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(Circulation Research. 1995;76:325-334.)
© 1995 American Heart Association, Inc.

Articles

Functional Consequences of Sulfhydryl Modification in the Pore-Forming Subunits of Cardiovascular Ca²⁺ and Na⁺ Channels

Nipavan Chiamvimonvat, Brian O'Rourke, Timothy J. Kamp, Roland G. Kallen, Franz Hofmann, Veit Flockerzi, Eduardo Marban

From the Division of Cardiology (N.C., B.O., T.J.K., E.M.), Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Md; the Institut für Pharmakologie und Toxikologie der Technischen Universität München (Germany) (F.H., V.F.); and the Department of Biochemistry and Biophysics and School of Medicine (R.G.K.), University of Pennsylvania, Philadelphia.

Correspondence to Eduardo Marban, MD, PhD, 844 Ross Bldg, The Johns Hopkins University School of Medicine, Baltimore, MD 21205.

	Abstract

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Abstract The structure and function of many cysteine-containing proteins critically depend on the oxidation state of the sulfhydryl groups. In such proteins, selective modification of sulfhydryl groups can be used to probe the relation between structure and function. We examined the effects of sulfhydryl-oxidizing and -reducing agents on the function of the heterologously expressed pore-forming subunits of the cloned rabbit smooth muscle L-type Ca²⁺ channel and the human cardiac tetrodotoxin-insensitive Na⁺ channel. The known sequences of the channels suggest the presence of three or four cysteine residues within the putative pores of Ca²⁺ or Na⁺ channels, respectively, as well as multiple other cysteines in regions of unknown function. We determined the effects of sulfhydryl modification on Ca²⁺ and Na⁺ channel gating and permeation by using the whole-cell and single-channel variants of the patch-clamp technique. Within 10 minutes of exposure to 2,2'-dithiodipyridine (DTDP, a specific lipophilic oxidizer of sulfhydryl groups), Ca²⁺ current was reduced compared with the control value, with no significant change in the kinetics and no shift in the current-voltage relations. The effect could be readily reversed by 1,4-dithiothreitol (an agent that reduces disulfide bonds). Similar results were obtained by using the hydrophilic sulfhydryl-oxidizing agent thimerosal. The effects were Ca²⁺-channel specific: DTDP induced no changes in expressed human cardiac Na⁺ current. Single-channel Ba²⁺ current recordings revealed a reduction in open probability and mean open time by DTDP but no change in single-channel conductance, implying that the reduction of macroscopic Ca²⁺ current reflects changes in gating and not permeation. In summary, the pore-forming (₁) subunit of the L-type Ca²⁺ channel contains functionally important free sulfhydryl groups that modulate gating. These free sulfhydryl groups are accessible from the extracellular side by an aqueous pathway.

Key Words: Ca²⁺ channels • Na⁺ channels • cysteine • sulfhydryl oxidation

	Introduction

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Ion channels are pore-forming proteins that open and close in response to defined stimuli, providing a passive conduit for ion transfer across the cell membrane.¹ Ion channels of many classes have now been cloned, sequenced, and functionally expressed; among these are the voltage-gated Na⁺ and Ca²⁺ channels.^{2 3} L-type Ca²⁺ channels were first purified from skeletal muscle and consist of five subunits: ₁, ₂, ß, , and .^{3 4} Na⁺ channels, in contrast, consist of one to three subunits: and variable tissue-specific coexpression of ß₁ and/or ß₂.^{5 6} The structure of the principal pore-forming subunit ( or ₁ subunit) of each of these channels is based on the same motif: four homologous domains, each containing six transmembrane segments surrounding a central pore.⁷ This principal subunit has been shown to confer full channel-forming function when expressed alone in heterologous systems.⁷

Sulfhydryl groups of cysteinyl residues of peptides and proteins are the most reactive of all amino acid side chains under physiological conditions.⁸ They may be readily alkylated, acylated, arylated, and oxidized. Two cysteine residues that are adjacent in the three-dimensional structure of a protein can form a disulfide bridge. This reaction requires an oxidative environment, and such disulfide bridges are usually not found in intracellular domains of proteins, which spend their lifetime in an essentially reductive environment.⁹ Disulfide bridges, however, do occur quite frequently in extracellular or transmembrane segments of proteins. Disulfide bridges stabilize the three-dimensional structure of the proteins, making them less susceptible to degradation. In some proteins, these bridges hold together different polypeptide chains, forming subunits of the protein, eg, the ₂ and subunits of the Ca²⁺ channel^{10 11} and and ß₂ subunits of the Na⁺ channel.^{12 13} In addition, cysteine residues have a high affinity for divalent cations of the group IIB series, including Cd²⁺ and Zn²⁺, in solution as well as in metal-binding proteins, such as the Zn²⁺ finger transcription factors.¹⁴ Recently, a critical cysteine residue within the putative pore region of the Na⁺ channel has been shown to confer isoform-specific divalent cation sensitivity and tetrodotoxin insensitivity.^{15 16}

