Enteropathogenic Escherichia coli (EPEC) causes severe, watery diarrhea in children. We investigated ATP release during EPEC-mediated killing of human cell lines and whether released adenine nucleotides function as secretory mediators. EPEC triggered a release of ATP from all human cell lines tested: HeLa, COS-7, and T84 (colon cells) as measured using a luciferase kit. Accumulation of ATP in the supernatant medium was enhanced if an inhibitor of 5'-ectonucleotidase was included and was further enhanced if an ATP-regenerating system was added. In the presence of the inhibitor/regenerator, ATP concentrations in the supernatant medium reached 1.5-2 microM 4 h after infection with wild-type EPEC strains. In the absence of the inhibitor/regenerator system, extracellular ATP was rapidly broken down to ADP, AMP, and adenosine. Conditioned medium from EPEC-infected cells triggered a brisk chloride secretory response in intestinal tissues studied in the Ussing chamber (rabbit distal colon and T84 cell monolayers), whereas conditioned medium from uninfected cells and sterile filtrates of EPEC bacteria did not. The short-circuit current response to EPEC-conditioned medium was completely reversed by adenosine receptor blockers, such as 8-(p-sulfophenyl)-theophylline and MRS1754. EPEC killing of host cells releases ATP, which is broken down to adenosine, which in turn stimulates secretion via apical adenosine A2b receptors. These findings provide new insight into how EPEC causes watery diarrhea.

We demonstrate that the C-terminal truncation of hIK1 results in a loss of functional channels. This could be caused by either (i) a failure of the channel to traffic to the plasma membrane or (ii) the expression of nonfunctional channels. To delineate among these possibilities, a hemagglutinin epitope was inserted into the extracellular loop between transmembrane domains S3 and S4. Surface expression and channel function were measured by immunofluorescence, cell surface immunoprecipitation, and whole-cell patch clamp techniques. Although deletion of the last 14 amino acids of hIK1 (L414STOP) had no effect on plasma membrane expression and function, deletion of the last 26 amino acids (K402STOP) resulted in a complete loss of membrane expression. Mutation of the leucine heptad repeat ending at Leu 406 (L399A/L406A) completely abrogated membrane localization. Additional mutations within the heptad repeat (L385A/L392A, L392A/L406A) or of the a positions (I396A/L403A) resulted in a near-complete loss of membrane-localized channel. In contrast, mutating individual leucines did not compromise channel trafficking or function. Both membrane localization and function of L399A/L406A could be partially restored by incubation at 27°C. Co-immunoprecipitation studies demonstrated that leucine zipper mutations do not compromise multimer formation. In contrast, we demonstrated that the leucine zipper region of hIK1 is capable of co-assembly and that this is dependent upon an intact leucine zipper. Finally, this leucine zipper is conserved in another member of the gene family, SK3. However, mutation of the leucine zipper in SK3 had no effect on plasma membrane localization or function. In conclusion, we demonstrate that the C-terminal leucine zipper is critical to facilitate correct folding and plasma membrane trafficking of hIK1, whereas this function is not conserved in other gene family members.

The role of the NH 2 -terminal leucine zipper and dileucine motifs of hIK1 in the assembly, trafficking, and function of the channel was investigated using cell surface immunoprecipitation, co-immunoprecipitation (Co-IP), immunoblot, and whole-cell patch clamp techniques. Mutation of the NH 2 -terminal leucine zipper at amino acid positions 18 and 25 (L18A/L25A) resulted in a complete loss of steady-state protein expression, cell surface expression, and whole-cell current density. Inhibition of proteasomal degradation with lactacystin restored L18A/L25A protein expression, although this channel was not expressed at the cell surface as assessed by cell surface immunoprecipitation and wholecell patch clamp. In contrast, inhibitors of lysosomal degradation (leupeptin/pepstatin) and endocytosis (chloroquine) had little effect on L18A/L25A protein expression or localization. Further studies confirmed the rapid degradation of this channel, having a time constant of 19.0 ؎ 1.3 min compared with 3.2 ؎ 0.8 h for wild type hIK1. Co-expression studies demonstrated that the L18A/L25A channel associates with wild type channel, thereby attenuating its expression at the cell surface. Co-IP studies confirmed this association. However, L18A/L25A channels failed to form homotetrameric channels, as assessed by Co-IP, suggesting the NH 2 terminus plays a role in tetrameric channel assembly. As with the leucine zipper, mutation of the dileucine motif to alanines, L18A/L19A, resulted in a near complete loss in steady-state protein expression with the protein being similarly targeted to the proteasome for degradation. In contrast to our results on the leucine zipper, however, both chloroquine and growing the cells at the permissive temperature of 27°C restored expression of L18A/L19A at the cell surface, suggesting that the defect in the channel trafficking is the result of a subtle folding error. In conclusion, we demonstrate that the NH 2 terminus of hIK1 contains overlapping leucine zipper and dileucine motifs essential for channel assembly and trafficking to the plasma membrane.The KCNN gene family is composed of four members, including the small conductance Ca 2ϩ -activated K ϩ channels (SK1, SK2, and SK3) 1 and the intermediate conductance Ca 2ϩ -activated K ϩ channel (IK1 or SK4). While the IK1 and SK channels share roughly 40% homology, their expression patterns and pharmacology are widely disparate, consistent with their unique physiological functions (1). The human IK1 channel (hIK1) is widely expressed, being found in salivary gland, colon, bladder, stomach, lung, smooth muscle, red blood cells, T-cells, and placenta (2, 3) where it plays a crucial role in a variety of physiological functions. Indeed hIK1 plays a seminal role in modulating Ca 2ϩ -dependent transepithelial ion transport (4, 5), has recently been confirmed to be the Gardos channel of red blood cells (6), and has been proposed to play a role in both vascular remodeling (7) and in modulating vascular tone (8). Because of these critical physiological roles, the ...

