From the Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104
The effects of sulfhydryl reduction/oxidation on the gating of large-conductance, Ca2+-activated K+
(maxi-K) channels were examined in excised patches from tracheal myocytes. Channel activity was modified by
sulfhydryl redox agents applied to the cytosolic surface, but not the extracellular surface, of membrane patches.
Sulfhydryl reducing agents dithiothreitol, -mercaptoethanol, and GSH augmented, whereas sulfhydryl oxidizing agents diamide, thimerosal, and 2,2
-dithiodipyridine inhibited, channel activity in a concentration-dependent
manner. Channel stimulation by reduction and inhibition by oxidation persisted following washout of the compounds, but the effects of reduction were reversed by subsequent oxidation, and vice versa. The thiol-specific reagents N-ethylmaleimide and (2-aminoethyl)methanethiosulfonate inhibited channel activity and prevented the
effect of subsequent sulfhydryl oxidation. Measurements of macroscopic currents in inside-out patches indicate
that reduction only shifted the voltage/nPo relationship without an effect on the maximum conductance of the patch, suggesting that the increase in nPo following reduction did not result from recruitment of more functional
channels but rather from changes of channel gating. We conclude that redox modulation of cysteine thiol groups,
which probably involves thiol/disulfide exchange, alters maxi-K channel gating, and that this modulation likely affects channel activity under physiological conditions.
Alterations in the redox state of cysteine residues constitute an important mechanism for the regulation of
cellular functions. The thiol group of cysteine residues
is the most reactive of any amino acid side chain, existing as free thiols, or, in the presence of appropriate
electron acceptors, disulfides formed between vicinal thiols (Creighton, 1984, 1993
). Thiol/disulfide redox
state exchanges are generally reversible (Gilbert, 1995
),
their proportions varying in response to changes in cellular redox potential, which in turn affects the biological activities of enzymes, receptors, transporters, and
transcription factors (Gilbert, 1990
).
Redox modification of cysteine sulfhydryl groups
may also be an important mechanism of controlling
ion channel function. Redox agents alter the function
of several channels including sarcoplasmic reticulum
Ca2+-release channels in skeletal muscle (Zaidi et al.,
1989), N-methyl-D-aspartate receptor channels in the
brain (Sucher and Lipton, 1991
), voltage-dependent (Ruppersberg et al., 1991
), ATP-regulated K+ channels
(Islam et al., 1993
), and nonselective cation channels in guinea pig ventricular myocytes (Jabr and Cole,
1995
) and in yeast Saccharomyces cerevisiae vacuolar
membranes (Bertl and Slayman, 1990
). These observations are characterized by the opposite actions of sulfhydryl reducing and oxidizing agents on channel function and the reciprocal reversal of their effects. In the
case of Kv1.4 potassium channels, fast inactivation of
the channel is dependent on the reduced redox status
of a cysteine residue in the ball-domain (Ruppersberg
et al., 1991
).
Large conductance, Ca2+-activated K+ (maxi-K)1 channels are present in a wide variety of cell types. In smooth
muscle cells, maxi-K channels are important determinants of vasomotor tone (Brayden and Nelson, 1992)
and of the cellular responses to hormones and neurotransmitters (Cole et al., 1989
; Kume et al., 1989
;
Toro et al., 1990
; Kume and Kotlikoff, 1991
; Anwer et
al., 1992
; Kume et al., 1994
). In experiments examining
the modulatory actions of protein kinases, protein
phosphatases, and G protein subunits, we observed a
marked stimulation of maxi-K channel activity by control
buffer solutions prepared to mimic those used to suspend the proteins. By a process of elimination, the active
components of these buffers were identified as
-mercaptoethanol (
-ME) and dithiothreitol (DTT), both
of which are reducing compounds commonly used to
prevent oxidation of protein sulfhydryl groups. We hypothesized that the activity of maxi-K channels is regulated by the redox state of critical sulfhydryl groups in
the channel protein or an associated regulatory protein, involving exchanges between free thiols and disulfides. In the present study, we examined the effects of
several types of sulfhydryl-modifying agents on maxi-K
channel activity in isolated membrane patches from
tracheal smooth muscle cells. We demonstrate that
channel activity is markedly affected by alterations in
cytosolic redox potential; channel activity is augmented
in reducing, and inhibited in oxidizing, conditions,
and the action of oxidizing agents is eliminated following alkylation of the sulfhydryl side chain. The mechanism of channel modulation appears to be an effect on
channel gating since the number of functional channels does not change after reduction.