The function of many cysteine-containing proteins critically depends on the oxidation state of one or more of the protein's sulfhydryl groups.^{17 18 19} If so, selective chemical modification of the sulfhydryl groups can help to localize functionally important regions of the molecule. In the present study, we examined the effects of sulfhydryl oxidizing and reducing agents on the function of the heterologously expressed pore-forming subunits of rabbit smooth muscle L-type Ca²⁺ channels²⁰ and human cardiac tetrodotoxin-insensitive Na⁺ channels (hH1).²¹ This approach has two unique advantages: the channels are of known structure, thus facilitating the interpretation of functional changes; likewise, the fact that only one subunit is expressed eliminates the known intersubunit disulfide bonds and significantly restricts the number of cysteines that must be considered.

	Materials and Methods

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Stable Transfection of ₁ Subunit of L-type Ca²⁺ Channel From Rabbit Lung in Chinese Hamster Ovarian Cells
The generation of the stable Chinese hamster ovarian (CHO) cell line expressing the ₁ subunit of L-type Ca²⁺ channel from rabbit lung (type 2b Ca²⁺ channel ₁ subunit,²² GenBank accession number X55763) has been previously described.²⁰ Cells were maintained at 37°C in Dulbecco's modified Eagle's medium containing 10% dialyzed fetal bovine serum supplemented with penicillin, streptomycin, and nonessential amino acids. Cells were passaged after they had grown to 90% confluence. Electrophysiological recordings were performed 24 to 48 hours after plating.

Transient Transfection of ₁ Subunit of Human Cardiac Na⁺ Channel in Human Embryonic Kidney Cells
Human embryonic kidney (HEK 293) cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mmol/L L-glutamine, and 1% penicillin and streptomycin. Cell cultures were kept at 37°C in a 5% CO₂ incubator. Cells were transfected by the calcium phosphate precipitation method.²³ The hH1 cDNA was cloned as previously described,²¹ and the coding region was ligated into the expression vector RBG4 (provided by Dr W.S. Agnew, Johns Hopkins University) at the EcoRI site. The calcium phosphate–DNA mixture was added when cells were 50% confluent and left for 24 hours. The medium was then removed, cells were washed with phosphate-buffered saline, and fresh medium was added. Electrophysiological recordings were done 48 to 72 hours after transfection.

Functional Expression of ₁ Subunit of Human Cardiac Na⁺ Channel in Xenopus Oocytes
In vitro transcription of complementary RNA was effected from an expression plasmid containing the cDNA encoding the human cardiac Na⁺ channel (pSP64T*). Stage V and VI oocytes were removed from adult female Xenopus laevis (Xenopus 1, Ann Arbor, Mich, or Nasco, Ft Atkinson, Wis) and isolated by two periods (30 to 45 minutes) of collagenase treatment (2 mg/mL, type IA, Sigma Chemical Co) in modified Barth's solution [mmol/L: NaCl 88, KCl 1, NaHCO₃ 2.4, Tris(hydroxymethyl)aminomethane 15, CaNO₃ · 4H₂O 0.3, CaCl₂ · 6H₂O 0.41, MgSO₄ · 7H₂O 0.82, sodium pyruvate 5, theophylline 0.5] supplemented with penicillin (100 U/mL), streptomycin (100 µg/mL), fungizone (250 ng/mL), and gentamicin (50 µg/mL). After digestion, oocytes were maintained in modified Barth's solution. Oocytes were then injected with 50 to 100 nL of mRNA with a 10-µL microinjector (Drummond Scientific Co) and used for electrophysiological recording 1 to 2 days after injection.

Electrophysiological Recordings in Mammalian Cells
Both whole-cell and cell-attached variants of the patch-clamp recording technique²⁴ were used to record Na⁺ and Ca²⁺ currents (I_Na and I_Ca, respectively) from transfected cells. Small (35-mm) plastic tissue-culture dishes were transferred to the stage of an inverted microscope (Nikon) and superfused with external solution at a rate of 1 to 2 mL/min. All chemicals were purchased from Sigma unless stated otherwise.

For whole-cell I_Na recording, the external solution had the following composition (mmol/L): NaCl 140, KCl 5, MgCl₂ 1, CaCl₂ 2, HEPES 10, and glucose 10, pH 7.4 with NaOH. For whole-cell I_Ca recording, the external solution contained the following (mmol/L): NaCl 140, MgCl₂ 1, CaCl₂ 5, tetraethylammonium chloride 5, 4-aminopyridine 2, HEPES 10, and glucose 10, along with 100 nmol/L tetrodotoxin, pH 7.4 with NaOH. Patch-clamp electrodes were filled with a solution of the following composition (mmol/L): CsCl 130, MgCl₂ 1, MgATP 5, BAPTA 10, and HEPES 10, pH 7.2 with CsOH. Stock solutions containing sulfhydryl-modifying reagents were made fresh for each experiment.

