ATP-dependent Activation of the Intermediate Conductance, Ca2+-activated K+ Channel, hIK1, Is Conferred by a C-terminal Domain*

Aaron C. Gerlach, Colin A. Syme, LeeAnn Giltinan, John P. AdelmanDagger , and Daniel C. Devor§

From the Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 and the Dagger  Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201

Received for publication, August 23, 2000, and in revised form, November 15, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 His299-Lys427. Furthermore, truncation of hIK1 at Leu414 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 Arg355-Ala413. 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 Arg355-Met368 of hIK1 into the corresponding region of rSK2 resulted in an ATP-dependent activation, which was ~50% of that of hIK1. These results demonstrate that amino acids Arg355-Ala413 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+.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The human intermediate conductance KCa channel, hIK1, is required for a variety of physiological processes including transepithelial ion transport (2-5), vasodilation (6, 7), T cell activation (8, 9), cell proliferation (9-11), and regulatory volume decrease (9, 10). In addition to demonstrating modulation by intracellular Ca2+, we and others have demonstrated that hIK1 activity can be dynamically regulated by phosphorylation (1, 9, 12-14). Several of these studies have demonstrated an ATP-dependent activation of hIK1 in excised, inside-out membrane patches that can be reversed by exogenous phosphatases and/or kinase inhibitors (1, 12, 13). Based on these observations, we speculated that the phosphorylation-dependent modulation of hIK1 plays a critical role in modulating the physiological processes in which hIK1 is involved.

In our previous study we demonstrated, in excised, inside-out patches, that addition of ATP (1 mM) resulted in, on average, a 3-fold increase in hIK1 activity (1), having an EC50 of 50 µM.1 The stimulatory effect of ATP exhibited a slow onset, requiring several minutes for the maximal response, was strictly Mg2+-dependent, and could be mimicked by neither hydrolyzeable (ADP, GTP, UTP, CTP, ITP) nor non-hydrolyzeable (AMP-PNP,2 AMP-PCP) ATP analogs. The ATP-dependent stimulation of hIK1 could be reversed by both alkaline and acid phosphatases, suggesting that ATP activates hIK1 via a membrane-delimited kinase. In patches both from T84 cells, which natively express hIK1, (1, 2, 4) and from the Xenopus oocyte heterologous expression system, inhibitors of cAMP-dependent protein kinase could partially reverse that ATP-dependent activation. However, mutation of the only cAMP-dependent protein kinase consensus phosphorylation site, serine 334, as well as of all four protein kinase C consensus phosphorylation sites (Thr101, Ser178, Thr329, Ser388) resulted in channels that remained sensitive to phosphorylation. These results suggested that hIK1 was itself not the target for phosphorylation.

In this article we demonstrate that unlike hIK1, the SKCa channels, which share both sequence and functional homology to hIK1, fail to respond to ATP in excised, inside-out patches. Therefore, we used a chimeric hIK1/rSK2 strategy to define an amino acid region, arginine 355 through alanine 413, within the C terminus of hIK1 that confers complete ATP dependence. In addition, we demonstrate that the first 14 amino acids of this region, residues Arg355-Met368, which have been shown to interact with calmodulin in a Ca2+-dependent manner, are critical to the ATP-dependent modulation of hIK1.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Xenopus laevis Oocyte Preparation

X. laevis care and handling procedures were in accordance with University of Pittsburgh guidelines. X. laevis were obtained from either Xenopus 1 (Dexter, MI) or Nasco (Fort Atkinson, WI). Frogs were anesthetized with 3-aminobenzoic acid ethyl ester, ovaries were surgically removed, and oocytes were dissected in modified Barth's solution containing the following: 88 mM NaCl, 2.4 mM NaHCO3, 1 mM KCl, 0.82 mM MgSO4, 0.33 mM Ca(NO3), 0.41 mM CaCl2, 10 mM HEPES, and 1% penicillin-streptomycin. The oocyte follicular cells were removed by incubation in 5 mg/ml collagenase (Life Technologies, Inc.) plus 0.5 mg/ml trypsin inhibitor in Ca2+-free ND-96 (in mM concentrations: 96 NaCl, 1 KCl, 1 MgCl2, 5 HEPES; pH adjusted to 7.5 with NaOH) at room temperature for ~60 min. The oocytes were then incubated in 100 mM K2HPO4 (pH adjusted to 6.5 with HCl) containing 1 mg/ml bovine serum albumin for 30 min to remove any remaining follicular cells. Stage 5 and 6 oocytes were pre-sorted and allowed to incubate overnight in modified Barth's solution at 20 °C prior to injection of cRNA.

