1Division of Cardiology, Department of Medicine, University of Louisville, Louisville, Kentucky; and Departments of 2Physiology and 3Pediatrics, Emory University School of Medicine, Atlanta, Georgia
Submitted 20 July 2004 ; accepted in final form 4 October 2004
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ABSTRACT |
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Shaker potassium ion channels; Kv subunits; patch clamp; aldo-keto reductase; COS-7 cells
The Kv proteins contain multiple cysteine residues that could respond to redox changes in the local environment. In agreement with this view, it has been shown that sulfhydryl reagents affect native Kv conductances (35, 40). Additionally, the redox sensitivity of Kv channels may be due to their ability to associate with ancillary -subunits. The Kv
proteins are members of the aldo-keto reductase (AKR) superfamily (27, 28, 51), and as shown in previous studies by our group (26), they bind pyridine nucleotides with high affinity. These proteins associate with the cytoplasmic domain of specific
-proteins in homotetrameric assembly (27, 51, 54). They affect the voltage dependence of Kv currents, induce inactivation in noninactivating currents such as those due to Kv1.1 and Kv1.5, and accelerate inactivation of self-inactivating currents, e.g., those generated by Kv1.4 (19, 27, 39, 51). Thus the oxidoreductase properties of Kv
proteins could affect the nature of their interactions with Kv
and could underlie the redox and metabolic sensitivity of native Kv currents.
We therefore examined how reduced and oxidized pyridine nucleotides affect Kv1.5 currents in a heterologous expression system. A prototypical member of the Shaker family, Kv1.5 is the molecular correlate of the ultrarapid delayed-rectifier K+ current (Ikur) in the human atrium (14) and is expressed at high density in resistance pulmonary arteries (55). Mice lacking Kv1.5 display impaired HPV (3), and a decrease in the expression of Kv
1.5 is associated with pulmonary hypertension (48). Recently, Kv
1.5 gene therapy has been shown to restore oxygen-sensitive K+ current and reduce pulmonary vascular resistance (37), indicating that Kv1.5 may be an important component of vascular oxygen sensing and a link between excitability and metabolism. Our results show that, although Kv
1.5 expressed alone in COS-7 cells was insensitive to changes in pyridine nucleotides, reduced and oxidized pyridine nucleotides differentially regulated inactivation of the currents generated by the Kv
1.5-
1.3 complex. On the basis of these data, we speculate that regulation by the redox state of pyridine coenzymes may be one mechanism linking metabolic activity to native Kv currents.
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MATERIALS AND METHODS |
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For Western blot analysis, COS-7 cells were grown to 60% confluence and were transfected with pGFP-Kv1.5 and/or pGFP-Kv
1.3 as indicated. After 48 h, the cells were scraped and lysed by sonication. The membrane and cytosolic fractions were obtained by ultracentrifugation at 120,000 g for 1 h. Cytoplasmic and membrane proteins were separated on SDS-PAGE, and Western blots were developed with a polyclonal anti-FLAG antibody and chemiluminescence detection. The expression of Kv
1.3 protein was examined with a polyclonal antibody (rabbit anti-human Kv
1.3) against full-length Kv
1.3.
Whole cell current measurements. The cells were perfused at 2.5 ml/min in a recording chamber with normal Tyrode solution containing (in mM) 135 NaCl, 1.1 MgCl2, 1.8 CaCl2, 5.4 KCl, 10 HEPES, and 10 glucose, pH 7.4, at room temperature. Voltage-clamp experiments were performed in the whole cell configuration as described previously (23), using patch pipettes filled with an internal solution containing (in mM) 100 K-aspartate, 30 KCl, 1 MgCl2, 5 HEPES, 5 EGTA, 5 Mg-ATP, and 5 Na2-creatine phosphate, pH 7.2. The transfected cells were observed for green fluorescence with a Olympus IX51 microscope with an epifluorescence attachment. The currents were recorded only from cells showing GFP fluorescence, with an excitation wavelength of 490 nm and an emission wavelength of 540 nm. For internal perfusion, a 2PK+ pipette perfusion system (Adams and List) with a perfusible pipette holder in which a polyethylene tube with quartz capillary (to reduce electrical noise) at the ends ran from a reservoir of a multireservoir carousel to the very end of the pipette tip (within 100 µm) as described previously (23). The cells were patched with the perfusible pipette containing normal internal solution with no pyridine nucleotides. After basal current was recorded, the internal solution was switched to a solution containing 1 mM NAD+ by switching the tube to the reservoir containing NAD+ internal solution and applying positive pressure in the pressure vessel, which was balanced by negative pressure at the outflow of the pipette holder.