We have investigated the role of the S4-S5 linker in the trafficking of the intermediate (human (h) IK1) and small (rat SK3) conductance K ؉ channels using a combination of patch-clamp, protein biochemical, and immunofluorescence-based techniques. We demonstrate that a lysine residue (Lys 197 ) located on the intracellular loop between the S4 and S5 domains is necessary for the correct trafficking of hIK1 to the plasma membrane. Mutation of this residue to either alanine or methionine precluded trafficking of the channel to the membrane, whereas the charge-conserving arginine mutation had no effect on channel localization or function. Immunofluorescence localization demonstrated that the K197A mutation resulted in a channel that was primarily retained in the endoplasmic reticulum, and this could not be rescued by incubation at 27°C. Furthermore, immunoblot analysis revealed that the K197A mutation was overexpressed compared with wild-type hIK1 and that this was due to a greatly diminished rate of channel degradation. Co-immunoprecipitation studies demonstrated that the K197A mutation did not preclude multimer formation. Indeed, the K197A mutation dramatically suppressed expression of wild-type hIK1 at the cell surface. Finally, mutation of this conserved lysine in rat SK3 similarly resulted in a channel that failed to correctly traffic to the plasma membrane. These results are the first to demonstrate a critical role for the S4-S5 linker in the trafficking and/or function of IK and SK channels.The human (h) 3 intermediate conductance Ca 2ϩ -activated K ϩ channel IK1 is a member of the KCNN gene family, which also includes SK1-3, the small conductance Ca 2ϩ -activated K ϩ channels. Within this channel family, there is ϳ40% amino acid sequence homology, with the greatest level of identity occurring in the pore and proximal C terminus, a region known to constitutively bind calmodulin (1, 2), thereby conferring Ca 2ϩ sensitivity to these channels. The hIK1 channel is now known to be the Gardos channel involved in red blood cell volume regulation (3). hIK1 is also known to be involved in the Ca 2ϩ -dependent regulation of Cl Ϫ secretion across intestinal and airway epithelia (4 -8). More recent work has demonstrated that the progression of breast cancer cells through the cell cycle is dependent on membrane hyperpolarization resulting from the activation of hIK1 channels (9). hIK1 has also been shown to play a role in both macrophage activation and T-lymphocyte proliferation (10 -12). The critical role that pharmacological modulation of hIK1 may play has been revealed by the demonstration that blockers of hIK1 reduce the autoimmune response to experimental encephalomyelitis in mice (13), reduce brain edema following traumatic brain injury (14), and prevent restenosis following balloon angioplasty (15). Also, openers have been shown to alter vascular tone and hence may modulate peripheral blood pressure (16,17). A great deal of research has focused on both the biophysical and pharmacological properties of hIK1 (18 -20)...

We previously demonstrated that the ATP/PKA-dependent activation of the human intermediate conductance, Ca 2+ -activated K + channel, hIK1, is dependent upon a C-terminal motif. The NH 2 -terminus of hIK1 contains a multi-basic 13 RRRKR 17 motif, known to be important in the trafficking and function of ion channels. While individual mutations within this domain have no effect on channel function, the triple mutation ( 15 RKR 17 /AAA), as well as additional double mutations, result in a near complete loss of functional channels, as assessed by whole-cell patch-clamp. However, cell-surface immunoprecipitation studies confirmed expression of these mutated channels at the plasma membrane. To elucidate the functional consequences of the 15 RKR 17 /AAA mutation we performed inside-out patch clamp recordings where we observed no difference in Ca 2+ affinity between the wild-type and mutated channels. However, in contrast to wild-type hIK1, channels expressing the 15 RKR 17 /AAA mutation exhibited rundown, which could not be reversed by the addition of ATP. Wild-type hIK1 channel activity was reduced by alkaline phosphatase both in the presence and absence of ATP, indicative of a phosphorylation event, whereas the 15 RKR 17 /AAA mutation eliminated this effect of alkaline phosphatase. Further, single channel analysis demonstrated that the 15 RKR 17 /AAA mutation resulted in a four-fold lower channel open probability (P o ), in the presence of saturating Ca 2+ and ATP, compared to wild-type hIK1. In conclusion, these results represent the first demonstration for a role of the NH 2 -terminus in the second messenger-dependent regulation of hIK1 and, in combination with our previous findings, suggest that this regulation is dependent upon a close NH 2 /C-terminal association.