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Heather M Jones

Publications and source records attributed to Heather M Jones.

6 recordsLinked to original sources
PubMed2005

Role of an S4-S5 linker lysine in the trafficking of the Ca(2+)-activated K(+) channels IK1 and SK3.

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 degrees 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.

Animals↗
PubMed2004

Role of the NH2 terminus in the assembly and trafficking of the intermediate conductance Ca2+-activated K+ channel hIK1.

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 whole-cell 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 degrees 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.

Acetylcysteine↗
PubMed2002

Trafficking of the Ca2+-activated K+ channel, hIK1, is dependent upon a C-terminal leucine zipper.

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 non-functional 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 degrees 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.

Base Sequence↗
PubMed2002

Release of ATP during host cell killing by enteropathogenic E. coli and its role as a secretory mediator.

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.

5'-Nucleotidase↗
PubMedDate not supplied

Evaluation of plasma resistant hollow fiber membranes for artificial lungs.

Hollow fiber membranes (HFMs) used in artificial lungs (oxygenators) undergo plasma leakage (or wetting) in which blood plasma slowly fills the pores of the fiber wall, plasma leaks into gas pathways, and overall gas exchange decreases. To overcome this problem plasma resistant fibers are being developed that are skinned asymmetric or composite symmetric versions of microporous oxygenator fibers. This report evaluates several candidate plasma resistant HFMs in terms of their gas permeance and plasma resistance as measured in a surfactant wet out test. Five candidate fibers were compared with each other and with a control fiber. CO2 and O2 gas permeance (in ml/s/cm2/cm Hg) in the plasma resistant fibers ranged from 3.15E-04 to 1.71E-03 and 3.40E-04 to 1.08E-03, respectively, compared with 1.62E-02 and 1.77E-02 for the control fiber. Maximum dye bleed through for the plasma resistant fibers in the forced wet out test were significantly less than for the control fiber. CO2 gas permeance of a plasma resistant fiber imposes the greatest constraint upon artificial lung design for sufficient gas exchange. However, our results suggest sufficient plasma resistance can be achieved using special skinned and composite HFMs while maintaining an acceptable CO2 gas permeance for a broad range of artificial lung applications.

Artificial Organs↗