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J. Biol. Chem., Vol. 281, Issue 22, 15194-15200, June 2, 2006
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From the Department of Physiology and Cellular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032
Received for publication, December 27, 2005 , and in revised form, March 23, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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subunit (Kv) of the Shaker family voltage-dependent potassium channels (Kv1) is a cytosolic protein that forms a permanent complex with the channel. Sequence and structural conservation indicates that Kv resembles an aldo-keto reductase (AKR), an enzyme that catalyzes a redox reaction using an NADPH cofactor. A putative AKR in complex with a Kv channel has led to the hypothesis that intracellular redox potential may dynamically influence the excitability of a cell through Kv. Since the AKR function of Kv has never been demonstrated, a direct functional coupling between the two has not been established. We report here the identification of Kv substrates and the demonstration that Kv is a functional AKR. We have also found that channel function is modulated when the Kv-bound NADPH is oxidized. Further studies of the enzymatic properties of Kv seem to favor the role of Kv as a redox sensor. These results suggest that Kv may couple the excitability of the cell to its metabolic state and present a new avenue of research that may lead to understanding of the physiological functions of Kv. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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, found in plants, insects, and mammals, attaches to the cytosolic face of the Kv1 channels to form a macromolecular complex (Fig. 1A) (1–5). There are three mammalian Kv genes (Kv1–3) (6, 7), and all Kvs have a conserved core region with sequence similarities to an AKR3 (8, 9). The structure of the conserved core of a Kv, Kv2 from rat, shows that it has a canonical AKR fold, with highly conserved catalytic residues in the correct geometry for catalysis to happen, and has a nicotinamide cofactor tightly bound (Fig. 1B) (3, 10, 11). These features suggest that Kv is a functional AKR. In many cells, acute changes in Kv channel current have long been observed when the cellular redox state is altered, for example, under hypoxic condition or oxidative stress (12–17), but the redox-sensing mechanism remains unknown. Could Kv be the missing link between cellular redox chemistry and Kv channel activities? In this study, we address the questions of whether Kv is a functional AKR and whether the AKR function is coupled to channel functions.
AKRs catalyze the reduction of an aldehyde to an alcohol by oxidizing an NADPH cofactor. Most AKRs have a broad substrate spectrum so that in addition to their native substrates, they also convert small molecule aldehydes such as benzaldehyde derivatives (18). The catalysis follows a kinetic mechanism shown in Scheme I. An AKR, E, binds sequentially an NADPH and an aldehyde substrate (19) to form a ternary complex, E-NADPH-aldehyde. The redox reaction then occurs when the enzyme helps transfer a hydride from the cofactor to the aldehyde. The product, an alcohol, dissociates from the enzyme and is followed by the oxidized cofactor NADP+. Many AKRs show high specificity to NADPH over its close relative, NADH (18). The hydride transfer step is usually fast, and the nucleotide exchange steps, or the protein conformational changes associated with them, are slow and rate-limiting (20, 21). The cycle in Scheme I is reversible so that an alcohol can be oxidized to an aldehyde accompanied by the conversion of an NADP+ to NADPH.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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2 (GenBankTM accession number: CAA54142
[GenBank]
, residues 36–363) was cloned into a pQE70 vector (Qiagen) between the SphI and BamHI sites with a C terminus His6 tag. For electrophysiological measurements, rat Kv1.4 (GenBankTM accession number: CAA34133
[GenBank]
) or Kv2 was cloned into a modified pBluescript vector for in vitro transcription. Mutations were made using the QuikChange (Strategene) kit and verified by sequencing through the entire coding region.
Protein Expression and Purification—XL-1Blue cells were used to express protein. Cells were grown in Luria broth at 37 °C to an OD of 
1.2 and induced with 0.5 mM isopropyl -D-thiogalactopyranoside (final concentration). Immediately after induction, the temperature was reduced to 20 °C, and cells were harvested 16 h after induction. Expressed protein was purified on a Talon Co2+ affinity column (BD Biosciences). Throughout the purification procedure, the following buffer was used: 20 mM Tris, pH 8.0, 300 mM KCl, 10% (v:v) glycerol, and 1mM -mercaptoethanol. Non-specifically bound protein was washed using 20 mM imidazole added to the above solution, and Kv2 protein was eluted with 300 mM imidazole. Immediately after elution, the affinity tag was removed by a brief incubation with trypsin at a Kv2 to trypsin ratio of 150:1 (w:w). The protein was then loaded onto a Superdex 200 column (Amersham Biosciences) for final purification. The column was equilibrated with the reaction buffer: 150 mM KCl, 20 mM Tris, pH 8.0. Protein concentration was determined using the BCA kit (Pierce).
