ABSTRACT
Pharmacology and site-based mutagenesis probe function of ion
channels by altering specific sites and observing changes in channel
properties. Previous studies by Spires and Begenisich (1990) showed that
external diethylpyrocarbonate (DEPC), a chemical modifier of histidine,
irreversibly reduces and slows potassium current (ly) in squid giant axons.
Examination of the amino acid sequence of the SqKvlA channel thought to
correspond to the axonal delayed rectifier K' channel reveals a putative
external DEPC target at histidine 351 (H351) near the pore. To examine the
role of this residue in the DEPC effect, various Kvl channels were transiently
expressed in HEK 293 cells and tested for DEPC sensitivity. It was
hypothesized that this histidine is necessary for the DEPC effect.
Although it was found that K' channels with the proposed critical
residue exhibited a reduction in peak current after DEPC application, similar
effects were seen in clones lacking the histidine near the external mouth of
the pore. These cloned channels did have other external histidines, however
Additionally, DEPC slowed the activation kinetics of nearly all tested clones,
in some cases without a corresponding decrease in current amplitude. It was
concluded that while modification of histidine residues in Kvl channels
affects functional properties, all effects cannot be attributed to modification of
the residue equivalent to H351 in squid.
INTRODUCTION
Kyl potassium channels are a diverse group of membrane-spanning
polypeptides found in most eukaryotic cells (Jan and Jan 1992). These
channels are voltage-gated delayed rectifiers and play a critical role in
termination of the action potential. Therefore, changing the functional
properties of these channels has implications for altering nerve signaling.
Voltage-gated potassium channels in the Kvl family share similar
structural features (Jan and Jan 1992). Kvl’s are formed by four identical a-
subunits. A schematic representation of an a-subunit is shown in Figure 1.
The cytoplasmic N-terminal and C-terminal portions are hydrophilic, and the
middle of the polypeptide comprises the hydrophobic core region. The
emerging view of the channel from hydrophobicity analysis includes six
putative membrane spanning domains in the core region and a pore-loop (P-
loop) contained within the membrane. Amino acid identity within the Kvl
subfamily is highest within these domains, particularly in S4-S6 and the P-
loop.
The ability to use molecular approaches has rapidly advanced our
understanding of structure-function relationships of these channels, as
summarized by Jan and Jan (1992). For example, mutagenesis experiments
have revealed several functional domains in the channel protein. The
fourth membrane-spanning sequence, S4, is thought to be the intrinsic
voltage sensor of the molecule due to its unusual arrangement of positively
charged amino acids at every third residue. The P region, which is too short
to be membrane-spanning, forms the pore and selectively allows the passage
of K ions.
Pharmacological approaches combined with knowledge of molecular
structure have also revealed much about K' channel function. Mutating sites
suspected to mediate specific pharmacological effects has proven to be a
successful method. Using this approach, it has been found that scorpion toxin
binding is affected by mutation of residues at either end of the Shaker F
sequence, which implicates the external mouth of the pore as the site of toxir
binding (Mackinnon et al 1990). Site based mutagenesis and pharmacology
studies on Shaker channels have shown that position F425 (corresponding to
H351 in squid) is critical for charybdotoxin binding, and when mutated to
histidine, toxin block becomes pH-sensitive (Perez, 1996).
Chemical modification can be used as an alternative to mutagenesis, if
the modification is highly specific and irreversible. A study by Spires and
Begenisich (1990) examined the effects of histidine-specific reagents on
potassium channels in squid giant axons. It was found that external
application of 20-500 uM diethylpyrocarbonate (DEPC) slowed opening of
potassium channels with no effect on closing rates. Steady-state currents were