Molecular Biology

In Vitro Transcription-- All cDNAs were subcloned into the oocyte expression vector pBF containing both 5' and 3' untranslated regions of the Xenopus beta -globin gene flanking the multi-cloning site. The plasmid was linearized using either PvuI or MluI (Roche Molecular Biochemicals), and 5' capped cRNAs were generated using SP6 polymerase (mMESSAGE mMACHINETM in vitro transcription kit, Ambion). cRNAs were evaluated both spectrophotometrically and by agarose gel electrophoresis with ethidium bromide staining. Oocytes were injected with 10-50 ng of cRNA 2-4 days prior to recording.

Site-directed Mutagenesis and Chimera Generation-- Site-directed mutants were generated using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Chimeras between hIK1 and rSK2 were generated by overlap extension polymerase chain reaction using either Taq (Stratagene) or Pfx (Life Technologies, Inc.) polymerase. The chimeras were subcloned into the pBF vector using the EcoRI and XhoI restriction sites. The fidelity of all mutations and chimeras was confirmed by sequencing (ABI PRISM 377 automated sequencer, University of Pittsburgh) and subsequent sequence alignment (NCBI Blast 2.0) with hIK1 (GenBankTM accession number AF022150) and/or rSK2 (GenBankTM accession number U69882).

To illustrate the chimera nomenclature utilized throughout the manuscript, we will use the SK-355IK construct as an example. In SK-355IK the N-terminal portion of the construct is derived from amino acids Met1-Val466 of rSK2. Based on published sequence alignments (15), amino acid Val466 of rSK2 corresponds to residue Val354 of hIK1. A chimeric junction is therefore introduced between amino acid residues Val466 of rSK2 and the next residue of hIK1, Arg355. Thus, in the SK-355IK construct, amino acids Met1-Val466 are derived from rSK2, at which point the hIK1 residues Arg355-Lys427 are substituted.

Electrophysiology

The oocyte vitelline membrane was mechanically dissected prior to patch clamping in a hypertonic solution containing the following (in mM concentrations): 200 K-gluconate, 20 KCl, 1 MgCl2, 10 EGTA, and 10 HEPES (pH adjusted to 7.4 with NaOH). Single-channel currents were recorded in the inside-out patch configuration using an Axon 200B amplifier (Axon Instruments) and stored on videotape for later analysis. Pipettes were fabricated from #0010 glass (World Precision Instruments) and heat-polished to resistances of 3-6 megohms. The pipette solution contained the following (in mM concentrations): 145 K-gluconate, 5 KCl, 2.5 MgCl2, 10 HEPES, and 1 EGTA (pH adjusted to 7.4 with KOH). The bath contained the following (in mM concentrations): 145 K-gluconate, 5 KCl, 2.5 MgCl2, 10 HEPES, and 1 EGTA (pH adjusted to 7.2 with KOH). Sufficient CaCl2 was added to obtain the desired free [Ca2+] (program kindly provided by Dr. Dave Dawson, University of Michigan). For experiments with no added Ca2+, Ca2+ was excluded from the bath, and EGTA was maintained at 1 mM (estimated free Ca2+ < 10 nM). Every chimera tested was strictly Ca2+-dependent, because Ca2+-free buffer eliminated all channel activity. Constructs that were not activated by ATP (Roche Molecular Biochemicals) were always tested in parallel with a chimera known to be modulated by ATP as a positive control. In addition, constructs not expressing current were tested minimally on three separate oocyte injections in parallel with constructs that did express current. All recordings were maintained at a holding potential of -100 mV. The voltage was referenced to the extracellular compartment, as is the standard method for membrane potentials. Recordings were acquired onto computer using Pclamp software (version 6.0.2, Axon Instruments) with a low pass filter frequency of 400 Hz and a sample frequency of 1 KHz. Digitized recordings were analyzed using Biopatch software (version 3.3, Bio-Logic). Diary plots were constructed by averaging current in pA over 15-30-s intervals of the experimental record.