Enzyme Kinetics Measurement—To measure the steady-state multiple turnover reaction rate, the reaction mixture (100 µl) contains 0.2 mM NADPH, 30 µg Kv2 protein, and desired concentration of substrates in the reaction buffer. The reaction mixture was incubated at 37 °C and NADPH (
340 = 6.22 mM–1 cm–1) absorption was monitored at 340 nm using a PharmaSpec UV-1700 (Shimadzu) equipped with a temperature controlling unit (CPS-240A). The reaction was initiated by the addition of a substrate. The initial turnover rate constant was calculated from the linear part of the decrease in the absorbance. Blank controls with no protein were incorporated routinely, and the background NADPH consumption was subtracted.
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-hydroxysteroid dehydrogenase protein (a gift from Dr. Trevor Penning, University of Pennsylvania) and measured its specific activity using the following conditions: 75 µM androsterone (dissolved in acetonitrile), 2.3 mM NAD+, 100 mM potassium phosphate, pH 7.0, and 4% acetonitrile (total). We initiated the reaction by adding the enzyme and monitored the absorbance at 340 nm over time. We obtained a specific activity of 1.4 ± 0.1 µM (n = 5) of androsterone oxidized per minute per milligram of protein, similar to the value of 1.6 µM min–1 mg–1 obtained in Dr Penning's laboratory.
To measure the single turnover hydride transfer reaction rate, Kv2 protein was concentrated to 3–10 mg/ml and mixed with proper concentrations of substrates, and the absorption at 363 nm was followed over time at 37 °C. The 23 nm red shift of the absorption peak for the bound NADPH is likely caused by interactions between the nicotinamide ring and the surrounding residues. Previous studies have demonstrated that the 363 nm peak is due to the bound NADPH (22). The fraction of NADPH remaining on Kv was then plotted versus time, and the data points were fit with a single exponential function in Origin (Microcal Inc.). The inverse of the exponential time constant is defined as the hydride transfer rate constant. When higher than 5 mM 4-carboxybenzaldehyde (4-CB) was used in either the multiple or single turnover measurement, 40 mM Tris was used to maintain the solution pH at 8.0.
Channel Expression and Electrophysiology—mRNA was prepared by in vitro transcription and purified using the Trizol reagent (Invitrogen). mRNAs were injected into Xenopus oocytes for channel expression. For co-expression, Kv1.4 and Kv2 mRNAs were injected at a ratio of 1:3 (w:w). The wild type Kv1.4 has a cysteine residue at position 13 that can be oxidized on inside-out patches to affect channel inactivation, and a C13S mutant or addition of dithiothreitol abolishes this effect (23). To eliminate concerns that the change in inactivation could be due to cysteine oxidation, we used the C13S Kv1.4 in this study. In parallel, we also tested 4-cyanobenzaldehyde (4-CY) and 4-CB on Kv1.4 wild type coexpressed with Kv2, in the presence of 5 mM dithiothreitol, and essentially the same results were obtained.
We recorded patch clamp currents from oocytes 3–5 days after injection. Electrodes were drawn from patch glasses (G85150T-4, Warner Instruments) and polished (MP-803, Narishige Co.) to a resistance of 0.6–1 M
. The pipette solution contained (in mM): 130 KCl, 2 MgCl2, and 10 KH2PO4 at pH 7.4. The bath solution contained (in mM): 80 KCl, 5 EGTA, and 50 KH2PO4 at pH 7.4. pH was adjusted with KOH. The higher buffer content in the bath solution is necessary to maintain the pH at 7.4 for 5 mM or higher 4-CB. K+ currents were elicited by holding the patch at –100mV for at least 30 s and stepping to +60 mV for 500 ms. The volume of the recording chamber (Warner Instruments) is 120 µl, and complete exchange of solution was achieved by perfusing 1 ml of new solution by gravity flow with a flow rate of 2–3 ml/min.