reduced in a voltage-dependent manner, but the shape of the instantaneous I¬
V curve did not change with DEPC treatment. The rate of action of DEPC was
consistent with a single class of histidine residues. Internally applied DEPC
(up to 2 mM) had no effect.
DEPC is the mostly widely used reagent for the specific modification of
histidine (Lundblad 1994). In the pH range of 5.5 to 7.5, DEPC reacts with
histidine residues. At a moderate excess of DEPC, there is a substitution at
one of the nitrogen positions on the imidazole ring of the histidine side
group (Figure 2). Treatment with neutral hydroxylamine is used to
regenerate histidine.
The results by Spires and Begenisich suggest the presence of a histidine
residue on the extracellular surface of squid axonal potassium channels that is
important for voltage-dependent gating and conductance properties of the
channel. SqkvlA is the putative delayed rectifier K' channel in the squid
giant axon (Rosenthal et al 1996). Examination of the SqKvlA amino acid
sequence shows that the only external histidine is at position 351 (Figure 3).
As mentioned earlier, this site has been shown to be crucial for mediating
pharmacological effects in related channels.
To determine the importance of H351 in the DEPC effect, various Kvl
channels were assayed for DEPC sensitivity, taking advantage of mammalian
clones varying with respect to the presence of histidine in the equivalent
position. It was postulated that there is a correlation between the presence of
the homologous H351 and DEPC sensitivity.
MATERIALS AND METHODS
Squid GFL Dissection
GFL cells were dissociated into primary culture from the posterior tip
of the giant fiber lobe (GFL) in the stellate ganglion of Loligo opalescens as
described in Mathes et al (1997). Briefly, the lobes were treated with protease
(type XIV; Sigma) and mechanically dissociated onto sterile concanavilin A¬
treated 35 mm tissue culture petri dishes. Cells were kept in filter-sterilized L¬
15 medium supplemented with salts to achieve an ionic composition similar
to that of sea water (additional 263 mmol NaCl, 4.6 mmol KCl, 4.25 mmol
Cacl2, 49.5 MgCl,) at 18°C. Cells were used within one week of isolation.
Mammalian Cell Transfection
Human embryonic kidney (HEK 293) cells were transfected with
various Kvl plasmids. Cultures were maintained in high glucose DMEM
(GIBCO/BRL) with 584 mg/L L-glutamine, 10% FBS (GIBCO/BRL) and 100
U/ml pen/strep. Cells were split with trypsin, suspended and plated in 35
mm petri dishes one day prior to transfection. Cells were « 10% confluent at
the time of transfection. Transfection was performed as follows: 7.55 ul of
2M CaCl, and 1 ug DNA per plate was added dropwise to 61 ul of 2X HEPES
(consisting of 273 mM NaCl, 1 mM Na,HPO,-7H,O and 55 mM HEPES). This
solution, made up to 120 ul with dH,O, was added dropwise to the dish of
cells after transfection. The medium was changed 18-24 hours after
transfection. Most recordings were taken 2 - 4 days after transfection.
Electrophysiology
Recordings from GFL neurons and 293 cells were performed with
standard whole-cell voltage clamp procedures at 12°C and 18°C respectively.
Holding potential was -80 mV in all experiments. Non voltage-dependent
currents were subtracted on line using a standard P/-4 technique. The
recording solutions were as follows (all concentrations in mM): 150 KGEL
internal: 20 KCl, 50 KF, 80 K-Glutamate, 10 Lysine-Hepes, 1 EGTA, 1 EDTA,
381 Glycine, 291 Sucrose, 4 Mg-ATP, 5 TMA-ÖH, pH 7.4. 20 KGFL external: 20
KCl, 480 Nacl, 20 MgCl2, 5 CaCl2, 10 Hepes, 10 MgSO4, pH 7.66, plus 300 nM
tetrodotoxin to block Na“ current. 293 internal: 90 KCl, 60 KF, 1 MgCl,, 10
Hepes, 10 EGTA, 23 KÖH, pH 7.0,320 mÖOSM. 293 external: 10 KCl, 140 NaCl, 5
MgCl,, 4 CaCl,, 10 Hepes, pH 7.2. External solutions contained DEPC where
noted. Solutions were changed with a flow through system, but constant
external perfusion was not employed.
RESULTS
Effects of DEPC on GFL Neurons
DEPC was applied to GFL neurons to confirm its effect on giant axon Iy
(Begenisich and Spires 1990). 0.5 mM DEPC applied externally to GFL cells
caused a reduction in amplitude of K' current (Figure 4A). The reduction in