Excised-patch recordings.
Single-channel currents were measured in excised inside-out patches from COS-7 cells expressing Kv or Kv
+ Kv
. For single-channel recording, cells were kept in 35-mm culture dishes in the incubator to allow them to adhere to the bottom of the dish. The cells were washed with patch pipettes in bath solution containing (in mM) 135 NaCl, 1.1 MgCl2, 1.8 CaCl2, 5.4 KCl, 10 HEPES, and 10 glucose, pH 7.2. Patch pipettes were filled with the same bath solution. With this solution the pipette resistance was 510 M
. After formation of a gigaohm seal (>50 G
), the bath solution was replaced with the test solution containing (in mM) 100 K-aspartate, 30 KCl, 1 MgCl2, 5 HEPES, 5 EGTA, 5 Mg-ATP, and 5 Na2-creatine phosphate, pH 7.2, and the pipette was pulled away to form an inside-out excised-patch configuration. Patches were hyperpolarized to 80 mV for 800 ms from the holding potential of 60 mV and then depolarized to voltages ranging from 20 to +90 mV for durations ranging from 4 to 10 s, followed by repolarization to 80 mV for 800 ms and then a return to the holding potential of 60 mV. After basal channel activity was recorded in the excised patch for 57 min, the bath solution was exchanged with the solution containing NAD+ or NADPH and single-channel activity was recorded.
Data acquisition and analysis.
For whole cell recordings, the currents were filtered at 2 kHz, acquired at 410 kHz, and stored digitally. Single-channel currents were sampled at 2 kHz and filtered at 1.1 kHz. Whole cell membrane capacitance was determined by compensating the capacitive current transient. For measuring current-voltage relationships, the cells were depolarized for 800 ms from a holding potential of 80 mV to potentials ranging from 60 mV to +60 mV in 10-mV voltage steps at 0.1 Hz. The peak outward current (Ipeak), measured as the difference between the peak current amplitude and the zero current, and the steady-state current (Iss), measured as the difference between the current amplitude at the end of the 800-ms depolarization pulse and the zero current, were normalized by dividing the currents by the peak current obtained at +50 mV for each cell. The extent of current inactivation was quantified by calculating percent inactivation as [1 (Iss/Ipeak)] x 100. The time course of decay was calculated with the relationship:
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Single-channel data were acquired with Clampex version 5.5 of pCLAMP and were analyzed with Fetchan version 6. To record unitary currents, the excised patches were depolarized to different potentials ranging from 20 mV to +90 mV in 20-mV steps for a 7-s duration after a 700-ms conditioning pulse at 80 mV from a holding potential of 60 mV. Outward currents are noted as upward transitions. Current amplitude histograms were made from stable and continuous recordings of >610 s at +80 mV. The open probability (Po) was used to measure the channel activity within a patch and was determined for the entire duration of the test pulse. NPo, the product of the number of channels and the open probability, was calculated by relative area under an all-points histogram and expressed as:
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Statistical analysis. Data are presented as means ± SE. Data were analyzed with SigmaStat with paired or unpaired t-test or ANOVA followed by Student-Newman-Keuls test for all pairwise comparisons. P values <0.05 were considered significant.
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RESULTS |
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Internal pipette perfusion.
Although the results presented above show that inclusion of NAD(P)+ in the patch pipette affects Kv-
currents, the major limitation in these experiments is that it is not possible to record control current before applying nucleotides. To circumvent this limitation, we used a pipette-perfusion system to deliver nucleotides to the cell after recording control currents (27, 32). For these experiments, cells were patched with a perfusible pipette containing normal internal solution. After basal recordings, the internal solution was switched to a solution containing the coenzymes and changes in the currents were examined. The Kv current recorded at +50 mV from a cell expressing Kv
1.5+
1.3 with a perfusible pipette displayed characteristic inactivation (Fig. 5A). However, switching the internal solution to a solution containing 1 mM NAD+ significantly decreased the extent of inactivation (from 34.3 ± 7.5% to 10.2 ± 4.3%, n = 3; P < 0.05) and shifted the voltage dependence of activation (Fig. 5) from Vh of 14.6 ± 0.7 mV to 7.4 ± 0.4 mV (n = 3; P < 0.05), consistent with the observations described above for internal application of NAD+. In contrast, internal perfusion with 0.1 mM NADPH had no significant effect on Kv
-
currents (data not shown). These results confirm that NADPH preserves, but NAD+ abolishes, the inactivation of Kv
-
currents.