Inactivation of Kv1.4 with or without Kv2 has been studied extensively (24–28). Current inactivation can be fit by an exponential function with two components. The faster component, which is also the predominant one, is mainly contributed by the N-type inactivation (29), and the slower component is thought to be mainly due to the C-type inactivation (27, 30). Here we follow the same tradition to quantify channel inactivation. For Kv1.4 co-expressed with the wild type Kv2, both the slow and fast inactivation time constants were modulated by 4-CY (Table 1). Since the N- and C-type inactivations are tightly coupled, we do not know yet if the change in the slow component is due to a direct effect on the C-type inactivation or a result of change in the N-type inactivation. We focused on the fast component, and we defined the inverse of the smaller time constant as the rate constant of channel inactivation and used the rate constant as a measure for the effect of Kv substrates.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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2 protein contains the reduced form of the cofactor (NADPH), as indicated by a 363 nm peak in the UV absorption spectrum (Fig. 2A), and the occupancy of NADPH is more than 90% (10, 22). Since NADP+ has little absorption at the same wavelength, the NADPH-to-NADP+ conversion eliminates the 363 nm peak. Utilizing this as a readout, we screened small molecule aldehydes, which are common AKR substrates. We have identified several potential Kv substrates and two of the compounds, 4-CB and 4-CY (Fig. 2B, inset), were used here to study the enzymatic activity of Kv and substrate-induced channel modulations.
Shown in Fig. 2B are UV spectra recorded at the indicated time points after mixing Kv2 protein with 5 mM 4-CB. The peak at 363 nm decreases over time, and the reduction of the peak reflects the oxidation of the Kv2-bound NADPH. Since no free NADPH was added, this is a single turnover reaction. When the fraction of NADPH remaining was plotted versus time (Fig. 2C), the data points were well fit by a single exponential function with a time constant of 8.4 ± 0.1 min (Fig. 2C), consistent with a single step hydride transfer reaction. Similar results were obtained when 5 mM 4-CY was used but with a smaller time constant of 2.3 ± 0.05 min (Fig. 2C), indicating that 4-CY at 5 mM concentration consumes the bound cofactor 3 fold faster than 4-CB.
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2 protein, and the change in NADPH absorption was monitored over time. A very slight decrease of NADPH absorption was observed over a period of 1 h (Fig. 2D), indicating that the reaction is greatly facilitated when the cofactor binds to the Kv2 protein.
Second, an active site residue, aspartate 85 (Fig. 1B), was mutated to an asparagine (Asn) and the D85N mutant protein was expressed and purified. Asp85 is highly conserved in all AKRs, and a Asp-to-Asn mutation reduces the rate of catalysis in other AKRs such as aldose reductase and 3-hydroxysteroid dehydrogenase (3-HSD) by 12- and 31-fold, respectively (31, 32). The purified D85N Kv2 protein has a bound NADPH cofactor, as indicated by an absorption peak near 360 nm. When the D85N mutant protein was mixed with 5 mM 4-CB, the absorption of the bound cofactor changed very slowly with a time constant of 330 ± 20 min (Fig. 2C; see also Fig. 3A). The time constant is 40-fold slower than that of the wild type Kv, indicating that the mutation slowed down the enzymatic reaction. This result supports the conclusion that Kv is a functional AKR.
Third, the reverse reaction, that is, transfer of a hydride from an alcohol to an NADP+, was tested. To do this, we purified the oxidized Kv2, i.e. Kv2 protein after its bound NADPH was consumed by 4-CY, by passing the reaction mixture through a size exclusion column. Interestingly, we found that the elution does not contain a peak corresponding to NADP+, which should be easily detected (Fig. 2E). This indicates that the oxidized cofactor (NADP+) stays tightly bound to Kv2. Using the oxidized Kv2, we screened small molecule alcohols for their ability to reduce the bound cofactor, and we found potential substrates. The result from one of them, 4-methoxybenzalcohol (4-MOB), is shown in Fig. 2F. When 4-MOB (10 mM) was mixed with the oxidized Kv2, absorption at 363 nm gradually increased (Fig. 2F, inset) and recovers to approximately the same level as freshly purified protein. The time course of NADPH generation was well fit with a single exponential function with a time constant of 61 ± 1 min (Fig. 2F), consistent with a single step hydride transfer reaction. The consumption and the re-generation of the Kv2-bound NADPH by small molecule aldehydes and alcohols clearly demonstrated that Kv is a functional AKR.