peak Ig following DEPC application occurred for the most part within 5
minutes and reached a non-zero steady-state (Figure 4B). Äfter washout of
DEPC, there was no recovery. In addition, external DEPC altered activation
kinetics. DEPC slowed the opening rate of the channel, as indicated by the
increased half-time to peak Iy(Figure 4C)
DEPC on Mammalian Clones
To determine whether H351 is required for DEPC sensitivity,
mammalian clones varying in occurrence of histidine at the corresponding
residue were expressed in HEK 293 cells and assayed for sensitivity to 0.5 mM
DEPC. A comparison of results from these clones is given in Figure 5. Kvl.1,
Kvl.4 and Kv1.5, which have a histidine at this residue, show significant
sensitivity to DEPC comparable to that of GFL Ig, which is thought to be due to
SqkvlA channels (Rosenthal et al, 1996) (Figure 5A). In contrast, Kv1.2 and
ShakerB failed to exhibit a significant change in Ig amplitude after 5 minute
exposure to DEPC (Fig. 5B). In the case of ShakerB, activation kinetics
appeared to be significantly affected. Kvl.3 and Kv1.6, however, showed
DEPC sensitivity even though they also lack the equivalent of H351. The
time courses of development of the DEPC-effects on Iy amplitude and
activation kinetics observed in each of these experiments are shown in
Figures 6 and 7, respectively. In all cases, effects of DEPC were irreversible.
CONCLUSION
MULTIPLE RESIDUES MEDIATE DEPC EFFECI
The hypothesis that H351 residue of the SgKvlA channel is the only
critical position responsible for the DEPC effect was not supported by the data.
Nearly all channels exhibited a significant DEPC effect, regardless of the
presence of histidine at the external mouth of the pore. Clearly, histidine
modification by DEPC alters some critical aspect of Kvl channel function, and
the complex nature of these results strongly suggest that more than one
residue can mediate similar DEPC effects.
OTHER EXTERNAL HISTIDINES MAY BE INVOLVED
It is possible that in channels with histidine in the 351 position, this
residue mediates the bulk of the DEPC effect. In SqKvlA there is only one
external histidine, making this residue the likely target. However, this is not
true of all the other +H351 Kvl’s which were tested for DEPC sensitivity (see
Figure 8 for histidine maps of tested Kvl’s), and it is possible that
modification of these alternative histidines by DEPC alters channel properties.
Kvl.3 and Kv1.6 were the two channels lacking histidine near the
mouth of the pore, which exhibited DEPC sensitivity. Examination of their
respective amino acid sequences reveals possible external DEPC targets. Kvl.3
has a histidine residue in the mouth of the pore (Gen Bank). When the
channel is in the inactivated state, this residue is exposed to the external
environment and only then accessible to modification by external cysteine¬
reagents (Liu et al 1996), and possibly also to DEPC. Pulsing puts the channel
in the inactivated state more frequently, consequently exposing the target
residue more often. Further studies on Kv1.3 should probe a possible
correlation between the DEPC effect and pulsing frequency. The Kv1.6 amino
acid sequence reveals two putative DEPC targets on the extracellular side of
the membrane (Gen Bank). DEPC modification of these histidines may alter
channel properties. Neither Kv1.2 nor Shaker B showed a significant DEPC
effect on Ig amplitude; these channels’ singular external histidine is located
on the S1-S2 loop. The apparent kinetic alteration in Shaker B should be
considered tentative until this effect has been confirmed.
CONSIDERATIONS FOR FUTURE DEPC STUDIES
There are some concerns which must be addressed before concluding
with certainty that H351 is not the only residue responsible for the DEPC
effect. It is possible that not all of the observed results were due to specific
modification of histidine residues. Rarely, DEPC can react with other
nucleophilic residues in proteins other than histidine, including sulfhydryl,
arginyl and tyrosyl, as well as o- and e-amino groups. Although it is, in
principle, possible to track these side effects spectrophotometrically, or by
