AJP - Heart Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 280: H2038-H2045, 2001;
0363-6135/01 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (26)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Potet, F.
Right arrow Articles by Baró, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Potet, F.
Right arrow Articles by Baró, I.
Vol. 280, Issue 5, H2038-H2045, May 2001

AKAP proteins anchor cAMP-dependent protein kinase to KvLQT1/IsK channel complex

Franck Potet1, John D. Scott2, Raha Mohammad-Panah1, Denis Escande1, and Isabelle Baró1

1 Laboratoire de Physiopathologie et de Pharmacologie Cellulaires et Moléculaires, Hôpital Hôtel-Dieu, Institut National de la Santé et de la Recherche Médicale, 44093 Nantes Cedex, France; and 2 Howard Hughes Medical Institute, Vollum Institute, Portland, Oregon 97201


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In cardiac myocytes, the slow component of the delayed rectifier K+ current (IKs) is regulated by cAMP. Elevated cAMP increases IKs amplitude, slows its deactivation kinetics, and shifts its activation curve. At the molecular level, IKs channels are composed of KvLQT1/IsK complexes. In a variety of mammalian heterologous expression systems maintained at physiological temperature, we explored cAMP regulation of recombinant KvLQT1/IsK complexes. In these systems, KvLQT1/IsK complexes were totally insensitive to cAMP regulation. cAMP regulation was not restored by coexpression with the dominant negative isoform of KvLQT1 or with the cystic fibrosis transmembrane regulator. In contrast, coexpression of the neuronal A kinase anchoring protein (AKAP)79, a fragment of a cardiac AKAP (mAKAP), or cardiac AKAP15/18 restored cAMP regulation of KvLQT1/IsK complexes inasmuch as cAMP stimulation increased the IKs amplitude, increased its deactivation time constant, and negatively shifted its activation curve. However, in cells expressing an AKAP, the effects of cAMP stimulation on the IKs amplitude remained modest compared with those previously reported in cardiac myocytes. The effects of cAMP stimulation were fully prevented by including the Ht31 peptide (a global disruptor of protein kinase A anchoring) in the intracellular medium. We concluded that cAMP regulation of IKs requires protein kinase A anchoring by AKAPs, which therefore participate with the channel protein complex underlying IKs.

A kinase anchoring protein; KCNQ1; KCNE1; slow delayed rectifier potassium current


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

HORMONAL STIMULATION OF THE HEART involves the control of Ca2+ and K+ currents. These events are triggered by a multistep pathway comprising receptor-mediated activation of adenylyl cyclase and the generation of the diffusable second messenger cAMP, resulting in the activation of protein kinase A (PKA) (25). In guinea pig ventricular myocytes, PKA stimulation produces an increase in the amplitude of the delayed rectifier K+ current (27, 28, 32). PKA activation also promotes a negative shift in its activation curve and an alteration of its deactivation kinetics (27, 28). It was previously shown that the slow component of the delayed rectifier K+ current (IKs) constitutes the K+ current component sensitive to beta -adrenergic stimulations (27). In the heart muscle, IKs is related to the activity of the KvLQT1 K+ channel protein in tandem with its regulator, termed IsK (or minK) (2, 22). KvLQT1 channel proteins but not IsK possess a putative consensus phosphorylation site by PKA between residues 24 and 27. Expression of the human KvLQT1 proteins alone in Xenopus oocytes underlies a voltage-dependent K+ current that is sensitive to PKA stimulation (31), whereas expression of IsK alone activates an endogenous KvLQT1-like protein that generates a cAMP-sensitive voltage-dependent K+ current similar to IKs (13, 21, 26, 31).

In the present study, we investigated the regulation of the human recombinant KvLQT1/IsK complex in mammalian cell lines maintained at physiological temperature. Surprisingly, these expression systems lacked key element(s) to permit cAMP activation of the IKs. cAMP regulation of IKs was restored by coexpression of KvLQT1/IsK together with A kinase anchoring proteins (AKAPs). AKAPs comprise a group of proteins with a proposed role in mediating the attachment of type II PKA to subcellular structures (20). We concluded that AKAPs are a key element for regulation of IKs by cAMP and may be additional protein components of the IKs channel functional complex.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Intranuclear injection of plasmids. COS-7 cells [American Type Culture Collection (ATTC); Manassas, VA] were cultured in Dulbecco's medium supplemented with 10% fetal calf serum and antibiotics (100 IU/ml penicillin and 100 µg/ml streptomycin; all from GIBCO-BRL; Paisley, Scotland) at 37°C in a humidified incubator. Swiss 3T3 fibroblasts and HEK293 cells were also obtained from the ATTC. Cultured cells were microinjected into the nucleus with plasmids at day 1 after plating. The protocol to microinject cultured cells using the Eppendorf ECET microinjector 5246 system has been previously reported (17). Briefly, plasmids were diluted in a buffer made of (in mmol/l) 40 NaCl, 50 HEPES, and 50 NaOH; pH 7.4 supplemented with 0.5% fluorescein isothiocyanate-dextran (150 kDa). Human cardiac KvLQT1 isoform 1, KvLQT1 isoform 2, and the cystic fibrosis transmembrane conductance regulator (CFTR) were subcloned into the mammalian expression vector pCI (Promega; Madison, VI) under the control of a cytomegalovirus enhancer/promoter. AKAP79, a fragment of cardiac AKAP (mAKAP), and AKAP15/18 were subcloned into the mammalian expression vector pCDNA3. Human IsK cDNA was subcloned into a pRC vector under the control of a cytomegalovirus promoter. CFTR cDNA was subcloned into a pCDNA3 plasmid. A green fluorescence protein pCI plasmid (a gift from Dr. Rainer Waldmann; Sophia-Antipolis, France) was used as an inert coplasmid to ensure that cells were always injected with a constant 15 µg/ml plasmid concentration.