Cell Dissociation
Smooth muscle cells were dissociated from tracheas obtained from horses killed by intravenous injection of pentobarbital sodium. The horses were killed for teaching purposes and euthanasia procedures were in accordance with the guidelines set by the Institutional Animal Care and Use Committee of the University of Pennsylvania. After dissecting away connective tissue on the adventitial side, a piece of trachealis (1.5 × 1.5 cm) was cut and cannulated with an 18-gauge needle between the mucosa and muscle layers. The tissue was tied on the needle, suspended in a warmed, jacketed chamber, and perfused with dissociation solution containing 1,750 U collagenase D (Boehringer Mannheim Corp., Indianapolis, IN), 25 U elastase (Worthington Biochemical Corp., Freehold, NJ), and 5 mg trypsin inhibitor (Sigma Chemical Co., St. Louis, MO) in 5 ml of medium M199. After 10-20 min perfusion, the tissue was transferred to a petri dish, and the softened muscle was dissociated by trituration, filtered through a nylon mesh, and centrifuged at 5°C and 1,000 rpm for 5 min.
Patch-clamp
Maxi-K channel activity was measured in inside-out and outside-out patches as previously described (Kume and Kotlikoff, 1991). Gigaohm seals were obtained using heat-polished borosilicate
glass pipettes with a resistance of 3-7 M
. For inside-out patches, the bath solution was (in mM): KCl 135.0, MgCl2 2.0, CaCl2 1.8 or
2.2, EGTA 3.0, HEPES 10.0, pH adjusted to 7.2 with KOH solution. Free Ca2+ in this solution was either 250 nM (CaCl2 = 1.8 mM) or 500 nM (CaCl2 = 2.2 mM) (Fabiato, 1988
). The pipette
solution contained (in mM): NaCl 136.0, KCl 5.0, CaCl2 1.8, MgCl2 1.0, HEPES 10.0, with pH adjusted to 7.4 with NaOH solution. Solutions were reversed for outside-out patches. Patches
were examined at a holding potential of 0-40 mV, depending on
the level of control channel activity. After a patch was obtained,
an equilibration period of ~5 min was allowed; patches that
showed large fluctuations in channel activity over this period
were discarded. Thereafter, channel activity was recorded continuously until the end of the experiment. Drug actions were observed for 5-10 min after addition to the bath solution either by
pipette (at 1% of the bath volume) or by perfusion (in a volume
of at least 10 times the bath volume). Unitary currents were amplified (EPC-7; List-Medical-Electronic, Darmstadt, Germany),
displayed on a computer (pClamp software; Axon Instruments,
Foster City, CA) and simultaneously stored on a modified Sony
Digital Audio Tape Deck (DC-700). The stored data were later re-digitized using Axotape or pClamp software (Axon Instruments)
at a sampling rate of 2.5 and 5 kHz after filtration (
3dB, 8-pole
lowpass Bessel filter; Frequency Devices, Inc., Haverhill, MA) at 1 and 2 kHz, respectively.
Reagents
DTT, -ME, thimerosal, diamide, 2, 2
-dithiodipyridine (DTDP),
and N-ethylmaleimide (NEM) were purchased from Sigma
Chemical Co. Reduced glutathione (GSH) was purchased from
Calbiochem-Novabiochem Corp. (La Jolla, California). (2-Aminoethyl)methanethiosulfonate hydrochloride (MTSEA) was purchased from Toronto Research Chemicals Inc. (North York, Ontario, Canada). Except for DTDP, all the agents were dissolved in
bath solution. DTDP was first dissolved in ethanol to a concentration of 500 mM, and then diluted with bath solution to the final
concentration of 50 µM. None of the agents had an effect on the
pH of the bath solution at the applied concentration ranges. The
effect of redox agents on free calcium in the solutions used was
determined using fura-2. DTT,
-ME, DTDP, NEM, thimerosal, and MTSEA had no effect on free calcium at the highest concentrations used. The fluorescence of GSH and diamide prevented a
similar determination.
Statistics
SigmaStat for Windows (version 1.0; Jandel Corp., San Rafael, CA) was used for statistical analyses. Significance was determined by either paired t tests or one way analysis of variance (ANOVA) for repeated measures, depending on the number of treatments that the patch received. When a significant effect was detected with ANOVA, Student-Newman-Keuls test was used for pair-wise comparisons. P < 0.05 was considered statistically significant. All results are expressed as mean ± SE.