resistance to reversal by hydroxylamine (Lundblad 1994), these methods do
not always provide definitive answers. Thus, the possibility that part of the
observed effects were due to non-histidine modifications must be considered.
DEPC itself is a highly sensitive molecule which is easily degraded or
modified by environmental conditions. It has been shown that DEPC should
be selective for histidine residues in proteins at pH 6 (Ovadi 1969). Studies on
pH dependence of the rate of reaction of DEPC with the imidazole ring have
calculated a pK of 6.95, indicating that only the unprotonated imidazole is
reactive (Holbrook and Ingram 1973). Also, N-carbethoxyhistidyl residues are
most stable at pH 6. All experiments in this study were performed at pH 7.2.
Therefore it is important that DEPC studies on these Kvl channels are
repeated at pH 6 to be within the calculated range of the unprotonated
imidazole.
DEPC has been studied over a wide range of concentrations, from 0.01
mM to 40 mM. Studies show that excess DEPC can undergo a second reaction
with histidyl residues which is not reversed by hydroxylamine. This leads to
the conclusion that use of excess DEPC may lead to imprecise quantitation
and hydroxylamine-resistant modification (Miles 1977). For this reason, it is
important that the lowest possible concentration of DEPC is used in
modification experiments. Concentration-dependence studies should be
done for each clone.
Site-specific mutagenesis studies of subtilisin have shown the
influence of neighboring charged groups on histidine ionization, and thus on
DEPC reactivity as well (Bycroft and Fersht 1988). This is another factor to
consider in designing future DEPC experiments on Kvl channels.
Additionally, future studies should test the effect of DEPC on the
cloned squid channel (SqKvlA) in a suitable heterological expression system
to confirm that SqkvlA is the delayed rectifier channel in the squid giant
axon.
What has been presented serves as a set of preliminary data which
inspire numerous further questions to be probed. Clearly histidine
modification leads to interesting changes in channel properties, but it seems
that the modification reactions are complex and involve multiple target sites.
It is hard to conclude with certainty that DEPC acts on particular residues
without addressing many concerns with regards to DEPC modification itself,
including the pH range for imidazole protonation, DEPC modification of
non-histidine residues, and concentration dependence of DEPC modification.
10
ACKNOWLEDGEMENTS
1 would like to thank the Gilly lab for tolerating my invasion of lab
space, not to mention my countless questions and mistakes. In particular.
thank you to Taylor Liu for getting squid and giving me tons of advice and
help. Special thanks to Mat Brock for having the patience to teach me voltage
clamping and basically everything else, from Igor Pro to tremolo. To my
advisor, Dr. Gilly, I am unbelievably grateful for the encouragement
throughout the quarter, and for the words of wisdom - " the angler must
entice, not command the reward" - l'm getting there!
FIGURE LEGENDS
Figure 1: Schematic representation of a voltage gated K' channel. The
hydrophobic core region has six stretches of mostly hydrophobic residues (S1.
82, S3, S4, P, S6) which span the membrane. The S4 sequence is thought to be
the intrinsic voltage sensor of the channel due its unusual arrangement of
basic residues at every third position. The P sequence, which contains about
20 amino acids, has been identified as forming part of the pore region.
Figure 2: Diethylpyrocarbonate reacts with histidyl residues in to yield an N-
carbethoxy-histidyl derivative. The figure shows the modification of
histidine by DEPC.
Figure 3: The general structure of the squid K channel (SqKvlA).
Approximate locations of histidine residues are shown with open circles.
11
Figure 4: The effect of DEPC on GFL neurons. Application of DEPC decreased
peak K' current over time. In (B) and (C), the bars indicate the time over
which 0.5 mM DEPC was applied. Washout of DEPC did not reverse the
decrease in current. (C) shows the effect of DEPC on channel opening rates.