Patch-clamp recordings. Whole cell currents were recorded as previously described (17). Cells were placed on the stage of an inverted microscope and continuously superfused with the standard extracellular solution. Patch pipettes with a tip resistance of 2.5-5 MOmega were electrically connected to a patch-clamp amplifier (Axopatch 200A, Axon Instruments; Foster City, CA). Stimulation, data recording, and analysis were performed through an analog-to-digital converter (Tecmar TM100 Labmaster, Scientific Solutions; Solon, OH) and Acquis1 software (Bio-Logic; Claix, France). A microperfusion system allowed local application and rapid change of the different experimental solutions warmed at 35°C. Current measurements were normalized using the cell capacitance. Patch-clamp measurements are presented as the means ± SE. Statistical significance of the observed effects was assessed by means of the paired t-test or two-way ANOVA when needed. A value of P < 0.05 was considered significant.

Solutions and drugs. The standard extracellular medium contained (in mmol/l) 145 NaCl, 4 KCl, 1 MgCl2, 1 CaCl2, 5 HEPES, and 5 glucose; pH was adjusted to 7.4 with NaOH. The intracellular medium contained (in mmol/l) 145 potassium gluconate, 5 HEPES, 2 EGTA, 2 hemimagnesium gluconate (free Mg2+: 0.1), and 2 K2ATP; pH 7.2 with KOH, whereas the extracellular medium used to record K+ currents contained (in mmol/l) 145 sodium gluconate, 4 potassium gluconate, 7 hemicalcium gluconate (free Ca2+: 1), 4 hemimagnesium gluconate (free Mg2+: 1), 5 HEPES, and 5 glucose; pH 7.4 with NaOH. Free activities were calculated using a software designed by G. L. Smith (University of Glasgow, Glasglow, UK). Intracellular cAMP was increased with a mixture made of 10 µmol/l forskolin plus 400 µmol/l 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) (both from Sigma; St. Louis, MO). The anchoring inhibitor peptide derived from Ht31 was used at concentration of 1 µmol/l in the intracellular solution (3, 19).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of cAMP on KvLQT1/IsK complex expressed in mammalian cells. With the objective of investigating the PKA-mediated regulation of recombinant IKs in a mammalian expression system, human KvLQT1 and IsK plasmids were injected into COS-7 cells. The whole cell configuration of the patch-clamp technique was used to assess the formation of functional channel complexes. Results obtained in cells injected with 2.5 µg/ml pCI-KvLQT1 and 2.5 µg/ml pRC-IsK are summarized in Fig. 1. As shown in Fig. 1, A and B, the IKs current elicited by a depolarizing step to +40 mV was totally insensitive to cAMP stimulation produced by adding 10 µmol/l forskolin plus 400 µmol/l cpt-cAMP to the extracellular medium. On average, the voltage-activated K+ current measured during the depolarizing step to +40 mV was 27.5 ± 7.2 pA/pF in control versus 28.9 ± 7.6 pA/pF under cAMP stimulation [n = 8; P = not significant (NS)]. In the same expression system, we have previously shown that intracellular cAMP increases at least 10-fold in response to 10 µmol/l forskolin, thereby allowing PKA-mediated activation of CFTR channels (12). Therefore, the absence of response to forskolin plus cpt-cAMP of KvLQT1/IsK complexes was unlikely to be caused by low intracellular cAMP. Deactivating tail currents at -40 mV were fitted by a single-exponential decay, which was extrapolated to time 0 so as to reliably measure tail current amplitude. As illustrated in Fig. 1, A and C, no difference was observed in either the tail current amplitude (9.6 ± 2.4 pA/pF in control vs. 9.5 ± 2.4 pA/pF with cAMP, respectively; n = 8) or deactivation kinetics [deactivation time constant (tau deact) was 95.4 ± 7.1 ms in control vs. 101.7 ± 10.6 ms with cAMP, n = 6; Fig. 1C] in the absence or presence of cAMP stimulation. The voltage-dependence of the IKs was also investigated. Neither the current-voltage relation for IKs (Fig. 1B) nor its activation (Fig. 1D) were affected by increased cAMP: the half-activation potential (V0.5) was 9.4 ± 3.4 mV in control conditions versus 6.2 ± 5.8 mV under cAMP stimulation (n = 5). Additional experiments were also carried out in other cell lines including HEK293 and 3T3 fibroblast cells. These experiments produced similar negative data as obtained in COS-7 cells (data not shown). Thus this first set of experiments demonstrated that KvLQT1/IsK channel complexes expressed in mammalian cells are insensitive to cAMP-mediated PKA activation. We next tested whether a regulating protein, absent in host mammalian cells, was involved in the sensitivity of KvLQT1/IsK complex to cAMP stimulation.