All experiments were performed at room temperature (22°C to 23°C) by using an Axopatch 200A patch-clamp amplifier (Axon Instruments) interfaced to a personal computer. Voltage commands and data collection were controlled by using custom-written software. For whole-cell current recordings, the cell capacitance and series resistance were compensated and measured during 20-mV depolarizing pulses from a holding potential of -80 mV. In general, 60% to 80% of the series resistance was compensated. I_Na recordings were filtered at 10 kHz by using a four-pole Bessel filter and digitized at a sampling frequency of 50 kHz. Whole-cell I_Ca recordings were filtered at 2 kHz and sampled at 10 kHz. Currents were leak-subtracted by a P/4 method. Data were stored in the computer for later analysis by using custom-written software.

For cell-attached single-channel I_Ca recordings, the bath solution contained the following (mmol/L): KCl 140, NaCl 10, MgCl₂ 1, HEPES 10, and glucose 10, along with 100 µmol/L EGTA, pH 7.4 with KOH. Patch electrodes had 10- to 15-M tip resistances when filled with the following solution (mmol/L): BaCl₂ 70 and HEPES 10, pH 7.4, with Ba(OH)₂. Bay K 8644 (4 µmol/L) was added to the solution in the patch electrodes for some experiments. Cell-attached patches were formed with seal resistances of 20 to 100 G. Currents were filtered at 2 kHz and sampled at 10 kHz. Leakage and capacity currents were subtracted from unitary current recordings by fitting a smooth template to null tracings. Unitary current amplitude was determined from long-lasting openings obtained in the presence of Bay K 8644. Amplitude histograms at a given test potential were generated and fitted to a single gaussian distribution by using a Levenberg-Marquardt algorithm to obtain the mean unitary currents. Leak-subtracted current recordings were idealized with a half-height criterion.²⁵ Idealized recordings were used to construct ensemble-averaged currents, to determine open probability, and to generate histograms for the distributions of open intervals. Single and biexponential probability density functions were fitted to all open intervals by using a nonlinear least-squares criterion. Open-time histograms were plotted as the complements of the distributions for illustration only. The number of channels in a patch was estimated by binomial analysis²⁵ or by the stacking of the unitary events. In most patches, multiple channels were observed. Therefore, closed intervals were not calculated. Peak open probability was determined from the ensemble current by using the measured single-channel current amplitude and the estimated number of channels in the patch.

Membrane Current Recordings in Xenopus Oocytes
A two-microelectrode voltage-clamp technique²⁶ was used to record whole-oocyte I_Na. Glass pipettes were filled with 3 mol/L KCl solutions having resistances of 0.2 to 1 M. Membrane potential was controlled by a two-electrode voltage-clamp amplifier (Warner Instrument Corp). Currents were recorded in the following solution (mmol/L): NaCl 96, MgCl₂ 1, and HEPES 10, pH 7.6 with NaOH. Signals were low-pass–filtered at 1 kHz by an eight-pole Bessel filter (Frequency Devices Inc) and digitized on-line at 10 to 20 kHz with 12-bit resolution onto a personal computer. All electrophysiological recordings were obtained at room temperature.

Statistics
Pooled data are presented as mean±SEM. Statistical comparison was effected by using Student's t test and ANOVA (where appropriate), with a value of P<.05 considered significant.

	Results

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Sulfhydryl Modification
Reactive disulfide compounds with a pyridyl ring adjacent to the disulfide bond, such as 2,2'-dithiodipyridine (DTDP), oxidize free sulfhydryl groups specifically via a thiol-disulfide exchange reaction leading to the production of mixed disulfide bonds with the protein and the stoichiometric production of thiopyridone²⁷ (Fig 1A). Effective concentrations range between 10 and 50 µmol/L.^{27 28} Thimerosal ([(O-carboxyphenyl)thio]ethylmercury sodium salt, Fig 1B) is a mercurial compound that also oxidizes free sulfhydryl groups.²⁹ However, in contrast to DTDP, thimerosal is hydrophilic and poorly membrane permeable.