Statistics

All data are presented as means ± S.E., where n indicates the number of experiments. Statistical analysis was performed using a Student's t test. A value of p < 0.05 is considered statistically significant and is reported.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ATP-dependent Activation Is Selective for hIK1-- We previously demonstrated that ATP activates hIK1 via a kinase-mediated process (1). Because hIK1 shares ~40% sequence homology with the SKCa family of K+ channels (15, 16), we determined whether the SKCa channels (rSK2, hSK1, and rSK3) could similarly be modulated by ATP in excised, inside-out patches from Xenopus oocytes. As shown for representative experiments in Fig. 1, following the generation of stable current, addition of ATP (1 mM) failed to activate rSK2 (B), hSK1 (C), and rSK3 (D). However, subsequent addition of the known hIK1 and rSK2 opener, 1-EBIO (300 µM) (3, 17, 18), induced a significant increase in current. In contrast to our results with the SKCa channels, the activity of hIK1 increased in response to ATP addition (Fig. 1A), as previously reported (1). The average current upon patch excision for the various constructs was as follows: rSK2, 95 ± 16 pA (n = 10); rSK3, 453 ± 132 pA (n = 6); hSK1, 117 ± 56 pA (n = 6). This current then decreased an average of 42.1 ± 6.0% (n = 22) to a steady-state level averaging as follows: rSK2, 35 ± 12 pA; rSK3, 186 ± 53 pA; hSK1, 90 ± 39 pA. Subsequent addition of ATP (1 mM) failed to activate the SKCa channels (rSK2, 36 ± 12 pA; rSK3, 171 ± 47 pA; hSK1, 56 ± 15 pA), whereas addition of 1-EBIO (300 µM) resulted in a significant activation (rSK2, 176 ± 40 pA; rSK3, 1311 ± 298 pA; hSK1, 352 ± 111 pA). In contrast to these results on the SKCa channels, following patch excision, current due to hIK1 activity decreased from 143 ± 47 to 43 ± 4 pA (n = 6), with the subsequent addition of ATP increasing current 3.0 ± 0.3-fold to 132 ± 21 pA. These data demonstrate that despite 40% sequence homology and relatedness in the mechanism of Ca2+-dependent gating among the SKCa channels and hIK1, ATP-dependent modulation is specific for hIK1.



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Fig. 1.   The effect of ATP (1 mM) on hIK1 and the homologous SKCa channels. Top, schematics of hIK1 and rSK2. Representative diary plots are shown for hIK1 (A), rSK2 (B), hSK1 (C), and rSK3 (D) in response to ATP (1 mM). Each construct was expressed heterologously in Xenopus oocytes and recorded in the excised, inside-out patch configuration at a holding potential of -100 mV in symmetric K+. For constructs not responding to ATP (rSK2, hSK1, rSK3), the known hIK1 and SK2 opener 1-EBIO (300 µM) was added as a positive control. Each diary plot is sampled at 30-s intervals.

The Cytoplasmic C-terminal Tail of hIK1 Confers ATP-dependent Activation-- Based on the observation that ATP activates hIK1 but has no effect on the SKCa family of channels, we used a chimeric strategy between hIK1 and rSK2 to define a motif within hIK1 that confers ATP-dependent activation. Initially, we generated the chimera IK199-SK (depicted schematically in Fig. 2A) in which the initiation codon through amino acid Tyr199 (start of the putative fifth transmembrane helix) was derived from hIK1, and the remainder of the construct was derived from rSK2 (amino acids Met307-Ser580). As shown for one experiment in Fig. 2A, addition of ATP (1 mM) to an excised, inside-out patch failed to modulate IK199-SK current flow, whereas the subsequent addition of 1-EBIO (300 µM) increased current. In 11 experiments, the base-line current averaged 62 ± 20 pA, unaffected by ATP (57 ± 17 pA), whereas the subsequent addition of 1-EBIO (300 µM) resulted in an increase in current to 126 ± 37 pA (p < 0.01). The failure of ATP to activate the IK199-SK channel suggests that the C-terminal tail of hIK1 is critical for conferring ATP-dependent activation.



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Fig. 2.   The C-terminal cytoplasmic tail of hIK1 confers ATP dependence. Schematics of both the IK199-SK (A) and the SK-299IK chimeras (B) in which filled circles represent amino acids derived from rSK2, and open circles represent amino acids from hIK1. Single letter amino acid nomenclature and arrows denote the hIK1 amino acid that defines the chimeric junction. Also shown are representative diary plots from chimeras IK199-SK (A) and SK-299IK (B) in response to ATP (1 mM); 1-EBIO (300 µM) is used as a positive control for the IK199-SK chimera that failed to respond to ATP. Chimeras were heterologously expressed in Xenopus oocytes and recorded in the excised, inside-out patch configuration at a holding potential of -100 mV in symmetric K+. The records were sampled at 30-s intervals.

To clarify the role of the C-terminal tail in the ATP-dependent activation of hIK1 we generated the chimeric channel SK-299IK, in which only amino acids His299-Lys427 were derived from hIK1 (see Fig. 2B for schematic). ATP robustly activated this chimera, as shown for a representative experiment in Fig. 2B. On average, after current rundown, perfusion of ATP stimulated a 2.9 ± 0.5-fold increase in the activity of this chimera (p < 0.05; n = 6) from a steady-state current of 121 ± 55 pA. The time course for this activation was similar to that observed for wild type hIK1. The fold increase in current of this chimera in response to ATP did not differ significantly from the 3.0-fold potentiation observed for wild type hIK1 (p = 0.88), suggesting that the region that defines the ATP dependence of hIK1 is fully contained within the C-terminal cytoplasmic tail.