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 1.   Effect of cAMP on recombinant slow component of delayed rectifier K+ current (IKs) in COS-7 cells recorded at physiological temperature. A, left: typical K+ currents recorded in a cell injected with 2.5 µg/ml pCI-KvLQT1 isoform 1 and 2.5 µg/ml pRC-IsK in control conditions (trace c) and after cAMP stimulation (trace a). The membrane potential was stepped from -80 mV to +40 mV and then to -40 mV (vertical bar, 200 pA; horizontal bar, 500 ms). Right: magnified tail currents (vertical bar, 50 pA; horizontal bar, 100 ms). B: current-voltage (I-V) relation for IKs in control conditions (open circle ) and under cAMP stimulation (black-triangle; n = 8). Inset: superimposed current traces elicited by test potentials between -100 and +60 mV (increment, 20 mV; holding potential, -80 mV; vertical bar, 250 pA; horizontal bar, 1,000 ms). C: superimposed normalized tail currents before and after cAMP stimulation. Deactivation kinetics were fitted to a single exponential [trace c: solid line, deactivation time constant (tau deact) = 103 ms; trace a: dashed line, tau deact = 104 ms; vertical bar, 20% maximum current (Imax); horizontal bar, 100 ms]. D: activation curves for IKs before (open circle ) and after (black-triangle, n = 5) cAMP stimulation. V0.5, half-activation potential.

Effects of cAMP on IKs in the presence of regulating proteins. KvLQT1 isoform 2 is a KCNQ1 gene alternative splice variant constitutively expressed in the adult human heart, which participates in the IKS channel complex (4). Isoform 2 has an NH2-terminal sequence 131 amino acids shorter than the cardiac KvLQT1 full-length isoform (isoform 1) (4) and thus lacks the consensus protein kinase phosphorylation site localized at amino acids 24-27 of isoform 1. In a previous work (4), we have shown that COS-7 cells expressing both KvLQT1 isoform 1 and isoform 2 in the presence of IsK exhibited a K+ current with a reduced amplitude compared with cells expressing KvLQT1 isoform 1 plus IsK. We decided to test whether coexpression of isoform 2 as in cardiac myocytes interferes with protein kinase phosphorylation of isoform 1. As shown in Fig. 2A, cells injected with KvLQT1 isoform 1, KvLQT1 isoform 2, and IsK plasmids exhibited an IKs that was also insensitive to cAMP stimulation (n = 7). Varying the concentration of IsK or KvLQT1 isoform 2 plasmids in the injection medium did not affect cAMP regulation of IKs.


View larger version (12K):
[in this window]
[in a new window]
  		Fig. 2.   Effect of cAMP on IKs in cells coexpressing KvLQT1 isoform 2 or the cystic fibrosis transmembrane conductance regulator (CFTR). A, left: K+ current recorded in a COS-7 cell injected with 5 µg/ml pCI-KvLQT1 isoform 1, 5 µg/ml pRC-IsK, and 2 µg/ml pCI-KvLQT1 isoform 2 (same protocol and abbreviations as in Fig. 1A) in control conditions (trace c) and after cAMP (trace a) (vertical bar, 100 pA; horizontal bar, 500 ms). Right: magnified tail currents recorded under same experimental conditions (vertical bar, 50 pA; horizontal bar, 100 ms). B, left: K+ currents recorded in a cell injected with 5 µg/ml pCI-KvLQT1 isoform 1, 5 µg/ml pRC-IsK, and 5 µg/ml pCDNA3-CFTR (same voltage protocol as in Fig. 1A). Vertical bar, 200 pA; horizontal bar, 500 ms. Right: vertical bar, 20 pA; horizontal bar, 100 ms.

We (14) have previously shown that CFTR possesses properties of K+ channel regulator in epithelial cells. In pancreatic epithelial cells, a K+ current is elicited by cAMP stimulation in the presence of a functional CFTR expression but not in its absence (14). Because KvLQT1 has also an epithelial expression (5) and because CFTR is also expressed in heart muscle, we tested the hypothesis of CFTR-dependent KvLQT1 regulation by PKA. We investigated the effects of cAMP stimulation on IKs recorded in COS-7 cells expressing the KvLQT1 isoform 1/IsK channel plus CFTR. Typical results from these experiments are illustrated in Fig. 2B. Again, in seven different cells, we did not observed any significant change in the IKs amplitude in response to cAMP stimulation. In these experiments, we checked for expression of functional CFTR by making use of the 6-methoxy-N-(3-sulfopropyl) quinolinium assay (12). Recently, it has been shown that AKAPs mediate the effects of PKA on various ion channels (1, 7, 11, 19). We thus explored whether the absence of AKAPs in our expression system was responsible for the failure of the KvLQT1/IsK channels to respond to cAMP.