View larger version (15K): [in this window] [in a new window]   Figure 1. A, Diagram showing thiol-disulfide exchange reaction between the free sulfhydryl group (SH) on cysteine residues (C) of channel proteins (P) and the sulfhydryl-oxidizing agent 2,2'-dithiodipyridine (DTDP, C10H8N2S2). The oxidizing agents attack the free sulfhydryl groups on the channel proteins to form mixed disulfides and thiopyridones. B, Diagram showing sulfhydryl oxidation by thimerosal ([(O-carboxyphenyl)thio]ethylmercury sodium salt), a hydrophilic compound. C, Diagram showing reduction of disulfide bond (S-S) on channel proteins by the reducing agent 1,4-dithiothreitol. The reaction disrupts intramolecular disulfide bonds and yields free sulfhydryl groups on channel proteins.

1,4-Dithiothreitol was used to reduce disulfide bonds (Fig 1C). The reagent has a very low redox potential, leading to a reaction yielding intramolecular disulfide bonds and free sulfhydryl groups on the channel proteins.³⁰ Effective concentrations are in the range of 1 to 5 mmol/L.^{28 29 31}

Effects of Sulfhydryl Oxidation by DTDP on L-type I_Ca
CHO cells, which were stably transfected with the ₁ subunit of the L-type Ca²⁺ channel, expressed I_Ca in >95% of the cells. Current density (peak current normalized to cell capacitance) was 8 to 10 pA/pF (external Ca²⁺ of 5 mmol/L). As previously reported, the currents were blocked by nitrendipine and could be potentiated by Bay K 8644 (results not shown).

Fig 2A shows representative whole-cell current recordings of a family of Ca²⁺ currents elicited by a series of depolarizing voltage-clamp steps from a holding potential of -80 mV during baseline and 10 minutes after superfusion with 50 µmol/L DTDP (Fig 2B). I_Ca activated with a threshold potential of -30 mV, and current peaked near +20 mV. Sulfhydryl oxidation led to a reduction in the macroscopic current magnitude, with no apparent shift in the voltage dependence of activation. This change in macroscopic current can be reversed by application of 5 mmol/L dithiothreitol (Fig 2C). Fig 2D shows the pooled current-voltage relations of I_Ca obtained from seven different cells at baseline (), after 8 to 10 minutes of superfusion with DTDP (), and after 3 minutes of superfusion with 5 mmol/L dithiothreitol ().

View larger version (19K): [in this window] [in a new window]   Figure 2. A, Representative family of Ca2+ currents elicited by a series of voltage-clamp steps from a holding potential of -80 mV during baseline in Chinese hamster ovary (CHO) cells stably transfected with the 1 subunit of the type 2b L-type Ca2+ channel from rabbit lung. Recordings were obtained by stepping the membrane potential to 0, +10, +20, and +30 mV. The cell membrane capacity was 32 pF, and the effective series resistance (after compensation) was 2 M. B, Current recordings obtained from the same cell after 10 minutes of superfusion with 50 µmol/L 2,2'-dithiodipyridine (DTDP). C, Current recordings obtained after 4 minutes of superfusion with 5 mmol/L 1,4-dithiothreitol. D, Graph showing current-voltage relations of the Ca2+ current obtained from seven different cells during baseline (), after 8 to 10 minutes of superfusion with 50 µmol/L DTDP (), and after 3 minutes of superfusion with 5 mmol/L 1,4-dithiothreitol ().

We next determined whether sulfhydryl modification by DTDP affects the time course of macroscopic I_Ca decay. Fig 3 shows the fraction of current remaining at 150 ms of depolarization in eight cells before and after 10 minutes of superfusion with DTDP. There were significant differences within each curve at various voltages, with inactivation being more complete at positive test potentials; nevertheless, the curves before and after exposure to DTDP were not significantly different. The current tracings in Fig 3B were obtained during voltage steps to +20 mV at baseline and after exposure to DTDP. The normalized tracing after the sulfhydryl modification (shown in stippled line) superimposes well on the current recording obtained during baseline. Thus, DTDP does not alter the kinetics of I_Ca inactivation.

View larger version (17K): [in this window] [in a new window]   Figure 3. A, Graph showing Ca2+ current at 150 ms during depolarizing voltage steps normalized by the peak inward current during baseline () and after 10 minutes of superfusion with 50 µmol/L 2,2'-dithiodipyridine (DTDP, ) (n=8 for each curve). B, Ca2+ current recordings from one cell from a holding potential of -80 to +20 mV during baseline () and after sulfhydryl modification (). Stippled line represents the current tracing after sulfhydryl modification normalized to the control tracing.

Effect of Sulfhydryl Modification by DTDP Is Ca²⁺ Channel Specific
The subunit of the Na⁺ channel architecturally resembles the L-type Ca²⁺ channel ₁ subunit. To determine whether the effects of DTDP are specific for Ca²⁺ channels, we examined the effects of this compound on expressed hH1 Na⁺ channels. The expression efficiency of I_Na in the transiently transfected HEK 293 cells was low; 10% of the cells exhibited current densities of 5 to 8 pA/pF (external Na⁺of 140 mmol/L). The voltage dependences of activation and inactivation were well described by single Boltzmann functions with half-activation and -inactivation voltages of -26.1±1.3 and -63.9±6.9 mV and slope factors of 6.9±1.4 and 7.1±0.5 mV, respectively.