The ATP-dependent Motif of hIK1 Resides Entirely within Amino Acids Arg355-Ala413 of the C-terminal Cytoplasmic Tail-- In an attempt to more narrowly define the domain of hIK1 responsible for the phosphorylation-dependent gating observed, we employed a truncation strategy. Unfortunately, truncation of only 26 amino acids of hIK1 Lys402STOP) resulted in channels that failed to express current (n = 0 of 19). Only when we truncated 14 amino acids (Leu414STOP) were functional channels observed. As shown for one experiment in Fig. 3A, L414Stop was robustly activated by ATP. In five experiments, L414Stop current increased 3.7 ± 0.3-fold upon perfusion of ATP from a steady-state level of 54 ± 16 pA (p < 0.05). These data demonstrate that hIK1 requires the majority of the distal C-terminal tail for functional expression, and amino acids Leu414-Lys427 are not required for ATP-dependent activation.



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Fig. 3.   The C-terminal motif, Arg355-Ala413, is necessary and sufficient for the ATP-dependent activation of hIK1. Schematics for the Leu414STOP (A), SK-321IK (B), SK-342IK (C), and SK-355IK (D) constructs. Filled circles represent amino acid residues derived from rSK2, open circles represent amino acid residues from hIK1, and a double line break represents the point of channel truncation. Single letter amino acid nomenclature and arrows denote the hIK1 amino acids that define the chimeric junction/truncation. Representative diary plots for each chimera in response to ATP (1 mM) are shown to the right of each schematic (A-D). Excised, inside-out patches from Xenopus oocytes expressing each construct were recorded at a holding potential of -100 mV in symmetric K+. Each record was sampled at 30-s intervals.

Because truncations of hIK1 failed to express functional channels, we generated three additional chimeras, SK-321IK, SK-342IK, and SK-355IK, in which progressively smaller portions of the C terminus of hIK1 were appended onto rSK2. Representative experiments, demonstrating the ATP-dependent activation of SK-321IK, SK-342IK, and SK-355IK, are shown in Fig. 3, B, C, and D, respectively. Each of these chimeras was activated by ATP (1 mM), averaging 2.7 ± 0.6-fold from a steady-state activity of 166 ± 76 pA (n = 6; SK-321IK), 2.8 ± 0.5-fold from a basal activity of 53 ± 24 pA (n = 3; SK-342IK), and 2.5 ± 0.5-fold from a steady-state current level of 108 ± 61 pA (n = 9; SK-355IK). The ATP-induced fold increase in current for these three chimeras did not significantly differ from the fold increase observed for hIK1 (p = 0.65, 0.77, and 0.29, respectively). These data indicate that the 59-amino acid region, Arg355-Ala413 of hIK1 wholly defines the domain responsible for conferring ATP dependence. The amino acid sequence of this region is shown in Fig. 4. In contrast to these results, the complimentary chimera to SK-355IK, i.e. IK354-SK, in which the distal C-terminal tail of rSK2 (amino acids Lys467-Ser580) was appended after Val354 of hIK1, failed to express functional channels and could not be evaluated. Similar to our truncations, this result suggests that the distal C terminus of hIK1 is essential to the functional expression of these channels.



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Fig. 4.   Amino acid sequence for Arg355-Ala413. The amino acid sequence is shown in single-letter code. The boxed region represents the 14-amino acid region, Arg355-Met368, that is critical for the phosphorylation-dependent activation of hIK1. The residues in bold type indicate serines and threonines that were mutated to alanines. The five leucines of the potential leucine zipper motif are italicized and underlined.

The ATP-dependent Regulation of hIK1 Does Not Depend upon Direct Phosphorylation of hIK1-- The amino acid sequence between residues Arg355 and Ala413 of hIK1 lacks consensus phosphorylation residues; however, the domain does contain 10 non-consensus serines and threonines. Only two of these residues are conserved between hIK1 and rSK2. Because kinases have been shown to phosphorylate ion channels at non-consensus sites, we mutated these non-conserved residues to alanines (shown in bold in Fig. 4). ATP (1 mM) activated the mutant channel S367A/S372A, increasing current from an average steady state of 32 ± 10 to 126 ± 13 pA (n = 4, p < 0.01). ATP also activated the triple mutation S386A/S387A/S388A from a basal activity of 56 ± 17 to 179 ± 47 pA (n = 5, p < 0.05). Finally, introducing the T407A/S411A/T412A mutations into hIK1 had no effect on the ability of ATP to activate the channel, because ATP increased current from 44 ± 6 to 137 ± 35 pA (n = 7, p < 0.05). These data demonstrate that ATP-dependent modulation of hIK1 is independent of non-conserved serines and threonines within amino acids Arg355-Ala413 of hIK1.