Effect of cAMP on IKs in the presence of AKAPs. COS-7 cells were coinjected with KvLQT1, IsK, and AKAP79 plasmids. In the absence of cAMP stimulation, the characteristics of IKs generated by the KvLQT1/IsK complex in the presence of AKAP79 were undistinguishable from those recorded in its absence. In cells expressing KvLQT1 plus IsK, the K+ current amplitude at +40 mV was 25.9 ± 5.1 pA/pF (n = 12), i.e., not different from the value obtained in cells coexpressing AKAP79 (26.9 ± 5.8 pA/pF; n = 10). Other parameters of IKs were not altered by AKAP79 expression including activation and deactivation kinetics and voltage dependence. In contrast, in cells expressing AKAP79, the amplitude of the K+ current related to KvLQT1/IsK expression was consistently enhanced by 10 µmol/l forskolin plus 400 µmol/l cpt-cAMP (Fig. 3, A and B). The tail current amplitude increased from 7.8 ± 2.6 to 8.6 ± 2.5 pA/pF (+15%, n = 6, P < 0.01) under cAMP stimulation. In addition, tau deact also increased from 104.5 ± 16 to 122.9 ± 17 ms (n = 6, P < 0.001; Fig. 3C). Figure 3D illustrates the time course of IKs tail current amplitude and tau deact changes as produced by cAMP enhancement. In native cardiac cells, phosphorylating agents shift the activation curve of IKs toward more negative potentials (28, 32). In accordance with this, in our cells, the IKs activation curve shifted toward more negative potentials under cAMP (Fig. 3E; V0.5 was 12.9. ± 6.5 mV under control vs. 3.6 ± 5.7 mV with cAMP, n = 6, P < 0.01). Thus AKAP79 expression in COS-7 cells restores regulation by cAMP of the expressed KvLQT1/IsK channel.


View larger version (23K):
[in this window]
[in a new window]
  		Fig. 3.   Effect of AKAP79 expression on IKs sensitivity to cAMP stimulation. A-C are as in Fig. 1. COS-7 cells were injected with 2.5 µg/ml pCI-KvLQT1 isoform 1, 2.5 µg/ml pRC-IsK, and 3.75 µg/ml pCDNA3-A kinase anchoring protein (AKAP)79. A, left: vertical bar, 500 pA; horizontal bar, 500 ms. Right: vertical bar, 100 pA; horizontal bar, 100 ms. B: vertical bar, 500 pA; horizontal bar, 1,000 ms. Inset: superimposed current traces elicited by test potentials between -100 and +60 mV (increment, 20 mV; holding potential, -80 mV; vertical bar, 250 pA; horizontal bar, 1,000 ms). C: deactivation kinetics fitted to a single exponential (trace c: solid line, tau deact = 105 ms; trace a: dashed line, tau deact = 127 ms; vertical bar, 20% Imax; horizontal bar, 100 ms). D: time course of IKs tail current amplitude (top) and IKstau deact (bottom). Horizontal bar, external application of the cAMP-activating cocktail. E: IKs activation curves before (open circle ) and after (black-triangle) cAMP stimulation (n = 6). *P < 0.05 and ***P < 0.001 as assessed by two-way ANOVA.

Recently, it has been reported that mAKAP tethers PKA to multiple subcellular compartments in cardiac myocytes (16, 30). As with AKAP79, the characteristics of IKs generated by the KvLQT1/IsK complex in the absence of cAMP stimulation were not modified by mAKAP expression. In cells coexpressing mAKAP, the K+ current amplitude at +40 mV was 21.3 ± 3.1 pA/pF (n = 18), i.e., not different from the K+ current amplitude in cells not expressing mAKAP. As illustrated in Fig. 4, coexpression of mAKAP restored cAMP sensitivity of IKs inasmuch as cAMP stimulation increased the current amplitude, slowed the deactivation kinetics, and shifted to more negative potentials the V0.5 from 13.9. ± 2.8 to 3.2 ± 3.3 mV (n = 10, P < 0.001). In a parallel study (9), we observed that AKAP15/18, another cardiac AKAP, was also capable of reconstituting cAMP regulation of the KvLQT1/IsK complex (Fig. 4F). Again, in the presence of AKAP15/18, the baseline characteristics of IKs were undistinguishable from those determined in its absence. In cells coexpressing AKAP15/18, the K+ current amplitude at +40 mV was 30.6 ± 5.8 pA/pF (n = 12), i.e., not significantly different from the K+ current amplitude in cells not expressing AKAP15/18. In AKAP15/18-expressing cells submitted to cAMP stimulation, the tail current amplitude increased from 9.4 ± 1.5 to 11.5 ± 1.6 pA/pF (+22%, n = 12, P < 0.001), and the V0.5 shifted from 17.3 ± 2.9 to 6.8 ± 2.3 mV (n = 8, P < 0.001).


View larger version (28K):
[in this window]
[in a new window]
  		Fig. 4.   Effects of a fragment of cardiac AKAP (mAKAP) or AKAP15/18 expression on IKs sensitivity to cAMP stimulation. A-E are as in Fig. 3. COS-7 cells were injected with 2.5 µg/ml pCI-KvLQT1 isoform 1, 2.5 µg/ml pRC-IsK, and 3.75 µg/ml pCDNA3-mAKAP. A, left: vertical bar, 200 pA; horizontal bar, 500 ms. Right: vertical bar, 50 pA; horizontal bar, 100 ms. B: vertical bar, 250 pA; horizontal bar, 1,000 ms. Inset: superimposed current traces elicited by test potentials between -100 and +60 mV (increment, 20 mV; holding potential, -80 mV; vertical bar, 250 pA; horizontal bar, 1,000 ms). C: deactivation kinetics fitted to a single exponential (trace c: solid line, tau deact = 92 ms; trace a: dashed line, tau deact = 117 ms; vertical bar, 20% Imax; horizontal bar, 100 ms). D: same as in Fig. 3. E: IKs activation curves before (open circle ) and after (black-triangle) cAMP stimulation (n = 6). F, left: K+ currents recorded in a cell injected with 3.75 µg/ml pCDNA3-AKAP15/18. Same protocol as Fig. 1A. Vertical bar, 200 pA; horizontal bar, 500 ms. Right: vertical bar, 50 pA; horizontal bar, 100 ms. *P < 0.005 and ***P < 0.001 as assessed by two-way ANOVA (n = 14).