In contrast to the effects of sulfhydryl oxidation of the L-type I_Ca, DTDP had no significant effects on I_Na. Fig 4A shows representative whole-cell I_Na elicited by a depolarizing step to +10 mV from a holding potential of -100 mV during baseline () and after 10 minutes of superfusion with 50 µmol/L DTDP (). There were no changes in magnitude or kinetics of the macroscopic current. The current-voltage relations elicited at baseline () and after drug () are shown in Fig 4B (n=5).

View larger version (16K): [in this window] [in a new window]   Figure 4. A, Whole-cell Na+ currents elicited by a voltage step to +20 mV from -120 mV during baseline () and after 10 minutes of superfusion with 50 µmol/L 2,2'-dithiodipyridine (DTDP, ) in a human embryonic kidney (HEK 293) cell transiently transfected with human cardiac tetrodotoxin-insensitive Na+ channel cDNA. The cell membrane capacitance was 45 pF, and effective series resistance (after compensation) was 2.3 M. B, Graph showing current-voltage relations obtained from five different cells at baseline () and after sulfhydryl modification ().

The effects of sulfhydryl modification with DTDP on I_Na were also assessed in Xenopus oocytes expressing hH1 channels. Similar results were obtained in two other myocytes (data not shown). In addition, the effects of DTDP were assessed in three oocytes that had been pretreated with 1,4-dithiothreitol. Just as in cells not preexposed to a reducing agent, there were no consistent effects of DTDP.

Effects on Macroscopic I_Ca of the Hydrophilic Sulfhydryl-Oxidizing Compound Thimerosal
Application of the hydrophilic sulfhydryl-oxidizing agent thimerosal produced effects on macroscopic I_Ca similar to those of the membrane-permeant DTDP. Fig 5 shows current-voltage relations of I_Ca obtained at baseline (), 10 minutes after superfusion with 10 µmol/L thimerosal (), and after reversal by dithiothreitol () (n=5). As with DTDP, sulfhydryl modification by thimerosal decreased whole-cell I_Ca but did not otherwise change the current-voltage relations. This effect was readily reversed by 1,4-dithiothreitol.

View larger version (14K): [in this window] [in a new window]   Figure 5. Graph showing current-voltage relations of Ca2+ current obtained during baseline (), after 10 minutes of sulfhydryl modification with 10 µmol/L thimerosal ([(O-carboxyphenyl)thio]ethylmercury sodium salt, ), and after 5 minutes of 5 mmol/L 1,4-dithiothreitol ().

Reduction in I_Ca by DTDP Was due to Oxidation of Free Sulfhydryl Group(s)
The effects of sulfhydryl oxidation cannot be reversed by simple washout using normal Tyrode's solution; the effects can only be reversed by using the sulfhydryl-reducing agent 1,4-dithiothreitol. Fig 6 shows the time course of peak I_Ca elicited at a test potential of +20 mV during baseline, superfusion with 50 µmol/L DTDP, washout with normal Tyrode's solution, and finally, exposure to 5 mmol/L 1,4-dithiothreitol. The reduction in peak I_Ca by DTDP was promptly reversed by the disulfide-reducing agent, despite the ineffectiveness of washing out with normal Tyrode's solution. Similar results were obtained in three other cells with DTDP and in two cells with thimerosal as the oxidizing agent (data not shown). These results suggest that chemically modified sulfhydryls are quite stable in the absence of reducing agents, consistent with previous results.^{29 31}

View larger version (14K): [in this window] [in a new window]   Figure 6. Peak Ca2+ current elicited by using a voltage-clamp step to +20 mV from a holding potential of -80 mV plotted as a function of time during baseline, during superfusion with 50 µmol/L 2,2'-dithiodipyridine (DTDP), during washout using Tyrode's solution, and during washout with 5 mmol/L 1,4-dithiothreitol (DTT). Current recordings shown below correspond to current tracings obtained from voltage steps at points 1, 2, 3, and 4 along the curve.

Effects of 1,4-Dithiothreitol on L-type Ca²⁺ Channels and Na⁺ Channels With No Prior Oxidizing Agents
In contrast to the sulfhydryl-oxidizing agents, the sulfhydryl-reducing agent 1,4-dithiothreitol had negligible effects on I_Ca on its own. Dithiothreitol changed neither the magnitude nor the time course of the macroscopic current (n=6, data not shown). Similar results were obtained with cardiac Na⁺ channels expressed in Xenopus oocytes (n=6, data not shown).