A 14-Amino Acid Region, Arg355-Met368, of hIK1 Confers Partial ATP Dependence to rSK2-- To further narrow the domain of hIK1 responsible for ATP-dependent regulation, we generated additional rSK2/hIK1 chimeras in which smaller segments of the distal C terminus of hIK1 were appended onto rSK2. These additional chimeras, SK-383IK and SK-369IK, overlap with the region of hIK1 that is critical for the Ca2+-dependent interaction of calmodulin (19) and rSK2 (20, 21). It is potentially for this reason that these constructs did not express functional channels as well as the chimeras outlined above. To increase our current signal, we studied these chimeras at 10 µM Ca2+, because we have previously shown that ATP robustly activates hIK1 even at this saturating level of Ca2+ (1). In 10 experiments, SK-383IK failed to respond to ATP, having an average current of 59 ± 11 pA, which continued to run down to 47 ± 9 pA in the presence of ATP. As a positive control, 1-EBIO (300 µM) increased the current to 121 ± 18 pA (p < 0.001). Similar to SK-383IK, SK-369IK failed to respond to ATP (1 mM), although 1-EBIO (300 µM) produced a significant increase in current (Fig. 5A). In seven experiments, the current averaged 113 ± 28 pA, which was not affected by ATP (108 ± 29 pA), whereas 1-EBIO increased current to 172 ± 44 pA (p < 0.05). These data suggest that the 14 amino acids of hIK1, Arg355-Met368, are critically important for the ATP-dependent regulation of the channel.



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Fig. 5.   Amino acids Arg355-Met368 of hIK1 confer partial ATP dependence. Schematics of the SK-369IK (A) and the SK+14IK (B) chimeras are shown. The filled circles represent amino acid residues derived from rSK2, whereas the open circles depict residues from hIK1. Single-letter amino acid nomenclature and arrows denote the hIK1 amino acids that define the chimeric junctions. A, as shown in the representative diary plot, SK-369IK failed to respond to ATP (1 mM); 1-EBIO (300 µM) was added as a positive control. B, a representative diary plot from the SK+14IK chimera showing activation in response to ATP (1 mM). Excised, inside-out patches from Xenopus oocytes were recorded at a holding potential of -100 mV in symmetric K+. Records were sampled at 30-s intervals.

To further define the role of this 14-amino acid domain in the ATP-dependent activation of hIK1, we generated a chimera in which we swapped amino acids Arg355-Met368 of hIK1 into the homologous region of rSK2 (amino acids Lys468-Leu481), SK+14IK. This 14-amino acid region is shown boxed in Fig. 4. As shown for one experiment in Fig. 5B, substituting these 14 amino acids from hIK1 into rSK2 conferred ATP sensitivity to rSK2. In 10 experiments the steady-state current in 10 µM Ca2+ averaged 105 ± 48 pA, and this increased 1.6 ± 0.1-fold in response to ATP (p < 0.05). This activation was ~50% of that seen for hIK1 in response to ATP (3.0-fold). These results demonstrate that, within amino acids Arg355-Ala413 of hIK1, the 14-amino acid region Arg355-Met368 is critical to conferring ATP-dependent activation.

We also generated chimeras in which we substituted similar small regions of rSK2 into the distal C terminus of hIK1 in an attempt to demonstrate loss of ATP-dependent activation in hIK1. Unfortunately, similar to what we observed both with hIK1 truncations and the IK354-SK chimera, these constructs failed to express functional channels.

The ATP-dependent Activation of hIK1 Is Ca2+-independent-- Previously, we demonstrated that phosphorylation potentiates hIK1 activity only in the presence of elevated Ca2+ (i.e. greater than 100 nM). However, these studies did not address the question of whether the ATP-dependent activation (phosphorylation) of hIK1 requires Ca2+ to proceed. To test this hypothesis we employed the following protocol, for which a representative experiment is shown in Fig. 6. Upon patch excision into a bath containing 10 µM free Ca2+, hIK1 current decreased to a steady-state level of activity (68 pA). Subsequently, Ca2+-free buffer was perfused for 1 min to guarantee complete washout of Ca2+. Note that current activity decreased to 0 pA, demonstrating that free Ca2+ had decreased to below the threshold for maintaining channel activity (<100 nM). At this point, Ca2+-free buffer containing ATP (1 mM) was perfused for 3 min. This amount of time is sufficient to obtain near maximal activation in the presence of elevated Ca2+ (see Figs. 1-3 and 5). Note that this failed to increase current flow, demonstrating that ATP cannot activate hIK1 in the absence of Ca2+. Following perfusion with ATP, Ca2+-free buffer without ATP was again perfused for 1 min. During this phase, 10 units/ml apyrase (tri- and diphosphatase, Sigma) was transiently added to the perfusate to ensure complete depletion of ATP from the bath. Finally, the initial solution containing 10 µM free Ca2+ was perfused again, resulting in a large activation of hIK1 to 215 pA. The result of this one experiment is consistent with an ATP-dependent phosphorylation event occurring in the absence of Ca2+, because the current flow observed in 10 µM Ca2+ subsequent to ATP was 3.16-fold greater than the initial steady-state level. In eight experiments, the initial steady-state current (prior to ATP) averaged 56.8 ± 9.9 pA, and this was completely inhibited upon removal of Ca2+. Re-addition of 10 µM Ca2+, following exposure to ATP in Ca2+-free buffer, increased current 3.0 ± 0.3-fold above the initial steady-state value to 171.0 ± 23.6 pA. This fold increase in activity is not significantly different from that observed when hIK1 was stimulated by ATP in the continued presence of Ca2+ (p = 0.96). These data demonstrate that ATP-dependent phosphorylation proceeds in the absence of Ca2+.