To further ensure that colocalization of the PKA to the inner membrane by AKAPs was responsible for cAMP sensitivity of the KvLQT1/IsK complex, the effects of Ht-31 peptide were investigated. Ht-31 peptide is a 24-amino acid peptide derived from a conserved amphipatic helix common to the family of AKAPs (3) that can adopt an alpha -helical conformation, binding to the PKA RII site and preventing PKA anchoring. The same experiments as conducted in cells coexpressing KvLQT1/IsK and mAKAP were repeated in the presence of 1 µmol/l Ht31 peptide in the intracellular pipette solution dialysing the cell. Results are summarized in Fig. 5. The basal current recorded under such conditions was not modified by Ht31 peptide. In the presence of Ht31 peptide, however, cAMP stimulation did not increase the K+ current amplitude (tail current amplitude was 8.2 ± 1.7 pA/pF in control vs. 7.5 ± 1.5 pA/pF under cAMP, n = 7, P = NS) did not modify tau deact (97.1 ± 5.9 vs. 108.5 ± 7.1 ms, n = 6, P = NS) and did not shift the activation curve (V0.5 was 10.3. ± 5.4 vs. 7.5 ± 4.2 mV, n = 5, P = NS). Thus preventing PKA anchoring to AKAPs with Ht31 peptide suppresses its activity on KvLQT1/IsK channel complexes.


View larger version (29K):
[in this window]
[in a new window]
  		Fig. 5.   Ht31 peptide prevents cAMP enhancement of IKs in cells expressing mAKAP. A-D are as in Fig. 1. COS-7 cells were injected with 2.5 µg/ml pCI-KvLQT1 isoform 1, 2.5 µg/ml pRC-IsK, and 3.75 µg/ml pCDNA3-mAKAP. Cells were dialyzed with 1 µmol/l Ht31 peptide diluted in the pipette solution. A, left: vertical bar, 200 pA; horizontal bar, 500 ms. Right: vertical bar, 50 pA; horizontal bar, 100 ms. B: I-V relation for IKs in control conditions (open circle ) and after cAMP (black-triangle, n = 11). Inset: vertical bar, 500 pA; horizontal bar, 1,000 ms. C: deactivation kinetics were fitted to a single exponential (trace c: solid line, tau deact = 97 ms; trace a: dashed line, tau deact = 104 ms; vertical bar, 20% Imax; horizontal bar, 100 ms). D: IKs activation curves before (open circle ) and after (black-triangle) cAMP stimulation (n = 6).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this report, we show that AKAP expression is required for cAMP regulation of recombinant IKs in mammalian cell lines. Recombinant KvLQT1/IsK complexes expressed in mammalians cells (COS-7, HEK293, or 3T3 cells) are insensitive to cAMP stimulation in the absence of AKAP. This observation contrasts with the well-known stimulatory effects of PKA on recombinant IKs in Xenopus oocytes (13, 21, 26, 31). However, Xenopus oocytes have been shown to express an endogenous KvLQT1-like protein, which complements with exogenous IsK to form functional channel complexes (2, 22). In the absence of exogenous IsK, endogenous KvLQT1-like channel activity cannot be detected in oocytes. KvLQT1 channel proteins but not IsK possess a putative consensus phosphorylation site for PKA. Therefore, when oocytes are injected with IsK mRNA to record recombinant IKs, the target for PKA is the KvLQT1-like protein settled in its natural environment. To our knowledge, only one study (31) showed that exogenous human KvLQT1 expressed in oocytes is also sensitive to PKA phosphorylation. In that situation, however, the exogenous KvLQT1 profits in the same environment as the endogenous KvLQT1-like protein. Our study, which was performed in an expression system containing no endogenous KvLQT1 expression, demonstrates that exogenous KvLQT1 is not sensitive to PKA. In this connection, it was previously shown that the sensitivity to regulation by PKA of Kv1.3 channels is partially lost when recombinant channels are expressed in mammalian HEK293 host cells (15). Similarly, Zong et al. (34) reported that the L-type Ca2+ channel reconstituted in HEK293 cells does not display the same extent of sensitivity to modulation by cAMP-dependent phosphorylation as the native channel in myocytes. Likewise, Gerlach et al. (8) found that hIK1 K+ channels are activated by PKA when endogenously expressed and that this regulation is recapitulated in Xenopus oocytes but not in HEK293 cells. Interestingly, cAMP regulation of KCNQ2 and KCNQ3 gene products, which are highly homologous to KvLQT1, does not require coexpression of an AKAP in mammalian cells (24).

