From the Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia 22908
Voltage-dependent channels respond to changes in the
transmembrane electric field by rearranging their voltage sensors, triggering increases in selective permeability. The channels reside in three types of conformationally distinct populations: closed states, open states, and
a variety of inactivated states. These populations are
closely interrelated and, while significant advances
have been made in understanding these relationships,
their quantitative and molecular description has occupied most of the waking hours of channel biophysicists
for the last five decades.
Initial studies of ion channel gating depended on the
ability of channels to conduct ions at rates of 106-107
ions/s, allowing for exquisitely sensitive ionic current
measurements both at the ensemble and single molecule levels (see Hille, 1992 Spectroscopic approaches, either global or local,
through reporter group techniques enable investigators (in principle) to sample most of the conformational space of a macromolecule. Application of this
idea to the study of voltage-dependent processes is not new. Hubbell and MacConnell (1968) first applied the
reporter group approach to the study of excitable
membranes by looking at the partition behavior of spin
labels incorporated into vagus nerves placed inside a
custom-made EPR resonator. The first global spectroscopic approach to the study of membrane excitation
was the measurement of changes in birefringence from
single axons (squid giant axons) or bundles of axons
(crab leg nerves) by Cohen and co-workers (Cohen et
al., 1968 Fast-forward 20-30 yr and introduce molecular cloning, heterologous expression systems, and site-directed
mutagenesis. By taking advantage of sulfhydryl chemistry to specifically attach fluorophores, and cleverly
timing channel membrane incorporation to minimize
background fluorescence levels, Manuzzu et al. (1996) made a seminal contribution to the measurement of
voltage-dependent conformational changes by providing direct physical evidence for voltage-dependent S4
movements in Shaker K+ channels. These movements
were detected as changes in fluorescence intensity of
probes attached at positions 356 and 359 in Shaker channels (at the extracellular surface and at the membrane-water interface of the S4 transmembrane segment), and were found to track the kinetics and voltage
dependence of gating current traces. This initial report
was followed and extended by the work of Cha and Bezanilla (1997) The key challenge of VCF, however, is not the detection of fluorescence changes after switching the transmembrane voltage, but the identification or correlation of these changes with specific steps along the activation/inactivation path of a channel. Two articles
appearing in this issue of The Journal (Cha and Bezanilla, 1998 Cha and Bezanilla (1998) Loots and Isacoff (1998) A structural perspective of the VCF studies in Shaker
K+ channels based on the results of both articles is
shown in Fig. 1. The positions of residue
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References
, for references). But, by definition, ionic currents give little or no information on
the nonconductive steps of the activation pathway, thus
limiting these studies to the transitions to and from the
open state. The first direct evidence of voltage-gated conformational changes, gating current measurements
(Armstrong and Bezanilla, 1973
; Keynes and Rojas,
1974
), opened a rich source of information on the multiple nonconductive states that channels populate before the final opening transition. The catch is, however, that gating currents can only monitor channel conformational changes involving charge movements or dipole rearrangements; no information is obtained from
electrically silent protein rearrangements, no matter
how massive they might be.
, 1971
). These experiments were later superseded by measurements of membrane birefringence
under voltage clamp by Landowne (1985)
, who observed voltage-dependent rearrangement of peptide
bonds correlating with Na+ channel gating current kinetics. Although exciting, these pioneering studies
were intrinsically limited by their inability to (a) study and target a specific molecular entity, and (b) to probe
a defined site within a given molecule.
, who looked at similar residues in S4 and detected additional fluorescence changes from positions at the extracellular end of S2 (residue T276) and
the pore (residues F425 and T449). Cha and Bezanilla
(1997)
also probed systematically the origins of the fluorescence changes, suggesting that these, in most cases,
derive from collisional quenching from other parts of
the channel. Isacoff and co-workers later coined the
term voltage-clamp fluorimetry (VCF) to define the simultaneous measurement of fluorescence from specifically attached probes and ionic or gating currents in
voltage-clamped cells (Xenopus oocytes, so far).
; Loots and Isacoff, 1998
) rise to the challenge and give additional insight into the physical and
functional relations between areas of the extracellular
face of Shaker K+ channels involved in activation and
slow inactivation. Although the driving force behind
each of the articles is different, they both show compelling evidence for a physical interaction between the
pore of the channel and its voltage sensor (S4 segment) during gating-related events. To this end, both
groups cleverly use the nonconducting mutant W434F
(Perozo et al., 1993
), which very handily eliminates ion
conduction but leaves gating currents intact, allowing a
direct comparison between fluorescence changes and
charge movements.
made the surprising discovery that manipulations affecting ion flow also have remote effects on S4 fluorescence changes. By comparing
voltage-evoked fluorescence changes in the presence
and absence of specific K+ channel blockers (TEA or
Agitoxin II), or by directly mutating the pore maintaining the W434F mutation, they detected a component of
the fluorescence signal that disappears in the absence
of ionic flow. A systematic analysis of this component
revealed that, although its voltage dependence was essentially identical to that of the conductance vs. voltage
curve, its kinetics were much slower than those of
equivalent ionic currents, as if presaging an entry into the slow inactivated state. The work by Cha and Bezanilla (1998)
is also significant from a purely technical
standpoint. Changes in their previous experimental set-up, including vastly different optics, produced a large
increase in the signal-to-noise ratio (10×), allowing for
routine spectral analysis of the fluorescence signals
and, for the first time, in situ membrane protein fluorescence anisotropy measurements.
also detected this pore-S4
interplay, taking it a step further by thoroughly dissecting the slow inactivation process through the study of
fluorescence signals during extended depolarizations.