Effects of Sulfhydryl Redox Agents on Maxi-K Channel Activity
Channel activity in inside-out patches was concentration-dependently augmented by exposure of the cyto-solic patch surface to the sulfhydryl reducing agents DTT
(10 µM to 1 mM, in logarithmic increments; Fig. 1), -ME
(10 µM to 10 mM, in logarithmic increments; Fig. 2),
and GSH (50 or 170 µM; see Figs. 6 and 8). At the highest concentrations applied, open-state probability (nPo)
increased 7.7-fold after addition of DTT (0.028 ± 0.009 to 0.216 ± 0.062, n = 9), 4.3-fold after
-ME (0.029 ± 0.014 to 0.125 ± 0.028, n = 6), and 8.4-fold after GSH
(0.049 ± 0.016 to 0.406 ± 0.126, n = 6). An augmenting effect was generally observed within 1 min of drug
application, followed by a continued increase in channel activity during the next 2 or 3 min; thereafter channel activity remained constant. The time course of a typical experiment in which DTT augmented channel activity is shown in Fig. 3; the stimulatory effect of DTT
was not reversed by perfusing the bath for 5 min with
10-20 times the bath volume.
Conversely, application of sulfhydryl oxidizing agents
to the patch membrane cytosolic surface reduced channel open-state probability. As shown in Fig. 4, concentrations as low as 5 µM diamide significantly inhibited
channel activity, and nPo was only 10% of the control
level at 5 mM of diamide (0.391 ± 0.085 to 0.040 ± 0.011, n = 7). Channel activity was similarly inhibited by DTDP; at a concentration of 50 µM, DTDP significantly reduced nPo from 0.383 ± 0.062 to 0.171 ± 0.033 (n = 7) (Fig. 5). To eliminate possible solvent effects, the same concentration (0.01%) of ethanol was
included in the bath solution during the control period, and DTDP was applied by perfusing at least 10 times the bath volume.
To confirm that the change in channel activity following a redox agent resulted from redox state modification, we tested whether alteration of channel activity
produced by sulfhydryl reduction could be reversed by
subsequent oxidation, and vice versa. As shown in Fig.
6, GSH (50 µM) markedly augmented maxi-K channel activity, which was reversed by diamide (500 µM). In
five experiments, nPo increased 4.5-fold after GSH
(0.022 ± 0.002 to 0.100 ± 0.020), and was then reduced to 8% of the stimulated value (0.100 ± 0.020 to
0.008 ± 0.002) following diamide. Likewise, inhibition
of channel activity following sulfhydryl oxidation was
reversed by sulfhydryl reduction. As shown on the left
panel of Fig. 7, the oxidizing agent thimerosal (10 µM)
applied to the cytosolic surface of inside-out patches reduced channel nPo, which persisted after washout but was reversed by DTT (1 mM). Three identically designed experiments using diamide instead of thimerosal produced similar results; nPo dropped from 0.342 ± 0.086 to 0.093 ± 0.024 following diamide (5 mM), and
remained at the low level after washing for 5 min (0.091 ± 0.037). Two of the three patches were exposed to DTT (1 mM) following diamide. In these
patches nPo increased by 14.4-fold and 21.1-fold, respectively. The new levels of nPo were 4.6 and 2.7 times
of the initial control values, respectively. The opposite actions of sulfhydryl reducing and oxidizing agents,
and the reciprocal reversal of their effects, suggest that
the activity of maxi-K channels is dependent on the redox status of one or more sulfhydryl groups on the
channel protein, or an associated regulatory protein.