Effects of Sulfhydryl Modification on Single-Channel Ca²⁺ Currents
Changes observed at the macroscopic current level due to effects of sulfhydryl modification on channel proteins can theoretically lead to a change either in permeation or in gating of the channel or both. To distinguish among these possibilities, the effects of sulfhydryl modification were further studied at the single-channel level. Effects of DTDP were assessed either by superfusion with the reagent or by including it in the pipette solution.

First, we studied the possible effects of sulfhydryl modification on the permeation pathway by determining single-channel conductance. Fig 7 shows consecutive single-channel current tracings obtained from a holding potential of -80 mV to a test potential of +10 mV during baseline (panel A) and after 10 minutes of superfusion with DTDP (panel B). To prolong openings, Bay K 8644 (4 µmol/L) was included in the pipette solution. Despite an obvious reduction in the number of channel openings, DTDP appears not to reduce unitary current amplitude. This impression is confirmed by Fig 7C, which shows pooled single-channel current-voltage relations obtained from four different control patches and four patches containing DTDP. The calculated single-channel conductances are 17.7 and 17.3 pS for control and sulfhydryl-modified patches, respectively.

View larger version (26K): [in this window] [in a new window]   Figure 7. A and B, Recordings showing effects of sulfhydryl modification on single-channel Ba2+ current from a cell-attached patch during baseline (A) and after 10 minutes of superfusion with 50 µmol/L 2,2'-dithiodipyridine (DTDP, B). Recordings were obtained using 180-ms steps to +10 mV from a holding potential of -80 mV. Bay K 8644 (4 µmol/L) was included in the patch pipette. C, Graph showing single-channel current-voltage relations obtained from control patches (, n=3) and sulfhydryl-modified patches (, n=3) (50 µmol/L DTDP was included in the patch pipettes). The calculated conductances were 17.7 and 17.3 pS for control and sulfhydryl-modified patches, respectively.

In contrast to the lack of effects on unitary current amplitudes, sulfhydryl oxidation by DTDP did produce a significant decrease in open probability and open time. Figs 8 and 9 show diaries of open probability versus sweep number, open-time histograms, and ensemble-averaged currents obtained from control and sulfhydryl-modified patches, respectively. Open times were fitted by using biexponential functions for both patches. Time constants obtained from the control patch in Fig 8 equaled 1.1 and 6.8 ms. In contrast, the time constants obtained from the sulfhydryl-modified patch were much shorter: 0.4 and 1.3 ms. Long openings typical of mode 2 that would ordinarily be favored by the presence of Bay K 8644³² became rare in the presence of DTDP. Similar results were obtained in three other patches.

View larger version (21K): [in this window] [in a new window]   Figure 8. Single-channel Ba2+current kinetics obtained from a control patch with Bay K 8644 (4 µmol/L) in the patch pipette. Current tracings were obtained by using 180-ms steps to +10 mV from a holding potential of -80 mV. A, Plot of open probability vs sweep number. B, Open-time histogram plotted in the form of complemented distribution. The solid line represents the biexponential fit to the data with time constants of 1.1 and 6.8 ms. C, Ensemble-averaged current constructed from idealized recordings.

View larger version (14K): [in this window] [in a new window]   Figure 9. Effects of sulfhydryl modification on single-channel Ba2+ current kinetics. Currents were obtained from a sulfhydryl-modified patch with 50 µmol/L 2,2'-dithiodipyridine in the pipette during 180-ms steps to +10 mV from -80 mV. Bay K 8644 (4 µmol/L) was also included in the pipette. A, Plot of open probability vs sweep number. B, Open-time histogram plotted as the complement of the distribution. The solid line represents a biexponential fit to the data with time constants of 0.4 and 1.3 ms. C, Ensemble-averaged current constructed from idealized recordings. Note different current scale from Fig 8C.

To rule out a possible chemical interaction between DTDP and Bay K 8644, further detailed analysis was performed in control and sulfhydryl-modified patches in the absence of any dihydropyridine agonist. There was a similar decrease in open probability in sulfhydryl-modified patches. The distribution of open times in the absence of Bay K 8644 could be fitted by a single exponential function, since long openings were rarely observed in our experiments either in control or sulfhydryl-modified patches. Time constants obtained from sulfhydryl-modified patches were significantly abbreviated by exposure to DTDP (0.16±0.016 versus 0.303±0.037 ms in sulfhydryl-modified and control patches, respectively). Calculated dead time for the experiments was 90 µs.²⁶ The Table summarizes the estimated mean open times from all control and sulfhydryl-modified patches. The reduction in open times by DTDP was consistent and statistically significant, both in the presence and absence of Bay K 8644 modification.