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Fig. 6.   hIK1 can be phosphorylated in the absence of Ca2+. Following patch excision into 10 µM free Ca2+, current due to hIK1 declined to a steady state. Perfusion of Ca2+-free buffer (1 mM EGTA) resulted in the complete loss of channel activity. Next, Ca2+-free buffer with 1 mM added ATP was perfused onto the patch for 3 min. Subsequently, Ca2+-free buffer was perfused for 1 min. During this period, addition of 10 units/ml apyrase was included to ensure complete removal of ATP. Finally, the initial buffer (10 µM Ca2+) was re-perfused, and the instantaneous current response was compared with control, steady-state channel activity. Excised, inside-out patches were recorded from Xenopus oocytes at a holding potential of -100 mV in symmetric K+. The diary plot was sampled at 15-s intervals



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We previously demonstrated that ATP activates hIK1 in a protein kinase A inhibitor 5-24-dependent manner in excised, inside-out patches. These results suggested a role for phosphorylation in the regulation of this KCa channel. However, mutation of the single consensus cAMP-dependent protein kinase phosphorylation site (S334A) failed to modify the response of hIK1 to ATP (1). In the present study, we employed hIK1/rSK2 chimeras and hIK1 truncations to identify amino acids Arg355-Ala413 as necessary and sufficient for the ATP-dependent regulation of hIK1. The Arg355-Ala413 domain is devoid of consensus phosphorylation sites, and mutation of all serines and threonines within this region that are present in hIK1 and not rSK2 resulted in channels that remained sensitive to ATP. The first 14 amino acids of this region, Arg355-Met368, play a critical role in the ATP-dependent modulation of hIK1, because substitution of these amino acids into rSK2 produced an ATP-dependent increase in current that was ~50% of the full hIK1 response.

Several mechanisms could account for the necessity of the Arg355-Ala413 domain in the ATP-dependent regulation of hIK1. One explanation is that ATP directly binds this region of the channel. This interaction could then induce a conformational change in the channel promoting phosphorylation of an hIK1 residue outside of the Arg355-Ala413 region. This mechanism cannot be dismissed, but we believe it to be unlikely, because Arg355-Ala413 lacks consensus ATP binding sites. As previously reported, hIK1 can be activated by ATP when all consensus phosphorylation sites are individually mutated (1). Thus, this mechanism would also require phosphorylation at a novel non-consensus site or at multiple residues. Alternatively, the stimulatory effect of ATP could depend upon phosphorylation of multiple non-consensus residues within the Arg355-Ala413 region. We believe this mechanism to be unlikely, because substitution of the smaller Arg355-Met368 region of hIK1 into rSK2 confers partial ATP sensitivity. This region does contain a single serine (Ser367), although mutation of this residue in hIK1 to alanine does not modulate the sensitivity of the channel to ATP.

Rather, we speculate that the phosphorylation-dependent regulation of hIK1 is mediated via an alternative (beta ) subunit that interacts with amino acids Arg355-Ala413. The role of beta -subunits in the kinase-dependent regulation of ion channels is well established, because these accessory molecules confer cell type-specific regulation. For example, the sensitivity of the hslo KCa channel to cAMP-dependent protein kinase is dependent upon co-expression with hKCNMB1. In the absence of the subunit, hslo activity is stimulated by cAMP-dependent protein kinase, whereas in the presence of hKCNMB1, hslo activity is inhibited (24). The literature suggests that a similar paradigm may be true for hIK1. We previously demonstrated a role for cAMP-dependent protein kinase-mediated phosphorylation of hIK1 in both endogenous (T84 cells) and heterologous (Xenopus oocytes) expression systems. However, when hIK1 was expressed in HEK293 cells, the channel was modulated via cAMP-dependent protein kinase-independent phosphorylation (1). Khanna et al. (9) also reported cell-specific phosphorylation-dependent modulation of hIK1. These authors showed that, when natively expressed in T lymphocytes, hIK1 activity was modulated via calmodulin-dependent kinases. In contrast, when hIK1 was expressed heterologously in Chinese hamster ovary cells, calmodulin-dependent kinase inhibitors had no effect on channel activity. Although our results are suggestive of the involvement of a beta -subunit in the ATP-dependent regulation of hIK1, this would require that Xenopus oocytes endogenously express this beta -subunit. Currently, we have no direct evidence that Xenopus oocytes express this proposed subunit.

