T cell receptor engagement results in the reorganization of intracellular and membrane proteins at the T cell-antigen presenting cell interface forming the immunological synapse (IS), an event required for Ca(2+) influx. KCa3.1 channels modulate Ca(2+) signaling in activated T cells by regulating the membrane potential. Nothing is known regarding KCa3.1 membrane distribution during T cell activation. Herein, we determined whether KCa3.1 translocates to the IS in human T cells using YFP-tagged KCa3.1 channels. These channels showed electrophysiological and pharmacological properties identical to wild-type channels. IS formation was induced by either anti-CD3/CD28 antibody-coated beads for fixed microscopy experiments or Epstein-Barr virus-infected B cells for fixed and live cell microscopy. In fixed microscopy experiments, T cells were also immunolabeled for F-actin or CD3epsilon, which served as IS formation markers. The distribution of KCa3.1 was determined with confocal and fluorescence microscopy. We found that, upon T cell activation, KCa3.1 channels localize with F-actin and CD3epsilon to the IS but remain evenly distributed on the cell membrane when no stimulus is provided. Detailed imaging experiments indicated that KCa3.1 channels are recruited in the IS shortly after antigen presentation and are maintained there for at least 15-30 min. Interestingly, pretreatment of activated T cells with the specific KCa3.1 blocker TRAM-34 blocked Ca(2+) influx, but channel redistribution to the IS was not prevented. These results indicate that KCa3.1 channels are a part of the signaling complex that forms at the IS upon antigen presentation.

Alanine scanning of the channel region linking the S4 and S5 transmembrane helices of the hIK channel revealed several residues critical for protein trafficking and function. Substitution of phenylalanine with alanine (F194A), produced a channel that was inactive. Placing a tryptophan at this site (F194W), produced a channel that was functional, but initial whole-cell patch-clamp experiments indicated that the hIK-F194W channel could no longer be stimulated by the channel opener DC-EBIO. We determined the properties of this S4–S5 linker mutant channel further using giant-patch and single-channel patch clamp electrophysiological techniques. Upon patch excision, hIK-WT channel activity decreased ~ 70% over 5 min while the hIK-F194W channel only decreased ~10%. In excised patches, dose response relationships to free calcium gave an EC50 of 304 ± 35.8 nM, Hill 3.3 ± 0.4 (n = 6) for the hIK-F194W which was shifted from an EC50 of 1.19 ± 0.11 μM, Hill 2.8 ± 0.2 (n = 8) for hIK-WT. After rundown, hIK-WT channel activity could be stimulated ~ 500% by ATP or DC-EBIO, while hIK-F194W channel activity was only stimulated ~ 15% by either maneuver. Non-stationary variance analysis on channel activity produced by rundown and reactivation or 0 to 10μM calcium steps suggested that the Po of hIK-WT was ~ 0.5 while the hIK-F194W was ~ 0.9. This observation explains the small ~10% increase in patch currents upon addition of DC-EBIO and ATP as channels are almost maximally open in 10μM Ca2+. We conclude that the S4-S5 linker region has a role in hIK channel gating and suggest that the hIK-F194W mutant channel may be useful in dissecting the mechanisms by which ATP and Ca2+ coordinate to regulate the activity of the hIK Ca2+ sensitive potassium channel.

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 (Lys197) 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 role of the NH2-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 NH2-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 NH2 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 degreesC 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 NH2 terminus of hIK1 contains overlapping leucine zipper and dileucine motifs essential for channel assembly and trafficking to the plasma membrane.

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 (1396A/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 degreesC. 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.

