivation of this A-type channel, blocks the inner, open mouth of a voltage-gated K+ channel present in the basolateral membrane of Necturus maculosa small intestinal enterocytes. We now demonstrate that antibodies to this "inactivating" peptide interact with proteins in solubilized and intact basolateral membranes from Necturus enterocytes. Asolectin vesicles reconstituted with the full complement of solubilized basolateral membrane proteins display 86Rb+ uptake that is inhibited by tetraethylammonium ion and abolished by immunoprecipitation with these antibodies. Furthermore, asolectin vesicles containing protein eluted from an antibody-affinity column display 86Rb+ uptake that is abolished by boiling. Finally, reconstitution of the immunoisolated protein into planar phospholipid bilayers disclosed a K+ channel whose single-channel properties are identical to those of the voltage-gated channel in the native basolateral membranes. Our data are consistent with the notion that a 150-kDa protein present in basolateral membranes of Necturus enterocytes possesses inwardly rectifying K+ channel activity and that this protein is antigenically similar to the type A K+ channel present in the flight muscles of Drosophila melanogaster and encoded by the Shaker B locus.

A peptide from the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membranes of Necturus enterocytes.

A synthetic peptide composed of the first 22 amino acid residues of the Drosophila Shaker K+ channel inhibits a voltage-gated K+ channel in basolateral membrane vesicles from Necturus enterocytes reconstituted in planar phospholipid bilayers when added to the solution bathing the inner surface of this channel but not when added to the solution bathing its outer surface. A modified peptide in which the leucine in the 7 position is replaced with phenylalanine is also an effective inhibitor, but replacement of the leucine-7 with lysine or glutamate, or digestion with trypsin, renders the peptide ineffective; replacement of the leucine-7 with glycine markedly reduces but does not abolish the effectiveness of the peptide as an inhibitor. These results are analogous to those reported for the Shaker K+ channel +ADHoshi, T., Zagotta, W.N. & Aldrich, R.W. (1990) Science 250, 533-538; and Zagotta, W.N., Hoshi, T. & Aldrich, R.W. (1990) Science 250, 568-571.+BD and suggest that the molecular anatomy of the receptor at the inner face of the Necturus K+ channel with which the peptide interacts to bring about inhibition of that channel may be similar to that of the Shaker K+ channel.