Interestingly, truncations involving only 25 amino acids of the hIK1 C terminus failed to express functional channels. In contrast, it has been reported that C-terminal truncations in excess of 100 amino acids of rSK2 have no overt functional consequences (20). In our present study, chimeric swaps in which portions of rSK2 were appended onto, or swapped into, hIK1 resulted in channels that failed to express functional channels, whereas chimeras in which portions of hIK1 were either swapped into, or appended onto, rSK2 had no affect on channel expression. Together, these data suggest that unlike rSK2, the region that confers phosphorylation dependence upon hIK1 is required for the functional expression of the channel. As first recognized by Joiner et al. (16), this region of both hIK1 and rSK2 contains a potential leucine zipper motif (leucines are italicized and underlined in Fig. 4). Because leucine zippers are known to stabilize a variety of protein-protein interactions (30), it will be important to evaluate the role of this leucine zipper in the ATP-dependent modulation and functional expression of hIK1.

We previously demonstrated that phosphorylation does not increase hIK1 activity in the absence of Ca2+ (1). Here we demonstrate that ATP-dependent phosphorylation occurs in the absence of Ca2+, although this phosphorylation event is insufficient to activate the channel until Ca2+ is increased above the threshold required for channel gating (Fig. 6). These data argue against a role for Ca2+-dependent kinases, such as calmodulin-dependent kinase, in the ATP-dependent activation of hIK1. These results are consistent with our previous report demonstrating that the peptide inhibitor of Ca2+/calmodulin kinase II, Ca2+/calmodulin kinase II inhibitor 281-309, failed to alter the ATP-dependent activation of hIK1. Although the region we define as critical for phosphorylation-dependent activation overlaps with the region of hIK1 that binds calmodulin in a Ca2+-dependent manner, our results demonstrate that hIK1 need not be in either a Ca2+/calmodulin-dependent or a conductive state for phosphorylation to occur.

In summary, using a series of truncations and hIK1/rSK2 chimeras, we have defined amino acids Arg355-Ala413 as necessary and sufficient for the ATP-dependent modulation of hIK1. The first 14 amino acids of this region, Arg355-Met368, are critical because they impart partial ATP dependence to rSK2. Mutation of all serines and threonines within the Arg355-Ala413 domain that are not conserved in rSK2 results in channels that remain sensitive to ATP-dependent phosphorylation. Thus, the ATP-dependent activation of hIK1 may be independent of direct phosphorylation of the hIK1 channel itself.


    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK54941-02 (to D. C. D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed: Dept. of Cell Biology and Physiology, S312 Biomedical Science Tower, University of Pittsburgh, 3500 Terrace St., Pittsburgh, PA 15261. Tel.: 412-383-8755; Fax: 412-648-8330; E-mail: dd2+@pitt.edu.

Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M007716200

1 A. C. Gerlach and D. C. Devor, unpublished observations.


    ABBREVIATIONS

The abbreviations used are: AMP-PNP, adenosine 5'-(beta ,gamma -imino)triphosphate; AMP-PCP, adenosine 5'-(beta ,gamma -methylenetriphosphate).