Redox Modulation Occurs at the Cytosolic Patch Surface
To determine whether the sulfhydryl group(s) involved
in the response to redox agents is located on the intra-cellular or extracellular side of the membrane, we compared the effects of reducing and oxidizing agents applied to the bath solution in inside-out and outside-out
patches. As shown in Fig. 7, thimerosal markedly inhibited channel activity when added to the bath solution of inside-out, but not outside-out, patches. Immediately
after application of thimerosal to the cytosolic surface
of inside-out patches, channel activity was markedly inhibited. In the experiment shown, nPo dropped to
about 10% of the control level within the first minute
after thimerosal application; the effect was not reversed
by washout of the oxidizing agent, but subsequent addition of DTT rapidly reversed the effect of the oxidizing
agent. By contrast, when thimerosal was applied to six
outside-out patches, nPo tended to decline slowly and
the effect was not significant after 8 min (Fig. 7 C). The
slow decline in channel activity likely reflects some
thimerosal permeation. Similarly, the relatively membrane-impermeant reducing agent GSH (DiPaola et al.,
1989) also was shown to be effective only in inside-out
patches. As shown in Fig. 8, GSH (170 µM) increased
the nPo of maxi-K channels by more than eightfold (n = 6) when applied to inside-out patches (n = 6) but had
no effect in outside-out patches (n = 4). The effect of
GSH on inside-out patches could be observed within
30 s in continuous traces. The configuration dependence and time course of the action of these relatively
impermeant oxidizing and reducing agents on channel
activity suggest that the cysteine residue(s) responsible
for redox regulation is located on the cytoplasmic aspect of the cell membrane.
Modification of Sulfhydryl Groups by NEM and MTSEA Inhibits Maxi-K Channel Activity and Prevents the Effect of Patch Oxidation
We reasoned that the action of redox agents on maxi-K
channel activity was due to the modification of the sulfhydryl group of cysteine residues, and the formation or
breakdown of one or more disulfide bonds. To confirm
that the modification of reactive thiols underlies the
observed effect of redox agents, we examined the response of maxi-K channels to oxidizing agents in NEM-treated patches. NEM alkylates free sulfhydryl groups
(Creighton, 1993). If free thiols are involved in the responses to oxidizing agents, covalent modification of
the free thiols by NEM should prevent disulfide bond
formation, and attendant alterations in channel activity, following exposure to oxidizing agents. As shown in Fig. 9 A, addition of NEM (1 mM) to the bath solution
rapidly inhibited maxi-K channel opening in inside-out
patches and prevented the subsequent inhibition of
channel activity upon exposure to diamide (0.5 mM).
The concentration of diamide applied was 100-fold greater than that required to significantly inhibit channel activity in nonalkylated patches (compare Fig. 4).
The results of six similar experiments are summarized
in Fig. 9 B.
We also examined the effect of MTSEA on channel
activity. MTSEA is a thiol-specific reagent that covalently modifies free thiol groups on cysteine residues
(Akabas et al., 1992; Stauffer and Karlin, 1994
). As
shown in Fig. 10, MTSEA (2.5 mM) itself markedly inhibited channel activity, and this effect was not reversed by washout. Moreover, MTSEA eliminated the
inhibitory action of thimerosal (10 µM). In the experiment shown, channel activity was increased by stepping
to positive voltages, so that inhibitory effects would not
be obscured by the low nPo following MTSEA. In MTSEA-treated patches (n = 3), subsequent exposure to thi-merosal had no effect on channel activity. Experiments
were also performed to determine whether MTSEA and
NEM, both covalent modifiers of cysteine thiol groups,
were functionally competitive. After channel inhibition by MTSEA, exposure to NEM did not further inhibit
channel activity. In four patches, nPo was 0.0105 ± 0.0041 before and 0.0113 ± 0.0078 after NEM (data
not shown). These data indicate that the inhibitory action of oxidizing agents on maxi-K channels requires
the presence of reactive sulfhydryl groups. Moreover,
the inhibition of maxi-K channel activity by NEM and
MTSEA provides further evidence of the relationship
between the state of critical cysteine sulfhydryl group(s)
and channel activity. Taken together, these results provide further evidence that the modulation of maxi-K
channel activity by redox reagents results from a chemical modification of sulfhydryl groups.
Mechanism of Channel Modulation by Sulfhydryl Redox Agents
The increase in maxi-K channel nPo after sulfhydryl reduction could occur either by the recruitment of maxi-K
channels that are unavailable for K+ conductance in
the oxidized state (increased n), or by an increase in
the open probability (Po) of available channels. To determine if an increase in available maxi-K channel
number is involved in the augmentation of channel activity after the reduction of cytosolic sulfhydryl groups,
the effect of -ME on macroscopic currents was examined in inside-out patches. The contribution of delayed
rectifier potassium channels to the macroscopic currents was minimized by holding patches at
20 mV and
by including 4-aminopyridine (5 mM) in the pipette solution (Boyle et al., 1992
). Macroscopic currents from
inside-out patches were averaged from voltage-clamp steps to 130 mV to determine whether the maximum
conductance of individual patches increased after sulfhydryl reduction. An example of such an experiment
and the normalized conductance for six patches before
and after reduction is shown in Fig. 11.