In mammalian cells transfected with human KvLQT1/IsK complexes, coexpression of AKAPs and biologically active fragments restored regulation of recombinant IKs by PKA. Functional analysis of AKAPs began when Rosenmund et al. (19) applied electrophysiological techniques in concert with the perfusion of Ht-31 peptide to assess the role of AKAPs in cultured hippocampal neurons. Since 1994, studies have shown that AKAPs are involved in ionic channel regulation in a variety of tissues (6). Cardiac AKAP15/18 has been shown to be required for PKA-dependent potentiation of L-type calcium channels (9, 11). In mammalian cells expressing AKAP79, mAKAP, or AKAP15/18, PKA increased the amplitude of recombinant IKs, shifted its activation curve, and slowed its deactivation kinetics. These effects recapitulate those produced by cAMP stimulation on endogenous IKs in cardiac myocytes (28, 32). However, in cardiac myocytes, cAMP stimulation with isoproterenol almost doubles the IKs tail amplitude (23, 28, 29, 32). In Xenopus oocytes expressing exogenous KvLQT1, the addition of 10 µmol/l forskolin plus 100 µmol/l IBMX increases the IKs peak current by 44% (31). In our study, 10 µmol/l forskolin plus 400 µmol/l cpt-cAMP increased the recombinant IKs tail amplitude by only 15% in COS-7 cells expressing AKAP79, by 16% in cells expressing mAKAP, and by 22% in cells expressing AKAP15/18. Although these effects were fully prevented by Ht31 peptide and although they were accompanied by significant modifications in the activation curve and deactivation kinetics, they remained modest in amplitude relatively to what is usually observed in myocytes. We interpret this difference as an indication that host mammalian cells are still missing an additional partner in the channel complex. Further investigation should be directed to identify this putative partner.

The main function of AKAPs is to contribute to PKA compartimentalization in the vinicity of PKA targets. KvLQT1 channel proteins are mainly expressed in cardiac and epithelial tissues but are not abundant in the central nervous system (5). Despite this tissue distribution, cAMP regulation of recombinant IKs was restored when either the neuronal AKAP79 or the cardiac mAKAP or AKAP15/18 were coexpressed, suggesting a low specificity for AKAPs to permit PKA regulation of KvLQT1. Critical RII-binding regions in several AKAPs have been identified and consist in each case of a single amphipathic helix (18). Interestingly, the AKAP binding of PKA in the heart may be regulated by RII autophophorylation (33). A short peptide containing the RII-binding amphipathic helix of the AKAP Ht31 has been shown to bind RII with nanomolar affinity and has been termed an "anchoring inhibitor peptide" on the basis of its ability to competitively inhibit RII-AKAP interactions. Microinjection of Ht31 peptide displaces the kinase from the anchoring sites and attenuates ion flow through skeletal muscle L-type Ca2+ channels (11) and alpha -amino-3-hydroxy-5-methyl-isotazole propione acid-kainate glutamate receptor ion channels (19).

Given the importance of IKs in cardiac action potential repolarization and its role in long Q-T syndrome, understanding the regulation of human IKs may shed light on its physiological and pathophysiological roles. The ability to up- or downregulate IKs is a means of controlling the duration of action potential in the human heart. IKs is generated by a complex protein assembly composed of KvLQT1 isoform 1 (the channel pore), KvLQT1 isoform 2 (a dominant negative protein), and IsK (the channel regulator). The present data provide support for the formation of signaling complexes in which protein kinases that regulate phosphorylation status of KvLQT1 isoform 1 are maintained in proximity to their target protein. Our results therefore suggest that, in the human heart, IKs is generated by a protein assembly composed of KvLQT1 isoform1, KvLQT1 isoform2, IsK, and a anchoring protein that permits its beta -adrenergic regulation.


    ACKNOWLEDGEMENTS

We thank Béatrice Leray, Marie-Joseph Louerat, Emilie Grelet, and Chloé Bellocq for expert technical assistance with cell cultures and plasmid amplifications.


    FOOTNOTES

This study was supported by Institut National de la Santé et de la Recherche Médicale Grant PROGRES 4P009D (to D. Escande) and National Institute of General Medical Sciences Grant GM-48231 (to J. D. Scott).

Address for reprint requests and other correspondence: D. Escande, Laboratoire de Physiopathologie et de Pharmacologie Cellulaires et Moléculaires, INSERM U533, Bât HNB, Hôpital Hôtel-Dieu, BP 1005, 44093 Nantes Cedex, France (E-mail: denis.escande{at}inserm.nantes.fr).

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.

Received 19 June 2000; accepted in final form 6 November 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ali, S, Chen X, Lu M, Xu JZ, Lerea KM, Hebert SC, and Wang WH. The A kinase anchoring protein is required for mediating the effect of protein kinase A on ROMK1 channels. Proc Natl Acad Sci USA 95: 10274-10278, 1998[Abstract/Free Full Text].

2.   Barhanin, J, Lesage F, Guillemare E, Fink M, Lazdunski M, and Romey G. KvLQT1 and lsK (minK) proteins associate to form the IKs cardiac potassium current. Nature 384: 78-80, 1996[Medline].

3.   Carr, DW, Hausken ZE, Fraser ID, Stofko-Hahn RE, and Scott JD. Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain. J Biol Chem 267: 13376-13382, 1992[Abstract/Free Full Text].

4.   Demolombe, S, Baró I, Pereon Y, Bliek J, Mohammad-Panah R, Pollard H, Morid S, Mannens M, Wilde A, Barhanin J, Charpentier F, and Escande D. A dominant negative isoform of the long QT syndrome 1 gene product. J Biol Chem 273: 6837-6843, 1998[Abstract/Free Full Text].

5.   Demolombe, S, Franco D, de Boer P, Kuperschmidt S, Roden D, Pereon Y, Jarry Y, Moormann A, and Escande D. Differential expression of KvLQT1 and its regulator IsK in mouse epithelia. Am J Physiol Cell Physiol 280: C359-C372, 2001[Abstract/Free Full Text].