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DISCUSSION |
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In general, pyridine nucleotides support oxidation-reduction reactions. However, accumulating evidence suggests that these nucleotides are regulators of several physiological processes. Sensitivity to the redox state of pyridine coenzyme nucleotides could couple individual processes to metabolism and respiration. This role of pyridine coenzymes is supported by the observations that the corepressor function of COOH terminal binding protein (57) and the DNA-binding activity of the transcription factors regulating the expression of clock genes, i.e., Clock:BMAL1 and NPAS2:BMAL1 (41), are regulated by NAD+-to-NADH ratio. In addition, NAD(P)H has also been shown to regulate the activity of the cystic fibrosis transmembrane conductance regulator (45). Our observation that oxidized and reduced pyridine nucleotides display antagonistic effects on Kv current, analogous to their effects on the transcription of clock genes, may be yet another example of the regulatory function of these coenzymes linking metabolic activity to membrane excitability.
Because inactivation could be removed by oxidized nucleotides, NAD+ and NADP+, but not by equimolar concentrations of NADPH or NADH, it appears that binding of NADPH as well as its permissive effects on inactivation could be selectively prevented by oxidized nucleotides. In the case of NAD+, this inhibition of inactivation appears at a concentration (0.51 mM) similar to that present in cells of most aerobic tissues (0.50.9 mM) (6, 16). Moreover, the half-maximal concentration of NAD+ required to remove inactivation (0.5 mM; Table 2) is similar to the NAD+ dependence of other oxidoreductases. For instance, Kd NAD+ of pig heart soluble malate dehydrogenase is 0.58 mM (13), the Kd of rabbit muscle lactate dehydrogenase is 0.91 mM (15), and the Kd of human aldehyde dehydrogenase is 0.1 mM (2). Hence, the Kv-
pair appears to be responding to NAD+ within the concentration range that affects other NAD+-utilizing enzymes. In addition to NAD+, NADP+ and NADH could also affect inactivation, but because in most aerobic cells the concentration of these nucleotides is one-third or one-fifth that of NADPH (24, 29), their basal contribution is likely to be small. Nonetheless, an increase in NADH during hypoxia or in NADP+ during oxidative stress could affect
-mediated inactivation of Kv currents.
The regulatory effects of pyridine nucleotides on Kv conductance appear to be mediated by Kv, because the Kv
currents by themselves were unaffected by either reduced or oxidized nucleotides. Indeed, previous work by our group (26) showed that Kv
displays a high affinity for both reduced and oxidized nucleotides. The Kv
proteins interact with Kv
via their conserved oxidoreductase domain, which contains the pyridine nucleotide binding site (18). Pyridine nucleotide binding to Kv
appears to be essential for imparting inactivation, because mutations at the coenzyme binding site prevent Kv
-mediated inactivation of Kv
currents (36). Binding of pyridine coenzymes to Kv
may be important for maintaining an appropriate conformation of the protein and for supporting oxidation-reduction reactions. Recent observations such as the rescue of Kv
-3.1-dependent inactivation of Kv1.5 in Xenopus oocytes by mutations in NADPH and substrate binding sites (5) and loss of Kv1.4 inactivation by Kv
2 due to mutations in the AKR active site tyrosine of the
-subunit (36) are consistent with a catalytic role of Kv
. However, no specific catalytic properties of the protein have been reported. On the basis of the relative affinities of the Kv
2 for pyridine nucleotides and their intracellular concentration, we had hypothesized that differential pyridine coenzyme binding could regulate Kv
-Kv
interactions (26). The evidence presented here is consistent with this view.
Fluorometric titrations of Kv show that protein preferentially binding to NADPH (26). Along with the observation that NADPH remains bound to crystallized Kv
2 protein (17), these results suggest that NADPH is the intrinsic ligand of the protein. This is consistent with the present data showing that, even with 100 µM NADPH in the patch pipette, Kv
-mediated inactivation was not affected, indicating that under basal conditions, most of Kv
is saturated with NADPH. The Kv
-mediated inactivation of Kv
currents was, however, abolished by oxidized nucleotides. That oxidized nucleotides can either prevent inactivation of Kv current when included in the patch pipette or instantaneously remove inactivation when introduced to the cell by internal perfusion suggests that oxidized nucleotides directly interfere with the ability of Kv
to inactivate Kv
. Moreover, the observation that NAD+ prevents Kv
-mediated inactivation of Kv current in excised patches is consistent with the notion that removal of inactivation by oxidized pyridine nucleotides is due to direct binding of these nucleotides to the
-subunit or is mediated by a membrane-delimited mechanism imparting pyridine nucleotide sensitivity to Kv
. Further experiments are required to distinguish between these possibilities.