There was an increase in time to half Lap or a slowing in the rate of channel
opening, and this effect was also irreversible. The apparent temporary
recovery immediately following the solution change is due to the elevated
temperature of the solution being applied which takes several minutes to be
cooled to 18°C in the recording chamber.
Figure 5: The effect of DEPC on Kvl’s, shown at different time points during
application. (A) represents channels with a histidine equivalent to H351. (B)
represents channels lacking this specific (see also Figure 8). DEPC reduced
steady-state Ig in nearly all clones. Kvl.2 and ShakerB were not nearly as
sensitive to DEPC as other channel types.
Figure 6: The effect of DEPC on amplitude of Iy in various Kvl’s over time.
DEPC reduced steady-state Ig over time in all clones except Kv1.2 and
ShakerB. In the channels which were DEPC-sensitive, there was no return to
pre-DEPC Ig levels with washout. Bar indicates presence of DEPC.
Figure 7: The effect of DEPC on activation kinetics of Kvl’s over time. In all
channels with DEPC-sensitivity described in Figure 6, as well as ShakerB,
there was a slowing of channel opening rates. Bar represents presence of
DEPC.
12
Figure 8: Some Kvl channel schematics with approximate locations of
histidine residues indicated by red circles.
REFERENCES
Bycroft, M. and Fersht, A.R., Assignment of histidine resonances in the 'H
NMR (500 MHz) spectrum of subtilisin BPN' using site-directed
mutagenesis, Biochemistry, 1527, 7390, 1988.
Chandy, K.G. 1991. Simplified gene nomenclature. Nature. 352.
Hille, B. 1992. lonic Channels of Excitable Membranes. 2nd Ed. Sinauer,
Sunderland, MA. 607P.
http:www2.ncbi.nlm.nih.gov/cgi-bin/genbank. National Center for
Biotechnology Information. Gen Bank.
Holbrook, J.J. and Ingram, V.A. 1973. lonic properties of an essential histidine
residue in pig heart lactate dehydrogenase.Biochem. J. 131: 729.
Jan, L. Y., and Y.N. Jan. 1992. Structural elements involved in specific K+
channel functions. Ann. Rev. Physiol. 54: 537-55
Liu, Y., Jurman, M.E., and Yellen, G. 1995. Dynamic rearrangement of the
13
outer mouth of a K channel during gating. Neuron 16: 859-867.
Lundblad, Roger. 1994. Techniques in Protein Modification. Boca Raton,
Florida: CRC Press.
Mackinnon, R., Heginbotham, L., Abramson, T. 1990. Mapping the receptor
site for charybdotoxin, a pore-blocking potassium channel inhibitor.
Neuron 5:767-71.
Mathes, C. et al. 1997. Fast Inactivation of Delayed Rectifier K Conductance in
Squid Giant Axon and Its Cell Bodies. J. Gen. Physiol. 109:1-14
Miles, E. W. 1977. Modification of histidyl residues in proteins by
diethylpyrocarbonate. Methods in Enzymology. 47:431 - 443.
Miller, C. 1995. The Charybdotoxin Family of K+ Channel-Blocking Peptides.
Neuron. 15: 1-10.
Naranjo, D., and Miller, C. 1996. A strongly interacting pair of residues on
the contact surface of charybdotoxin and a Shaker K+ Channel.
Neuron 16, 23-130.
Ovadi, J. and Keleti, T. 1969. The effect of diethylpyrocarbonate on the
14
conformation and enzymatic activity of d-glyceraldehyde-3-phosphate
dehydrogenase.Biochim. Biophys. Acta 4: 365.
Paulmichl, M. et al. 1991. Cloning and expression of a rat cardiac delayed
rectifier potassium channel. Proc. Natl. Acad. Sci. USA. 88: 7892-7895.
Perez, P., 1996. C-type inactivation in mutant Shaker potassium channels.
PhD thesis. University of Rochester.
Rogawski. 1993. Tityustoxin-K-alpha, a structurally novel and highly potent K
channel peptide toxin, interacts with the a-dendrotoxin binding site
on the cloned Kvl.2 K channel. Mol. Pharm. 44: 430-436.
Rosenthal, J.J.C., et al. 1996. Molecular Identification of SqKvlA A Candidate
for the Delayed Rectifier K Channel in Squid Giant Axon. J. Gen.
Physiol. 108: 207-219.
Spires, S., and T. Begenisich (1990). Modification of potassium channel
kinetics by histidine-specific reagents. J. Gen. Physiol. 96, 757-775.
Stocker, M. et al. Swapping of functional domains in voltage-gated K+
channels. 1991. Proc. Royal Soc. Lond. B. 245: 101-107.
15
Stuhmer, W. et al (1989). Molecular basis of functional diversity of voltage-
gated potassium channels in mammalian brain. The EMBO J. 8(11),
3235-3244.
5
2