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES


1. Gerlach, A. C., Gangopadhyay, N. N., and Devor, D. C. (2000) J. Biol. Chem. 275, 585-598[Abstract/Free Full Text]
2. Devor, D. C., and Frizzell, R. A. (1993) Am. J. Physiol. 265, C1271-C1280[Abstract/Free Full Text]
3. Devor, D. C., Singh, A. K., Frizzell, R. A., and Bridges, R. J. (1996) Am. J. Physiol. 271, L775-L784[Abstract/Free Full Text]
4. Warth, R., Hamm, K., Bleich, M., Kunzelmann, K., von Hahn, T., Schreiber, R., Ulrich, E., Mengel, M., Trautmann, N., Kindle, P., Schwab, A., and Greger, R. (1999) Pfluegers Arch. Eur. J. Physiol. 438, 437-444[CrossRef][Medline] [Order article via Infotrieve]
5. Dharmsathaphorn, K., and Pandol, S. J. (1986) J. Clin. Invest. 77, 348-354[Medline] [Order article via Infotrieve]
6. Rapacon, M., Mieyal, P., McGiff, J. C., Fulton, D., and Quilley, J. (1996) Br. J. Pharmacol. 118, 1504-1508[Abstract]
7. Mieyal, P., Fulton, D., McGiff, J. C., and Quilley, J. (1998) J. Pharmacol. Exp. Ther. 285, 659-664[Abstract/Free Full Text]
8. Jensen, B. S., Odum, N., Jorgensen, N. K., Christophersen, P., and Olesen, S. P. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 10917-10921[Abstract/Free Full Text]
9. Khanna, R., Chang, M. C., Joiner, W. J., Kaczmarek, L. K., and Schlichter, L. C. (1999) J. Biol. Chem. 274, 14838-14849[Abstract/Free Full Text]
10. Vandorpe, D. H., Shmulker, B. E., Jiang, L., Lim, B., Maylie, J., Adelman, J. P., De Franceschi, L., Capellini, M. D., Brugnara, C., and Alper, S. L. (1998) J. Biol. Chem. 273, 21542-21553[Abstract/Free Full Text]
11. Wulff, H., Miller, M. J., Hansel, W., Grissmer, S., Cahalan, M. D., and Chandy, K. G. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 8151-8156[Abstract/Free Full Text]
12. Pellegrino, M., and Pellgrini, M. (1998) Pfluegers Arch. Eur. J. Physiol. 436, 749-756[CrossRef][Medline] [Order article via Infotrieve]
13. Tabcharani, J. A., Boucher, A., Eng, J. W., and Hanrahan, J. W. (1994) J. Membr. Biol. 142, 255-266[Medline] [Order article via Infotrieve]
14. Roch, B., Baro, I., Hongre, A.-S., and Escande, D. (1995) Pfluegers Arch. Eur. J. Physiol. 426, 355-363
15. Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11651-11656[Abstract/Free Full Text]
16. Joiner, W. J., Wang, L. Y., Tang, M. D., and Kaczmarek, L. K. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11013-11018[Abstract/Free Full Text]
17. Jensen, B. S., Strobaek, D., Christophersen, P., Jorgensen, T. D., Hansen, C., Silahtaroglu, A., Olesen, S. P., and Ahring, A. K. (1998) Am. J. Physiol. 275, C848-C856[Medline] [Order article via Infotrieve]
18. Syme, C. A., Gerlach, A. C., Singh, A. K., and Devor, D. C. (2000) Am. J. Physiol. 278, C570-C581[Abstract/Free Full Text]
19. Fanger, C. M., Ghanshani, S., Logsdon, N. J., Rauer, H., Kalman, K., Zhou, J., Beckingham, K., Chandy, K. G., Cahalan, M. D., and Aiyar, J. (1999) J. Biol. Chem. 274, 5746-5754[Abstract/Free Full Text]
20. Xia, X. M., Fakler, B., Rivard, A., Wayman, G., Johnson-Pais, T., Keen, J. E., Ishii, T., Hirschberg, B., Bond, C. T., Lutsenko, S., Maylie, J., and Adelman, J. P. (1998) Nature 395, 503-507[CrossRef][Medline] [Order article via Infotrieve]
21. Keen, J. E., Khawaled, R., Farrens, D. L., Neelands, T., Rivard, A., Bond, C. T., Janowsky, A., Fakler, B., Adelman, J. P., and Maylie, J. (1999) J. Neurosci. 19, 8830-8838[Abstract/Free Full Text]
22. Deleted in proof
23. Deleted in proof
24. Dworetzky, S. I., Boissard, C. G., Lum-Ragan, J. T., McKay, M. C., Post-Munson, D. J., Trojnacki, J. T., Chang, C. P., and Gribkoff, V. K. (1996) J. Neurosci. 16, 4543-4550[Abstract/Free Full Text]
25. Weiger, T. M., Holmqvist, M. H., Levitan, I. B., Clark, F. T., Sprague, S., Huang, W.-J., Ge, P., Wang, C., Lawson, D., Jurman, M. E., Glucksmann, M. A., Silos-Santiago, I., DiStefano, P. S., and Curtis, R. (2000) J. Neurosci. 20, 3563-3570[Abstract/Free Full Text]
26. Xia, X. M., Ding, J. P., and Lingle, C. J. (1999) J. Neurosci. 19, 5255-5264[Abstract/Free Full Text]
27. Xia, X. M., Ding, J. P., Zeng, X. H., Duan, K. L., and Lingle, C. J. (2000) J. Neurosci. 20, 4890-4893[Abstract/Free Full Text]
28. Behrens, R., Nolting, A., Reimann, F., Scwartz, M., Waldschutz, R., and Pongs, O. (2000) FEBS Lett. 474, 99-106[CrossRef][Medline] [Order article via Infotrieve]
29. Meera, P., Wallner, M., and Toro, L. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 5562-5567[Abstract/Free Full Text]
30. Kobe, B., and Diesenhofer, J. (1994) Trends. Biochem. Sci. 19, 415-421[CrossRef][Medline] [Order article via Infotrieve]


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