Our results show that, in addition to removing inactivation, NAD(P)+ also prevents Kv-mediated depolarizing shift in the half-activation potential of Kv
currents and prevents the acceleration of Kv activation induced by Kv
. These observations suggest that NAD(P)H not only supports inactivation but also regulates the kinetics and the voltage dependence of activation. Inactivation of Kv
currents appears to be primarily determined by the NH2 terminus of the Kv
proteins, because deletion of the NH2 terminus prevents them from inactivating Kv
currents (27, 39, 51). In contrast, effects on the kinetics and voltage dependence of Kv
activation may be mediated by the binding of the COOH terminus of Kv
to the T1 domain of Kv
. Hence, the effects of oxidized nucleotides on the kinetics and the voltage dependence of activation suggest that in addition to affecting the NH2 terminus inactivating peptide, pyridine nucleotides also affect other interactions between the
- and
-subunits. However, oxidized coenzymes do not seem to completely abolish
-
interactions, because NAD+ was unable to prevent the increase in single-channel conductance of Kv
induced by Kv
binding (Fig. 6). In contrast to previous studies on Kv
-
interactions in Xenopus oocytes showing that the unitary conductance of Kv1.2 is not affected by Kv
1.2 or Kv
1.3 (1, 50), we found a twofold increase in single-channel conductance of Kv
1.5 on coexpression with Kv
1.3. Mechanisms by which association with an auxiliary subunit such as Kv
1.3 increases Kv
1.5 conductance are unclear but may be similar to the three- to sixfold increase in the conductance of KCNQ1 on interaction of the channel with its ancillary KCNE1 (minK) subunit (38, 43, 52). For KCNQ1, it has been suggested that KCNE1 stabilizes the open state of the channel by altering the interaction between the pore helix and the S5/S6 domains (43). Whether Kv
1.3-induced changes in the NH2 terminus T0 or T1 domain of Kv1.5 affect the ion-conducting pore is unknown, but the reported increase in the single-channel conductance of Kv1.3 due to mutations in the T0 domain (53) supports the possibility that Kv
1.3-induced changes at the NH2 terminus could indirectly affect the pore region. Nevertheless, the inability of NAD+ to affect single-channel conductance suggests that the NH2-terminal association between Kv
and
may be insensitive to the oxidation state of pyridine nucleotide. However, further experiments are required to address this possibility. Additional interactions at the COOH terminus are also possible. It was recently reported that binding of Kv
to the T1 domain induces a conformation change in the COOH-terminal peptide of Kv
(44). It was proposed that in the
-
complex the COOH terminus of Kv
is located in the crevice close to the Kv
nucleotide binding site (44), suggesting the possibility that the redox state of the cofactor bound to Kv
could affect the conformation of the COOH terminus of Kv
and thereby alter the voltage sensitivity and gating characteristics of the channel.
The Kv-mediated regulation of Kv inactivation by reduced and oxidized pyridine coenzymes may be a significant mechanism regulating the redox sensitivity of native Kv channels. Thus changes in the redox ratio of pyridine coenzyme caused by alterations in cellular metabolism or hypoxia could influence the Po of native Kv channels and alter their voltage dependence. Although extensive investigations are required to establish the role of pyridine nucleotides in metabolic regulation of Kv currents, the observation that NAD+ abolishes inactivation is consistent with greater inactivation of these channels during hypoxia and metabolic inhibition (4, 10, 30, 56), which decrease tissue NAD+ levels. Additionally, even though it remains to be determined whether other Kv
-
complexes respond similarly to the Kv
1.5-
1.3 couple studied here, it is tempting to speculate that such redox-sensitive mechanisms of Kv channel regulation may be operative during synaptic stimulation that increases oxidative activity and superoxide generation (7, 49).
In summary, our results demonstrate that oxidized and reduced pyridine coenzymes differentially regulate Kv channel activity via the -subunits. The sensitivity of the Kv
-
complex to pyridine nucleotide may be a novel mechanism of redox regulation of native Kv currents. These observations could form the basis of future investigations into the role of pyridine nucleotides in regulating the activity of native Kv channels as a function of cellular metabolism and respiration.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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