We previously demonstrated that the endogenously expressed human intermediate conductance, Ca2+-activated K+ channel (hIK1) was inhibited by arachidonic acid (AA) (Devor, D. C., and Frizzell, R. A. (1998) Am. J. Physiol. 274, C138-C148). Here we demonstrate, using the excised, inside-out patch-clamp technique, that hIK1, heterologously expressed in HEK293 cells, is inhibited 82 +/- 2% (n = 16) with 3 muM AA, being half-maximally inhibited (IC50) at 1.4 +/- 0.7 muM. In contrast, AA does not inhibit the Ca2+-dependent, small conductance K+ channel, rSK2, another member of the KCNN gene family. Therefore, we utilized chimeric hIK1/rSK2 channels to define the AA binding domain on hIK1 to the S5-Pore-S6 region of the channel. Subsequent site-directed mutagenesis revealed that mutation of Thr(250) to Ser (T250S) resulted in a channel with limited sensitivity to block by AA (8 +/- 2%, n = 8), demonstrating that Thr(250) is a key molecular determinant for the inhibition of hIK1 by AA. Likewise, when Val(275) in S6 was mutated to Ala (V275A) AA inhibited only 43 +/- 11% (n = 9) of current flow. The double mutation T250S/V275A eliminated the AA sensitivity of hIK1. Introducing the complimentary single amino acid substitutions into rSK2 (S359T and A384V) conferred partial AA sensitivity to rSK2, 21 +/- 3% and 31 +/- 3%, respectively. Further, introducing the double mutation S359T/A384V into rSK2 resulted in a 63 +/- 8% (n = 9) inhibition by AA, thereby demonstrating the ability to introduce this inhibitory AA binding site into another member of the KCNN gene family. These results demonstrate that AA interacts with the pore-lining amino acids, Thr(250) and Val(275) in hIK1, conferring inhibition of hIK1 by AA and that AA and clotrimazole share similar, if not identical, molecular sites of interaction.

Vasoactive intestinal peptide (VIP) stimulates active Cl- secretion by the intestinal epithelium, a process that depends upon the maintenance of a favorable electrical driving force established by a basolateral membrane K+ conductance. To demonstrate the role of this K+ conductance, we measured short-circuit current (I-SC) across monolayers of the human colonic secretory cell line, T84. The serosal application of VIP (50 nM) increased I-SC from 3 +/- 0.4 pA/cm(2) to 75 +/- 11 muA/cm(2) (n = 4), which was reduced to a near zero value by serosal applications of Ba2+ (5 mM). The chromanol, 293B (100 mum), reduced I-SC by 74%, but charybdotoxin (CTX, 50 nM) had no effect. We used the whole-cell voltage-clamp technique to determine whether the K+ conductance is regulated by cAMP-dependent phosphorylation in isolated cells. VIP (300 nM) activated K+ current (131 +/- 26 pA, n = 15) when membrane potential was held at the Cl- equilibrium potential (ECl- = -2 mV), and activated inward current (179 +/- 28 pA. n = 15) when membrane potential was held at the K+ equilibrium potential (EK- = -80 mV); however, when the cAMP-dependent kinase (PKA) inhibitor, PKI (100 nm), was added to patch pipettes, VIP failed to stimulate these currents. Barium (Ba2+, 5 mm), but not 293B, blocked this K+ conductance in single cells. We used the cell-attached membrane patch under conditions that favor K+ current flow to demonstrate the channels that underlie this K+ conductance. VIP activated inwardly rectifying channel currents in this configuration. Additionally, we used fura-2AM to show that VIP does not alter the intracellular Ca2+ concentration, [Ca2+](i). Caffeine (5 mM), a phosphodiesterase inhibitor, also stimulated K+ current (185 +/- 56 pA, n = 8) without altering [Ca2+](i). These results demonstrate that VIP activates a basolateral membrane K+ conductance in T84 cells that is regulated by cAMP-dependent phosphorylation.