-ME shifted
the conductance-voltage relationship of the evoked
currents by
17.7 mV, without altering either the maximum conductance or the slope values (14.8 and 16.6 for control and
-ME, respectively) of the Boltzmann
fits (n = 6). It is apparent that sulfhydryl reduction has
no influence on available channel numbers, and the
major response to it is a shift in the voltage-Po relationship resulting from changes in channel gating. Typical
traces illustrating single channel kinetics before and after treatment with thiol reagents are shown in Fig. 12.
The traces indicate that reduction increased open-state
probability primarily by decreasing the mean shut intervals between openings, and that the open-state duration was not substantially altered. Conversely, patch oxidation decreased nPo by increasing mean shut intervals (Fig. 12, bottom). Kinetic analysis indicated that
mean open times were not different after exposure to
thiol modifying agents, whereas the duration of shut intervals was decreased after sulfhydryl reduction (GSH)
and increased after sulfhydryl oxidation (thimerosal)
and alkylation (NEM) (data not shown). While the
presence of multiple channels in patches of smooth
muscle membranes complicates the kinetic analysis
(particularly the interpretation of closed times), these
results together with the findings illustrated in Fig. 11
suggest that reducing agents increase open-state probability by increasing the probability that a shut channel
will open, rather than altering the open-state dwell time.
Our results demonstrate that maxi-K channels in smooth muscle cells are regulated by agents that alter the redox state of sulfhydryl groups. We have shown that sulfhydryl reduction increases nPo of the channel whereas oxidation has the opposite effect. Maxi-K channels in patches pulled from smooth muscle cells appear to exist in a mixed redox state, since either reduction or oxidation markedly affected channel activity. This mixed redox state could occur either because some channels in the patch exist in the reduced state whereas others are in the oxidized state, or because each channel has more than one redox modulatory site existing in different redox states. Since membrane patches from airway myocytes always contain multiple channels, we could not differentiate between these two possibilities.
We compared the normalized conductance-voltage
curves constructed from macroscopic currents in response to step-depolarization during control and after
sulfhydryl reduction to determine whether reduction
would lead to an increase in the maximum conductance of the patch. The conductance-voltage curve was
shifted leftward following -ME, but there was no increase in the maximal conductance, suggesting that the
increase in nPo resulted from an increase in the open
probability of initially available channels (Fig. 11) and
that reduction does not lead to the opening of previously silent channels.
The thiol specificity of the redox agents was confirmed by experiments with NEM and MTSEA. NEM is
a thiol-alkylating agent, which is commonly used to trap
protein thiols in their existing redox states (Creighton,
1984; Gilbert, 1995
). Alkylation by NEM removes free
thiols and should therefore prevent the formation of
disulfides in response to sulfhydryl oxidizing agents. In
the present study, maxi-K channel activity was inhibited
after exposure to NEM. In addition, the inhibitory effect of diamide was abolished by NEM pretreatment, as
would be predicted for a thiol-specific action (Petronilli et al., 1994
). We did not test the effect of reducing
agents following NEM pretreatment because NEM alkylates free thiols but not disulfides, and, therefore, reducing agents may still work after NEM pretreatment
in channels existing in "mixed" redox states. We also
used MTSEA, which reacts specifically and rapidly with
thiols to form mixed disulfides (Akabas et al., 1992
;
Stauffer and Karlin, 1994
), to investigate the thiol-specific nature of redox modulation. As with NEM, exposure of inside-out patches to MTSEA inhibited maxi-K
channel activity and blocked the modulatory action of
oxidizing agents. After treatment of inside-out patches
with MTSEA, thimerosal no longer inhibited channel
activity (Fig. 10). Moreover, the inhibitory actions of MTSEA and NEM were mutually exclusive, in that following treatment with MTSEA, NEM no longer modulated channel activity. Taken together, these experiments strongly support the hypothesis that the inhibition of channel activity by oxidizing agents results from
reactions involving one or more protein thiol groups.