O
E
DNIA
HIOZVGIMI



—




2



E


O(
0
)
(C
C
FIGURE 4
Confirming the Effect of DEPC
on GFL Cells
before DEPC

5 min in DEPC
20 min in DEPC
+--------- -- --- -- -----1
—
5 ms
0.5 mM DEPC

1.0-
0.8-

0.6 -
0.4-
0.2-
0.0-
1000 2000 3000
time (s)
0.5 mM DEPC
2.5-
2.0-
15
1.0-
0.5—
0.0 +
1000 2000 3000
time (s)
Figure 5
The Effect of DEPC on Kyl's
A: +His351
B: -His351
GFL (SqKVIA)
before
Kv1.2
before
DEPC
5 min
in DEPC
J---------------

—
5 ms
Kvl.3
before
Kvl.1 before

20



Kvl.4
Kv1.6
before
before

2o

P
ShakerB
Kv1.5
before
-before

2
time (ms)
Figure 6
Effect of 0.5 mM DEPC on Kvl's
-H351
+H351
—
12
1.0

1.0-
0.8 -
0.8 -
e
0.6 -
0.6 -
0.4—
0.4 -
0.2-
0.2-
Kv1.2
GF
0.0+
0.0 +
1000 2000 3000
0 500 1000 1500 2000 2500
—
*..
1.0+ .
10... . ...
..
0.8 -
0.8 -
0.6-
0.6 -
3 0.4-
0.4 -

0.2-
0.2
KV1.1
KV1.3
0.0-
0.0-
500 1000 1500 2000 2500
500 1000 1500 2000
1.2-
10

0.8-
0.8 —
2
0.6 -
0.6-
0.4 -
a*
0.4-
0.2
2
KV1.4
0.2
KV1.6
0.0-
0.0-1
1000 2000 3000
1000 1500 2000 2500 3000
1.0
1.0...
... ......

0.8-
0.8 -
.
0.6-
0.6 -
0.4 -
0.4 -
0.2-
0.2:
Kv1.5
ShakerB
0.0—
0.0-
500 1000 1500 2000 2500
800 1000 1200 1400 1600 1800
time (s)
FIGURE 7
The Effect of DEPC on Kinetics of Kyl's
0.5 mM DEPC
• .900
2.5-
1.2-
2.0-
0.8-
15
0.4-
1.0-
Kv1.2
0.5-
0.0 -
0.0+
400 800 1200 1600
1000 200 3000

6-
30-
0°
20
. ..
3 -
1.0-
Kvi.1
Kvl.3
00-
1000 2000
500 1000 1500 2000
.
8-
5-
2
. *
4
3
....
44.
Kv1.6
Kv1.4
1000 2000 3000
1000 2000 3000
6-
30
20

1.0-
ShakerB
Kvl.5
0.0-
500 1000 1500
2000
1000
time (s)
Flguke 6

Histidine Maps of Kvl's
SqKvIA
Kv1.2

S

p;

KvI.1
Kv1.3





C
KV1.4
KV1.6





D

ShakerB
KV1.5
S



8