View this table: [in this window] [in a new window]   Table 1. Mean Open Times, Peak Open Probability, and Estimated Number of Channels Obtained From Control and Sulfhydryl-Modified Patches in the Presence and Absence of 4 µmol/L Bay K 8644

The reduction in the overall open probability in multichannel patches from sulfhydryl modification could be due to an alteration in the open probability of individual channels and/or the number of functional channels. Therefore, functional channel numbers were estimated by using the binomial theorem. Fig 10 shows the probability that a given number of channels was open (P_n) plotted against the number of channels open (n). Data were obtained from the same patch as in Fig 7 by using a voltage-clamp step to +20 mV from a holding potential of -80 mV during baseline (Fig 10A) and 10 minutes after superfusion with 10 µmol/L DTDP (Fig 10B). There was a good fit of the model predicted by the binomial theorem to the experimental data:

where N and P_o represent the number of functional channels and the open probability of individual channels, respectively. During baseline, the predicted open probability of an individual channel was .232 with N=6. Both of these values were reduced after sulfhydryl modification, with a predicted peak open probability of .086 and N=2, consistent with a reduction in the individual open probability with some of the channels entering a long-lived nonconducting state (ie, an apparent decrease in N). The values of the number of functional channels agree well with the estimation from the stacking of the unitary events.

View larger version (8K): [in this window] [in a new window]   Figure 10. Bar graphs showing the probability that a given number of channels was open (Pn) vs the number of channels (n) obtained from the same patch as in Fig 7 during baseline (A) and after 10 minutes of superfusion with 50 µmol/L 2,2'-dithiodipyridine (DTDP) (B). Single-channel currents were elicited by using 180-ms steps to +20 mV from -80 mV. The columns represent the experimental data using the measured values for each unitary current level; *Probability (Pn) predicted by the binomial theorem with predicted probability that an individual channel is open (Po)=.232 and number of functional channels (N)=6 at baseline and Po=.0865 and N=2 after superfusion with DTDP.

The Table shows the peak open probabilities (open probabilities at the peak current) determined from channel numbers and the measured single-channel currents. Peak open probability decreased significantly in the sulfhydryl-modified patches compared with control patches, as did the estimated numbers of channels in the patch, both in the presence and absence of dihydropyridine agonist.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we assessed the effects of sulfhydryl modification on the function of the pore-forming subunits of Ca²⁺ and Na⁺ channels expressed in heterologous systems. Such an approach enables the examination of each particular subunit of interest in isolation from other subunits. Therefore, this system lends itself well to the study of the structure-function relation of ion channel proteins. We have demonstrated that sulfhydryl modification of L-type Ca²⁺ channels resulted in a reduction in whole-cell I_Ca, which could be readily reversed by disulfide reduction. At the single-channel level, this reduction in macroscopic current was mediated by a decrease in open probability and open time and an apparent decrease in the number of functional channels, with no change in single-channel conductance, consistent with changes in gating but not permeation. The effects of sulfhydryl modification were Ca²⁺ channel specific, with no detectable changes in I_Na.

Presence of Accessible Free Sulfhydryl Groups in the Pore-Forming Subunit of the Ca²⁺ Channel
Voltage-activated Ca²⁺ channels can be viewed as having three modes of gating behavior: no openings (mode 0), brief repetitive openings (mode 1), and long-lasting openings with brief closures (mode 2).^{32 33} The dihydropyridine agonists (eg, Bay K 8644) enhance I_Ca by promoting mode 2, whereas the antagonists favor mode 0. The mechanism of transitions between modes is not known. Inhibition of L-type Ca²⁺ channels by dihydropyridine antagonists may be analogous to C-type inactivation previously described for K⁺ channels.³⁴ In the present study, sulfhydryl modification mimics the effects of dihydropyridine antagonists, promoting transition of the channel to mode 0 and mode 1, with a resultant reduction in open time and open probability and an apparent decrease in functional channel number. The results are consistent with the presence of free sulfhydryl groups on the Ca²⁺ channel, which are accessible from the extracellular side and are important in the gating of the channel. Previous biochemical studies have suggested the involvement of disulfide bonds and free sulfhydryl groups in the binding of dihydropyridine to the L-type Ca²⁺ channel in heart muscle.³⁵ Our findings that oxidation of free sulfhydryl groups of the Ca²⁺ channel leads to a channel with similar characteristics as that after treatment with dihydropyridine antagonists are certainly consistent with this interpretation.