Single Versus Multiple Turnover Enzymatic Reactions—To further characterize the hydride transfer reaction, we measured the rate constant of the bound NADPH consumption, i.e. the single turnover reaction, at different 4-CY or 4-CB concentrations. We found that the rate constant increases with either 4-CB or 4-CY concentration and does not reach saturation even at 20 mM of a substrate (Fig. 3A, left panel). As a control, we used the D85N Kv2 protein and measured its hydride transfer rate at three different 4-CB concentrations (Fig. 3A, right panel). The rate is much slower than that of the wild type at all three substrate concentrations (2, 5, and 10 mM), supporting the conclusion that that Kv is a functional AKR.
It is remarkable that the rate constant does not saturate with increasing substrate concentrations. This indicates that both substrates have low occupancies on the enzyme so that binding is not saturated, something that is expected because both compounds are not native substrates. More importantly, it indicates that Kv is capable of converting the bound NADPH at a rate equal to or faster than 1.9 ± 0.06 min–1 (20 mM 4-CY), a time scale that is physiologically relevant in terms of redox sensing in a cell (33).
The hydride transfer step is only a half-reaction in the enzymatic reaction cycle, so we next measured the steady-state turnover rate constant for the cycle shown in Scheme I. The turnover rate constant was plotted versus 4-CB or 4-CY concentrations (Fig. 3B), and the data points were well fit by a Michaelis-Menten equation with a maximum turnover rate constant of 0.073 ± 0.0016 and 0.082 ± 0.0011/min and a Km of 3.2 ± 0.17 and 2.7 ± 0.12 mM, for 4-CB and 4-CY, respectively. When NADPH was substituted with NADH, no turnover was observed (data not shown), indicating that Kv has evolved to use specifically NADPH for catalysis. The steady-state turnover rate constant for both 4-CB and 4-CY is approximately the same but is much slower than the hydride transfer rate constant, especially for 4-CY. This is consistent with a reaction mechanism in which the cofactor exchange steps are rate-limiting. Since the cofactor exchange steps are not substrate-dependent, it suggests that the steady-state turnover rate constant is similar even when Kv catalyzes its native substrate(s). This property may have important physiological implications (see "Discussion").
As an additional control and to further support the conclusion that Kv is an AKR, we mutated another highly conserved catalytic site residue, Lys118 (Fig. 1B), to a methionine. A Lys-to-Met mutation in rat 3-HSD reduces the multiple turnover rate by more than 1000-fold (32). We expressed and purified the K118M mutant Kv2 and measured the multiple turnover rate constant at three different 4-CY concentrations (Fig. 3B). At 5 mM 4-CY, the turnover rate constant ((1.04 ± 0.21) x 10–4 min–1) was 500-fold slower than that of the wild type (0.053 ± 0.003 min–1). The rate is essentially indistinguishable from that of free NADPH oxidation without the presence of a Kv protein. This result indicates that the conserved lysine is important for catalysis, and reinforces the conclusion that Kv is a functional AKR.
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Substrates—That Kv is a functional AKR naturally leads to the question of whether the redox reaction on Kv affects channel function. To address this question, we co-expressed Kv1.4 with Kv2 and monitored channel function on inside-out patches before and after applying a Kv substrate.
Fig. 4A shows currents from a typical inside-out patch excised from an oocyte co-expressing Kv1.4 and Kv2. When 4-CY was perfused on the intracellular side of the membrane, channel inactivation rate decreased from 81 ± 1.9 s–1 to 46 ± 1.4 s–1 (Fig. 4, A and E, and Table 1; n = 31 patches from eight batches of oocytes), and both the peak current and the steady-state current levels increased significantly (Fig. 4A). 4-CB induced an almost identical response (data not shown). We have observed the substrate-induced change of channel current consistently on all the inside-out patches, and the change remained after the substrates were washed away.
When 4-CY was perfused to patches expressing only Kv1.4, the rate of channel inactivation changed slightly from 35 ± 2.3 to 34 ± 2.7 s–1 (Fig. 4, B and E, and Table 1; n = 10 patches from four batches of oocytes), and the current level remained essentially unchanged, indicating that the substrates modulate channel function through Kv.