To test the hypothesis that Na+ transport in human bronchial epithelial (HBE) cells is regulated by a protease-mediated mechanism, we investigated the effects of BAY 39-9437, a recombinant Kunitz-type serine protease inhibitor, on amiloride-sensitive short-circuit current of normal [non-cystic fibrosis (CF) cells] and CF HBE cells. Mucosal treatment of non-CF and CF HBE cells with BAY 39-9437 decreased the short-circuit current, with a half-life of approximately 45 min. At 90 min, BAY 39-9437 (470 nM) reduced Na+ transport by approximately 70%. The inhibitory effect of BAY 39-9437 was concentration dependent, with a half-maximal inhibitory concentration of approximately 25 nM. Na+ transport was restored to control levels, with a half-life of approximately 15 min, on washout of BAY 39-9437. In addition, trypsin (1 microM) rapidly reversed the inhibitory effect of BAY 39-9437. These data indicate that Na+ transport in HBE cells is activated by a BAY 39-9437-inhibitable, endogenously expressed serine protease. BAY 39-9437 inhibition of this serine protease maybe of therapeutic potential for the treatment of Na+ hyperabsorption in CF.

We previously demonstrated that hIK1 is activated directly by ATP in excised, inside-out patches in a protein kinase A inhibitor 5-24 dependent manner, suggesting a role for phosphorylation in the regulation of this Ca2+-dependent channel. However, mutation of the single consensus cAMP-dependent protein kinase phosphorylation site (S334A) failed to modify the response of hIK1 to ATP (Gerlach, A. C., Gangopadhyay, N. N., and Devor, D. C. (2000) J. Biol Chem. 275, 585-598). Here we demonstrate that ATP does not similarly activate the highly homologous Ca2+-dependent K+ channels, hSK1, rSK2, and rSK3, To define the region of hIK1 responsible for the ATP-dependent regulation, we generated a series of hIK1 truncations and hIK1/rSK2 chimeras. ATP did not activate a chimera containing the N terminus plus S1-S4 from hIK1. In contrast, ATP activated a chimera containing the hIK1 C-terminal amino acids His(299)- Lys(427). Furthermore, truncation of hIK1 at Leu(414) resulted in an ATP-dependent channel, whereas larger truncations of hIK1 failed to express. Additional hIK1/rSK2 chimeras defined the minimal region of hIK1 required to confer complete ATP sensitivity as including amino acids Arg(355)-Ala(413). An alanine scan of all nonconserved serines and threonines within this region failed to alter the response of hIK1 to ATP, suggesting that hIK1 itself is not directly phosphorylated. Additionally, substitution of amino acids Arg(355)-Met(368) Of hIK1 into the corresponding region of rSK2 resulted in an ATP-dependent activation, which was similar to 50% of that of hIK1, These results demonstrate that amino acids Arg(355)-Ala(413) within the C terminus of hIK1 confer sensitivity to ATP. Finally, we demonstrate that the ATP-dependent phosphorylation of hIK1 or an associated protein is independent of Ca2+.

The diseases of cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) are characterized by mucus-congested airways. Agents that stimulate the secretion of Cl- are anticipated to facilitate mucociliary clearance and thus be of benefit in the treatment of CF and COPD. Recently 1-EBIO (1-ethyl-2-benzimidazolinone or 1-ethyl-1,3-dihydro-2H-benzimidazol-2-one) was shown to stimulate chloride secretion albeit at relatively high concentrations (0.6-1 mM). The studies reported here were undertaken to develop a more potent benzimidazolone. Structure activity studies with 30 benzimidazolone derivatives revealed that ethyl and hydrogen groups at the 1 and 3 nitrogen positions, respectively, were critical for the activation of hIK1 K+ channels and that other alkyl groups were not tolerated at these positions without some loss in potency. Substitutions at the 5 and 6 positions improved the potency of 1-EBIO. Compared with 1-EBIO, the most potent of these derivatives, DCEBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one) was severalfold better in a 86Rb+ uptake assay, 20-fold better in short circuit current measurements on T84 monolayers, and 100-fold better in patch-clamp assays of hIK1 activity. Short circuit current studies revealed DCEBIO stimulates Cl- secretion via the activation of hIK1 K+ channels and the activation of an apical membrane Cl- conductance. The improved potency of DCEBIO strengthens the possibility that compounds in this class may be of therapeutic benefit in the treatment of CF and COPD.