The mechanisms of action of the redox agents used
are well known. Reducing and oxidizing agents exert
their effects through two sequential thiol-disulfide exchanges with a mixed disulfide of the redox agent and
a protein cysteine residue as the intermediate (Creighton, 1993; Brocklehurst, 1979
; Kosower and Kosower,
1995
). The opposite actions of sulfhydryl reducing and
oxidizing agents and the reciprocal reversal of their actions on nPo are consistent with a mechanism of thiol/
disulfide exchange.
It is not clear how changes in redox state regulate
maxi-K channel activity. Since the activity of this channel is highly dependent on Ca2+, one possibility for the
change in nPo is that thiol/disulfide exchanges are associated with changes in protein conformation that influence channel Ca2+ binding affinity, which determines the rate constant for channel gating. Free protein thiols can exist either in the reduced state (-SH) or
as thiolate anions (-S), depending on pH and the pKa
of the thiol under consideration. One possibility is that
one or more sulfhydryl groups close to the Ca2+-binding region of the channel exists in the anion form, contributing to the Ca2+ binding affinity. The formation of
disulfides, or side chain modification by NEM or MTSEA,
would eliminate the negative charge and lower the
Ca2+ binding affinity. Although cysteine thiols have an
intrinsic pKa in the range of 9.0-9.5 (Creighton, 1993
),
thiols attached to protein molecules may deviate from
this typical value by many orders of magnitude, due to
electrostatic interactions within the protein (Gilbert,
1990
). For example, the pKa of one of the two thiols in
the active site of thioredoxin reductase has been estimated as ~7.0 while the pKa of the comparable active
site thiol of lipoamide dehydrogenase is <5.5 (Gilbert,
1990
).
Thiol/disulfide exchange involves covalent modifications, which occur only when appropriate electron acceptors (oxidizing agents) or donors (reducing agents) are present. We found that alterations of channel activity after redox modification were not reversed by wash-out of the redox agents, but were rapidly reversed by exposure to the counteracting reagents. The covalent nature of the modification likely underlies the fact that channel rundown is not commonly observed following patch excision, even though the reducing power of cytosolic solution is likely stronger than routine patch-clamp solutions.
We believe that the modulatory actions reported
here are likely to be of physiological relevance. The intracellular concentration of GSH ranges from 0.1 to 10 mM (Meister, 1995), and in the present study GSH augmented channel activity significantly at a concentration as low as 50 µM, and increased channel activity over
eightfold at 170 µM (Figs. 6 and 8), suggesting that
shifts in GSH concentration in the physiological range
are likely to alter maxi-K channel activity. In addition,
our study has important implications for patch-clamp
experiments examining the regulatory features of maxi-K channels. Reducing agents such as DTT and
-ME are
included in many protein preparations in order to protect free sulfhydryl groups. Our results predict that
stimulatory effects on maxi-K channels, associated with
the presence of the reducing agent, will be observed in
experiments utilizing common protein preparations such
as kinases and phosphatases, and will therefore tend to
confuse the interpretation of these experiments. This
complication can be particularly pernicious, since we
have observed that the augmenting effect of DTT on
maxi-K channels is removed by boiling the buffer solution. Therefore experiments using protein preparations
containing sulfhydryl reducing agents should be interpreted with caution, and boiling of the protein preparation alone is not adequate to rule out buffer actions.
In summary, we report the modulation of maxi-K
channel gating by alterations in the redox state of the
patch. The site of redox modulation is likely a cytosolic
cysteine residue or residues(s), since the poorly membrane-permeant reducing agent GSH and oxidizing agent thimerosal altered maxi-K channel activity only
when applied to the intracellular side of the patch
membrane. Alkylation of sulfhydryl groups ablated the
redox modulation. We have recently performed experiments on the recombinant -subunit of maxi-K channels expressed in Xenopus oocytes and shown that the
expressed channels are modulated by redox agents in a
similar manner. Experiments are currently underway to
locate the cysteine residue(s) responsible for the redox
modulation.
Original version received 21 February 1997 and accepted version received 14 April 1997.
Address correspondence to Dr. M.I. Kotlikoff, Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104-6046. Fax: 215-898-9923; E-mail: mik{at}vet.upenn.edu
The authors express their appreciation to Mrs. Kim Bowers and Ms. Laura Lynch for technical assistance, and to Dr. Owen B. McManus for his helpful comments on an earlier version of the manuscript.Supported by NIH grant HL41084 (M.I. Kotlikoff) and NRSA 1 F32HL09294 (Z.-W. Wang)