To find out if the change in channel function is due to the redox reaction on Kv, we tested 4-CY on Kv1.4 co-expressed with either the D85N or the K118M mutant Kv2. In the case of Kv1.4 paired with the D85N mutant, the inactivation rate constant changes from 61 ± 4.1 to 58 ± 4.0 s–1 (Fig. 4, C and E, and Table 1; n = 9, four batches of oocytes). In the case of Kv1.4 paired with the K118M Kv2, it changes from 59 ± 2.4 to 57 ± 2.5 s–1 (Fig. 4, D and E, and Table 1; n = 6, three batches of oocytes). In both cases, 4-CY only induced a small change in channel current level and altered the rate of inactivation slightly, indicating that 4-CY is less efficient in modulating channel inactivation when the mutant Kv is present. Combined, the electrophysiological studies indicate that the substrates modulate channel functions through Kv2 and most likely through inducing the redox reaction.
Oxidation of the Bound NADPH Modulates Channel Function—How does a Kv substrate modulate channel function? On an excised patch, Kv very likely contains an NADPH, and when a substrate is applied to the patch, the Kv2-bound NADPH is oxidized. The NADPH-to-NADP+ conversion induces a change in channel inactivation. If this hypothesis is true, we can make the following two predictions and test them experimentally.
First, we reasoned that perfusing a patch with NADPH after channel modulation should reverse the 4-CY/4-CB effect because it would reload Kv2 with the reduced cofactor. Indeed the 4-CY effect was slowly reversed when the patch was perfused with 0.2 mM of NADPH (Fig. 4F, left panel). Recovery of current was observed in all the patches (n = 8), and in two of them the current recovered to the level before 4-CY application. In sharp contrast, no change in channel current was observed when NADPH was perfused to patches (n = 5) expressing Kv1.4 only (Fig. 4F, right panel). These experiments suggest that Kv on an inside-out patch can complete an enzymatic cycle, when 4-CY was perfused to oxidize its bound cofactor and followed by fresh NADPH to re-prime the enzyme. Changes in channel function served as a readout.
Second, we found that when a patch expressing both Kv1.4 and Kv2 was exposed to a substrate, channel current increases gradually over time and reaches a steady state (Fig. 5A). This suggests that although we do not know how the NADPH-to-NADP+ conversion induces a change in channel current, we can nevertheless follow the time course of channel modulation by measuring the current level as a function of time after a substrate was applied. Since 4-CB reacts with the bound NADPH at a rate 3-fold slower than 4-CY (Fig. 2C), we predict that channel modulation by 4-CB may also be slower if the cofactor oxidation is required for channel modulation. To test this prediction, we measured channel modulation using 4-CY and 4-CB, both at 5 mM concentration. Shown in Fig. 5B are the fractions of the total current change plotted versus substrate exposure time. The data points were well fit by single exponential functions (Fig. 5B, solid curves), with a time constant of 8.5 ± 0.4 min (8 patches) and 2.6 ± 0.2 min (11 patches), for 4-CB and 4-CY, respectively. Therefore 4-CY modulates channel function at 3-fold faster than 4-CB, similar to the difference observed in the hydride transfer reaction. This result strongly supports the notion that channel modulation is induced by the Kv bound NADPH-to-NADP+ conversion.
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Is Assembled with Kv1—Since the patch clamp studies were done at room temperatures (20–23 °C), we then measured the hydride transfer reaction at 22 °C so that the time constant of channel modulation can be directly compared with that of NADPH consumption in a test tube. We found that 5 mM 4-CB and 4-CY oxidized the Kv2-bound NADPH with a time constant of 19.8 ± 0.3 min (n = 3) and 6.0 ± 0.1 min (n = 3), respectively. Both time constants are 2.5-fold larger than these measured at 37 °C so that 4-CY remains 3-fold faster than 4-CB in oxidizing the NADPH. Compared with the time constants of channel modulation, both time constants are larger than these measured in patch clamp studies. It is likely that the hydride transfer rate is faster on a patch because Kv is now assembled with a Kv1 channel, although we cannot rule out other possibilities, for example, Kv expressed in an oocyte may have different kinetic properties from that expressed in bacteria. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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is a functional AKR, and we have shown that Kv substrates modulate channel inactivation through oxidizing the bound NADPH cofactor. These results indicate that the functions of the two proteins are dynamically coupled. It is well known that co-expression of Kv with Kv1 channels increases channel surface expression level (26, 34) and alters kinetic properties of channel activation and inactivation (25, 35–37). With the demonstration that Kv is a functional enzyme, it is now possible to test if other aspects of channel functions are directly coupled to the oxidation of the bound NADPH cofactors as well.