We previously demonstrated that hIK1 is activated directly by ATP in excised, inside-out patches in a protein kinase A inhibitor 5-24 dependent manner, suggesting a role for phosphorylation in the regulation of this Ca(2+)-dependent channel. However, mutation of the single consensus cAMP-dependent protein kinase phosphorylation site (S334A) failed to modify the response of hIK1 to ATP (Gerlach, A. C., Gangopadhyay, N. N., and Devor, D. C. (2000) J. Biol. Chem. 275, 585-598). Here we demonstrate that ATP does not similarly activate the highly homologous Ca(2+)-dependent K(+) channels, hSK1, rSK2, and rSK3. To define the region of hIK1 responsible for the ATP-dependent regulation, we generated a series of hIK1 truncations and hIK1/rSK2 chimeras. ATP did not activate a chimera containing the N terminus plus S1-S4 from hIK1. In contrast, ATP activated a chimera containing the hIK1 C-terminal amino acids His(299)-Lys(427). Furthermore, truncation of hIK1 at Leu(414) resulted in an ATP-dependent channel, whereas larger truncations of hIK1 failed to express. Additional hIK1/rSK2 chimeras defined the minimal region of hIK1 required to confer complete ATP sensitivity as including amino acids Arg(355)-Ala(413). An alanine scan of all non-conserved serines and threonines within this region failed to alter the response of hIK1 to ATP, suggesting that hIK1 itself is not directly phosphorylated. Additionally, substitution of amino acids Arg(355)-Met(368) of hIK1 into the corresponding region of rSK2 resulted in an ATP-dependent activation, which was approximately 50% of that of hIK1. These results demonstrate that amino acids Arg(355)-Ala(413) within the C terminus of hIK1 confer sensitivity to ATP. Finally, we demonstrate that the ATP-dependent phosphorylation of hIK1 or an associated protein is independent of Ca(2+).

We previously demonstrated that 1-ethyl-2-benzimidazolone (1-EBIO) directly activates basolateral membrane calcium-activated K(+) channels (K(Ca)), thereby stimulating Cl(-) secretion across several epithelia. In our pursuit to identify potent modulators of Cl(-) secretion that may be useful to overcome the Cl(-) secretory defect in cystic fibrosis (CF), we have identified chlorzoxazone [5-chloro-2(3H)-benzoxazolone], a clinically used centrally acting muscle relaxant, as a stimulator of Cl(-) secretion in several epithelial cell types, including T84, Calu-3, and human bronchial epithelium. The Cl(-) secretory response induced by chlorzoxazone was blocked by charybdotoxin (CTX), a known blocker of K(Ca). In nystatin-permeabilized monolayers, chlorzoxazone stimulated a basolateral membrane I(K), which was inhibited by CTX and also stimulated an apical I(Cl) that was inhibited by glibenclamide, indicating that the G(Cl) responsible for this I(Cl) may be cystic fibrosis transmembrane conductance regulator (CFTR). In membrane vesicles prepared from T84 cells, chlorzoxazone stimulated (86)Rb(+) uptake in a CTX-sensitive manner. In excised, inside-out patches, chlorzoxazone activated an inwardly-rectifying K(+) channel, which was inhibited by CTX. 6-Hydroxychlorzoxazone, the major metabolite of chlorzoxazone, did not activate K(Ca), whereas zoxazolamine (2-amino-5-chlorzoxazole) showed a similar response profile as chlorzoxazone. In normal human nasal epithelium, chlorzoxazone elicited hyperpolarization of the potential difference that was similar in magnitude to isoproterenol. However, in the nasal epithelium of CF patients with the DeltaF508 mutation of CFTR, there was no detectable Cl(-) secretory response to chlorzoxazone. These studies demonstrate that chlorzoxazone stimulates transepithelial Cl(-) secretion in normal airway epithelium in vitro and in vivo, and suggest that stimulation requires functional CFTR in the epithelia.