From the known sequences of the two channels, there are three and four cysteine residues within the putative pore regions of the Ca²⁺ and Na⁺ channels, respectively.^{36 37 38} Modification of some of these cysteine residues might be expected to alter channel permeation.³⁹ However, these cysteine residues may not be accessible to modification when using the two different sulfhydryl-oxidizing agents. These residues may be located deep within the pore in a region inaccessible to the bulky modifying agents. For example, the cysteine residue in the first domain of the Na⁺ channel was localized to 20% of the distance down the electric field¹⁵ and is adjacent to the proposed selectivity filter of the channel. Interestingly, the smaller alkylating agent iodoacetic acid does gain access to this site.³⁹

The effects of sulfhydryl modification were specific for Ca²⁺ channels. Examination of the amino acid sequences in the Ca²⁺ and Na⁺ channels reveals a total of 41 and 42 cysteine residues. However, if only those cysteine residues that are predicted to be accessible from the extracellular aqueous pathway are considered, there are a total of 12 residues in each of the two channels; three and four of these are those previously mentioned, which fall within the pore-lining regions of the Ca²⁺ and Na⁺ channels, respectively. Comparison of the sequence alignments among different classes of Ca²⁺ channels⁴⁰ shows conservation of all the putatively external cysteine residues within different dihydropyridine-sensitive Ca²⁺ channels. However, one residue in the S5-S6 linker of domain IV is different between Ca²⁺ and Na⁺ channels. An isoleucine (position 1720 in hH1) is found instead of cysteine (position 1472 in type 2b L-type Ca²⁺ channel) in the aligned sequences of the Na⁺ channel compared with the Ca²⁺ channel. Of interest, the linker region between S5 and S6 in domain IV has recently been shown to be involved in dihydropyridine action.⁴¹ In addition, C-type inactivation in K⁺ channels has been shown to be mediated by an amino acid in the transmembrane S6 in the fourth domain.³⁴ Site-directed mutagenesis of these conserved cysteine residues may yield a definitive answer regarding which particular residue is responsible for the effects of sulfhydryl modifiers as well as the mechanisms by which the residue regulates gating in the channel.

Absence of Effects of Disulfide-Reducing Agent on the Pore-Forming Subunit of the Ca²⁺ Channel
Dithiothreitol has no effect on whole-cell I_Ca. Although results are consistent with the idea that there are no accessible disulfide bonds in the native ₁ subunit of Ca²⁺ channels, reduction of a disulfide bond will not necessarily affect the whole-cell current. Nonetheless, this finding may not come as a surprise. Although the presence of disulfide bonds between subunits has previously been documented (namely, those between ₂ and subunits), the presence of disulfide bonds within the ₁ subunit has not been proposed.

Previous Studies on Other Ion Channels
The oxidation state of sulfhydryl groups has previously been shown to be important in the function of several channel proteins. Sulfhydryl oxidation of the rat brain I_A K⁺ channels (rapidly inactivating K⁺ channels) expressed in oocytes led to the abolition of fast inactivation of the channel.³¹ This loss of inactivation was shown to be due to the oxidation of a critical cysteine residue located near the ball domain. Sulfhydryl oxidation induced a rapid and reversible closure of the ATP-regulated K⁺ channel in pancreatic ß cells.²⁹ Sulfhydryl oxidation of the Ca²⁺ release channel triggered Ca²⁺ release from sarcoplasmic reticulum (SR), whereas disulfide reduction led to a rapid re-uptake of Ca²⁺.²⁸ The effects were mediated by an increase in SR Ca²⁺ release channel open probability with no change in channel conductance.⁴² It was hypothesized that a free sulfhydryl group on the Ca²⁺ release channel can be oxidized, leading to the opening of the channel, and that the coupling of this channel to the voltage-gated Ca²⁺ channel on the membrane is critically dependent on the oxidation of this sulfhydryl group.⁴³ These studies, as well as ours, confirm the idea that the structure and function of many proteins, including ion channels, are critically dependent on the oxidative state of the sulfhydryl groups.

Pathological Implications
Previous studies have documented an increase in intracellular Ca²⁺ in guinea pig cardiac myocytes by sulfhydryl oxidation due to the release of Ca²⁺ from sarcoplasmic reticulum.⁴⁴ From our observations, this increase in intracellular Ca²⁺ is not likely to be due to an increase in Ca²⁺ entry through L-type Ca²⁺ channels. It seems reasonable to speculate that the dependence of L-type Ca²⁺ channels on the redox state of the cellular environment may exert protective effects during cell injury in ischemia and reperfusion, since oxidative stress would favor a reduction in voltage-dependent Ca²⁺ entry.

	Acknowledgments

 
This study was supported by the National Institutes of Health (grant RO1 HL-52768 to Dr Marban), by the Heart and Stroke Foundation of Canada and the Medical Research Council of Canada (Research Fellowship to Dr Chiamvimonvat), and by the Howard Hughes Medical Institute (Postdoctoral Research Fellowship for Physicians to Dr Kamp).

Received May 25, 1994; accepted November 1, 1994.

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