In a generic AKR, there are four highly conserved catalytic residues (Fig. 1B). Of the four, the catalytic aspartate, tyrosine, and lysine are almost 100% conserved among all AKR families including Kv, while the histidine is conserved in some of the AKR families, and in Kv it is an asparagine. In a 3-HSD when the histidine was mutated to a glutamate, it has little impact on the turnover rate constant but increased the Km by a factor of 10 (38). Similarly, mutating the histidine to an asparagine in an aldehyde reductase reduced the turnover rate constant by only a factor of 3 but substantially increased the Km value of the substrates (39). These results indicate that the histidine residue may play an important role in substrate recognition, and the corresponding asparagine residue in Kv could be evolved for the recognition of its specific native substrate.
The multiple enzymatic turnover rate constant of Kv2 is 0.08 min–1 and is almost the same for the two substrates. In comparison, 3-HSD catalyzes a very similar substrate, 4-nitrobenzaldehyde, at a rate of 61.4 min–1 (40), almost 760-fold faster than Kv2. On the other hand, the single turnover rate constant of Kv is at least 1.9 min–1. Compared with a 3-HSD, the native substrate 5-dihydrotestosterone oxidizes the bound NADPH at a limiting rate of 26 min–1 (41), which is 14-fold faster than Kv. These results indicate that Kv is a slow enzyme mainly because it has a slow cofactor exchange rate.
Is Kv a redox sensor that detects change in cellular chemistry and modulates channel function or an enzyme whose catalytic activity is regulated by channel activities? Data presented here cannot distinguish between the two possibilities but seem to favor somewhat the redox sensor mechanism. The enzymatic turnover rate constant appears too slow if the function of Kv is to catalyze substrate turnover but not so if it is to sense cellular redox changes, which would require only the hydride transfer step and occurs over the time scale consistent with our experiments. Furthermore, earlier structural studies have shown that the bound NADPH was oxidized over a period of 2 weeks during the crystallization process, but nevertheless the NADP+ still was present in the structure of Kv2 (10). The tight association is partly due to a flexible loop stretching over the NADPH binding site and restricting the dissociation of the cofactor (marked in yellow in Fig. 1B) (3, 10, 11). The structure of Kv suggests that the tight Kv-NADPH complex is built to respond to a redox signal quickly and keep the channel in a modulated state for a prolonged period of time. Consistent with this, Kv has been implicated in channel modulation during oxidative stress and hypoxic conditions, and we have found that 4-oxononenal, a lipid peroxidation product generated during oxidative stress (42, 43), oxidizes the Kv-bound NADPH and also modulates channel functions in a similar way (data not shown). However, a definitive answer to the question will require understanding the detailed mechanisms of the functional coupling and discovering the physiological substrate(s) of Kv.
The functional interactions between the two proteins demonstrate a simple way of coupling intracellular redox states to the excitability of a cell. The results presented here bring us one step forward to understanding the physiological functions of Kv.
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FOOTNOTES |
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This article was selected as a Paper of the Week.
1 These authors contributed equally to this work.
2 To whom correspondence should be addressed: Ming Zhou, Dept. of Physiology and Cellular Biophysics, Columbia University College of Physicians and Surgeons, 630 West 168th St., New York, NY 10032. Tel.: 212-342-3722; Fax: 212-305-5775; E-mail: mz2140{at}columbia.edu.
3 The abbreviations used are: AKR, aldo-keto reductase; 4-CB, 4-carboxybenzaldehyde; 4-CY, 4-cyanobenzaldehyde; 3-HSD, 3-hydroxysteroid dehydrogenase; 4-MOB, 4-methoxybenzalcohol.
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ACKNOWLEDGMENTS |
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-HSD protein. We thank Drs. C. Deutsch, S. Marx, C. Miller, and G. Pitt for critical comments on the manuscript. Finally, we thank J. Riley for expert technical assistance. |
REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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1 and 
) and 4-CY (
)) and the D85N mutant (
) Kv
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, 4-CY on the K118M mutant Kv