6.   Fraser, ID, and Scott JD. Modulation of ion channels: a "current" view of AKAPs. Neuron 23: 423-426, 1999[ISI][Medline].

7.   Gao, T, Yatani A, Dell'Acqua ML, Sako H, Green SA, Dascal N, Scott JD, and Hosey MM. cAMP-dependent regulation of cardiac L-type Ca2+ channels requires membrane targeting of PKA and phosphorylation of channel subunits. Neuron 19: 185-196, 1997[ISI][Medline].

8.   Gerlach, AC, Gangopadhyay NN, and Devor DC. Kinase-dependent regulation of the intermediate conductance, calcium-dependent potassium channel, hIK1. J Biol Chem 275: 585-598, 2000[Abstract/Free Full Text].

9.   Gray, PC, Johnson BD, Westenbroek RE, Hays LG, Yates JR, 3rd, Scheuer T, Catterall WA, and Murphy BJ. Primary structure and function of an A kinase anchoring protein associated with calcium channels. Neuron 20: 1017-2106, 1998[ISI][Medline].

10.   Gray, PC, Tibbs VC, Catterall WA, and Murphy BJ. Identification of a 15-kDa cAMP-dependent protein kinase-anchoring protein associated with skeletal muscle L-type calcium channels. J Biol Chem 272: 6297-6302, 1997[Abstract/Free Full Text].

11.   Johnson, BD, Scheuer T, and Catterall WA. Voltage-dependent potentiation of L-type Ca2+ channels in skeletal muscle cells requires anchored cAMP-dependent protein kinase. Proc Natl Acad Sci USA 91: 11492-11496, 1994[Abstract/Free Full Text].

12.   Leblais, V, Demolombe S, Vallette G, Langin D, Baró I, Escande D, and Gauthier C. beta3-Adrenoceptor control the cystic fibrosis transmembrane conductance regulator through a cAMP/protein kinase A-independent pathway. J Biol Chem 274: 6107-6113, 1999[Abstract/Free Full Text].

13.   Lo, CF, and Numann R. Independent and exclusive modulation of cardiac delayed rectifying K+ current by protein kinase C and protein kinase A. Circ Res 83: 995-1002, 1998[Abstract/Free Full Text].

14.   Loussouarn, G, Demolombe S, Mohammad-Panah R, Escande D, and Baró I. Expression of CFTR controls cAMP-dependent activation of epithelial K+ currents. Am J Physiol Cell Physiol 271: C1565-C1573, 1996[Abstract/Free Full Text].

15.   Martel, J, Dupuis G, Deschenes P, and Payet MD. The sensitivity of the human Kv1.3 (hKv1.3) lymphocyte K+ channel to regulation by PKA and PKC is partially lost in HEK 293 host cells. J Membr Biol 161: 183-196, 1998[ISI][Medline].

16.   McCartney, S, Little BM, Langeberg LK, and Scott JD. Cloning and characterization of A-kinase anchor protein 100 (AKAP100). A protein that targets A-kinase to the sarcoplasmic reticulum. J Biol Chem 270: 9327-9333, 1995[Abstract/Free Full Text].

17.   Mohammad-Panah, R, Demolombe S, Riochet D, Leblais V, Loussouarn G, Pollard H, Baró I, and Escande D. Hyperexpression of recombinant CFTR in heterologous cells alters its physiological properties. Am J Physiol Cell Physiol 274: C310-C318, 1998[Abstract/Free Full Text].

18.   Newlon, MG, Roy M, Hausken ZE, Scott JD, and Jennings PA. The A-kinase anchoring domain of type IIalpha cAMP-dependent protein kinase is highly helical. J Biol Chem 272: 23637-23644, 1997[Abstract/Free Full Text].

19.   Rosenmund, C, Carr DW, Bergeson SE, Nilaver G, Scott JD, and Westbrook GL. Anchoring of protein kinase A is required for modulation of AMPA/kainate receptors on hippocampal neurons. Nature 368: 853-856, 1994[Medline].

20.   Rubin, CS. A kinase anchor proteins and the intracellular targeting of signals carried by cyclic AMP. Biochim Biophys Acta 1224: 467-479, 1994[Medline].

21.   Salata, JJ, Jurkiewicz NK, Jow B, Folander K, Guinosso PJ, Jr, Raynor B, Swanson R, and Fermini B. IK of rabbit ventricle is composed of two currents: evidence for IKs. Am J Physiol Heart Circ Physiol 271: H2477-H2489, 1996[Abstract/Free Full Text].

22.   Sanguinetti, MC, Curran ME, Zou A, Shen J, Spector PS, Atkinson DL, and Keating MT. Coassembly of K(V)LQT1 and minK (IsK) proteins to form cardiac I(Ks) potassium channel. Nature 384: 80-83, 1996[Medline].

23.   Sanguinetti, MC, Jurkiewicz NK, Scott A, and Siegl PK. Isoproterenol antagonizes prolongation of refractory period by the class III antiarrhythmic agent E-4031 in guinea pig myocytes. Mechanism of action. Circ Res 68: 77-84, 1991[Abstract/Free Full Text].

24.   Schroeder, BJ, Kubisch C, Stein V, and Jentsch TJ. Moderate loss of function of cyclic-AMP-modulated KCNQ2/KCNQ3 K+ channels causes epilepsy. Nature 396: 687-690, 1998[Medline].

25.   Trautwein, W, and Hescheler J. Regulation of cardiac L-type calcium current by phosphorylation and G proteins. Annu Rev Physiol 52: 257-274, 1990[ISI][Medline].

26.   Varnum, MD, Busch AE, Bond CT, Maylie J, and Adelman JP. The min K channel underlies the cardiac potassium current IKs and mediates species-specific responses to protein kinase C. Proc Natl Acad Sci USA 90: 11528-11532, 1993[Abstract/Free Full Text].

27.   Walsh, KB, and Kass RS. Regulation of a heart potassium channel by protein kinase A and C. Science 242: 67-69, 1988[Abstract/Free Full Text].

28.   Walsh, KB, and Kass RS. Distinct voltage-dependent regulation of a heart-delayed IK by protein kinases A and C. Am J Physiol Cell Physiol 261: C1081-C1090, 1991[Abstract/Free Full Text].

29.   Washizuka, T, Horien M, Watanuki M, and Sasayama S. Endothelin-1 inhibits the slow component of cardiac delayed rectifier K+ currents via a pertussis toxin-sensitive mechanism. Circ Res 81: 211-218, 1997[Abstract/Free Full Text].

30.   Yang, J, Drazba JA, Ferguson DG, and Bond M. A-kinase anchoring protein 100 (AKAP100) is localized in multiple subcellular compartments in the adult rat heart. J Cell Biol 142: 511-522, 1998[Abstract/Free Full Text].

31.   Yang, WP, Levesque PC, Little WA, Conder ML, Shalaby FY, and Blanar MA. KvLQT1, a voltage-gated potassium channel responsible for human cardiac arrhythmias. Proc Natl Acad Sci USA 94: 4017-4021, 1997[Abstract/Free Full Text].

32.   Yazawa, K, and Kameyama M. Mechanism of receptor-mediated modulation of the delayed outward potassium current in guinea-pig ventricular myocytes. J Physiol (Lond) 421: 135-150, 1990[Abstract/Free Full Text].

33.   Zakhary, DR, Moravec CS, and Bond M. Regulation of PKA binding to AKAPs in the heart: alterations in human heart failure. Circulation 101: 1459-1464, 2000[Abstract/Free Full Text].

34.   Zong, X, Schreieck J, Mehrke G, Welling A, Schuster A, Bosse E, Flockerzi V, and Hofmann F. On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation. Pflugers Arch 430: 340-347, 1995[ISI][Medline].

Am J Physiol Heart Circ Physiol 280(5):H2038-H2045
0363-6135/01 $5.00 Copyright © 2001 the American Physiological Society


This article has been cited by other articles:


Home page
 Biophys. JHome page
H. S. Jensen, M. Grunnet, and S.-P. Olesen
Inactivation as a New Regulatory Mechanism for Neuronal Kv7 Channels
Biophys. J., April 15, 2007; 92(8): 2747 - 2756.
[Abstract] [Full Text] [PDF]

Home page
 NeuroscientistHome page
Y. Li, S. Y. Um, and T. V. Mcdonald
Voltage-Gated Potassium Channels: Regulation by Accessory Subunits
Neuroscientist, June 1, 2006; 12(3): 199 - 210.
[Abstract] [PDF]

Home page
 Circ. Res.Home page
K. L. Dodge-Kafka, L. Langeberg, and J. D. Scott
Compartmentation of Cyclic Nucleotide Signaling in the Heart: The Role of A-Kinase Anchoring Proteins
Circ. Res., April 28, 2006; 98(8): 993 - 1001.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
T. Jespersen, M. Grunnet, and S.-P. Olesen
The KCNQ1 Potassium Channel: From Gene to Physiological Function
Physiology, December 1, 2005; 20(6): 408 - 416.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurophysiol.Home page
C. Distler, P. K. Rathee, K. S. Lips, O. Obreja, W. Neuhuber, and M. Kress
Fast Ca2+-Induced Potentiation of Heat-Activated Ionic Currents Requires cAMP/PKA Signaling and Functional AKAP Anchoring
J Neurophysiol, May 1, 2003; 89(5): 2499 - 2505.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
P. E. MacDonald, W. El-kholy, M. J. Riedel, A. M. F. Salapatek, P. E. Light, and M. B. Wheeler
The Multiple Actions of GLP-1 on the Process of Glucose-Stimulated Insulin Secretion
Diabetes, December 1, 2002; 51(90003): S434 - 442.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
P. E. MacDonald, A. M. F. Salapatek, and M. B. Wheeler
Glucagon-Like Peptide-1 Receptor Activation Antagonizes Voltage-Dependent Repolarizing K+ Currents in {beta}-Cells: A Possible Glucose-Dependent Insulinotropic Mechanism
Diabetes, December 1, 2002; 51(90003): S443 - 447.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
S. Kupershmidt, I. C.-H. Yang, M. Sutherland, K.S. Wells, T. Yang, P. Yang, J. R. Balser, and D. M. Roden
Cardiac-enriched LIM domain protein fhl2 is required to generate IKs in a heterologous system
Cardiovasc Res, October 1, 2002; 56(1): 93 - 103.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
M. S. Kapiloff
Contributions of Protein Kinase A Anchoring Proteins to Compartmentation of cAMP Signaling in the Heart
Mol. Pharmacol., August 1, 2002; 62(2): 193 - 199.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (26)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Potet, F.
Right arrow Articles by Baró, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Potet, F.
Right arrow Articles by Baró, I.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online