They find that changes in fluorescence at either the
NH2-terminal end of the S4 segments (residue A359) or in the pore region (residue A424) closely follow the
development and recovery of slow inactivation. These
long depolarizations produce no further changes in
fluorescence at position 424, but affect the recovery
from the inactivated state. At position 359, a small but
measurable decrease in fluorescence intensity was observed. Based on these and other findings, Loots and
Isacoff (1998)
suggest that the development of the slow
inactivated state involve two sequential rearrangements
affecting a single inactivation gate located in the external mouth of the pore. In the first rearrangement, the
inactivation gate closes within a few seconds, abolishing ion conduction. This state is reminiscent of the "P-inactivation" described by De Biasi et al. (1993)
. In the second step, the inactivation gate is stabilized by its interaction with the S4 segment, thus generating a shift in
the voltage dependence of the gating charge movement, which defines the "true" C-inactivated state. The
suggestion of a sequential relationship between entry
into the P- and C-inactivated states correlates well with
earlier findings by Olcese et al. (1997)
, who showed
that gating charge immobilization during slow inactivation is likely to develop through a sequential mechanism. The results of Loots and Isacoff (1998)
also support the notion put forward by Yang et al. (1997)
that
the W434F mutation locks the channel into an inactive
state equivalent to the P inactivation.
carbons
showing large voltage-dependent fluorescence changes
(spheres) are shown against the
carbon tracing (black
trace) from a model of the last two transmembrane segments of Shaker, constructed based on the crystal structure of the Streptomyces lividans K+ channel (Doyle et al.,
1998
). (Segments S2 and S4 added arbitrarily at the periphery of the channel core, gray traces.) Positions with
signals that correlate with channel activation are shown as light spheres: T276 in S2 (Cha and Bezanilla, 1997
),
M356 in S4 (Cha and Bezanilla, 1997
, 1998
; Loots and
Isacoff, 1998
; Mannuzzu et al., 1996
), and F425 in the
pore region (Cha and Bezanilla, 1997
). A position that
mostly tracks the slow inactivation process is shown as
black spheres: A424 in the pore region (Loots and Isacoff, 1998
). Finally, positions that respond to both activation and slow inactivation events are shown as dark
gray spheres: A359 in S4 (Cha and Bezanilla, 1997
,
1998
; Loots and Isacoff, 1998
; Mannuzzu et al., 1996
),
and T449 in the pore (Cha and Bezanilla, 1997
). In the
absence of direct structural information on the conformation of the S4 segment and the extracellularly located S3-S4 loop, it is impossible to establish the nature and extent of the physical connection between the
S4 segment and the pore responsible for the cross talk
evidenced by the fluorescence measurements. However, the residues in the pore (A424, F425, and T449)
are spatially clustered but nevertheless report differentially on activation, slow inactivation, or both, which implies the existence of very specific interactions between
the voltage sensor and the extracellular inactivation
gate.
View larger version (22K):
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Fig. 1.
Structural correlates of conformationally active positions during activation and slow inactivation mechanisms. The putative locations of Shaker K+ residues showing voltage-dependent
fluorescence changes were mapped on a structural model of the
Shaker K+ based on the crystal structure of the Streptomyces K+ channel. Shown are the traces for the core of the channel (black lines)
and the putative locations of the S2 and S4 transmembrane segments (gray traces). The labeled spheres correspond to the position
of the
carbons of residues able to follow activation conformational changes (light gray spheres), slow inactivation conformational
changes (black spheres), or both (dark gray spheres).
Although the results and conclusions in the two articles tend to agree on the broad strokes, there are a
number of discrepancies that need to be addressed in
future work. Key among them is the accessibility of
the residues along the S3-S4 loop that show strong changes in fluorescence upon depolarization. The Isacoff group favors the notion that the extracellular portions of S4 and the adjacent S3-S4 loop are somewhat
buried at rest but exposed during activation (Larsson
et al., 1996; Mannuzzu et al., 1996
; Baker et al., 1998
).
In contrast, the anisotropy measurements of Cha and
Bezanilla (1998)
indicate larger motional constraints at
the extracellular end of S4 (residues M356 and A359)
than in its putatively membrane embedded regions
(residue 363), with M356 becoming more constrained
and A359 less constrained upon depolarization. This led Cha and Bezanilla (1998)
to invoke the presence of
a protein vestibule at the extracellular end of the S4
segment, which would interact differentially with S4 as
the voltage sensor reorients with changes in the transmembrane electric field. D2O accessibility studies indicate that this vestibule is always water filled, regardless of the state of the channel, a suggestion that would
challenge earlier data by Manuzzu et al. (1996), who
hypothesized that the S4 rearrangements were accompanied by changes in the hydrophobicity of S4's microenvironment.
Currently, there are few drawbacks in the use of VCF
to dissect voltage-dependent events on ion channels.
The main limitation of VCF lies in its inability to study
fluorescence changes from the intracellular portions of
channels. This is because, so far, it has been impossible to label intracellularly exposed cysteine-containing channels without also labeling the large number of
intrinsic cysteines in an oocyte. A plausible alternative
could be the selective incorporation of unnatural
amino acids, as introduced by Noren et al. (1989) and
Mendel et al. (1992)
, and recently adapted to the oocyte expression system by Nowak et al. (1995)
. Also,
there are concerns regarding the size of the maleimide
tether (7-8 Å): that the fluorophores are relatively
large and the fluorescence signal from these reporter molecules is intrinsically delocalized. Consequently,
the position of the fluorophore may not correlate spatially with the putative position of the residue, imposing clear restrictions on carefree interpretations of the
fluorescence changes. Still, the intrinsic capability of
VCF to monitor protein conformational changes in real time, in vivo, and under voltage control clearly outweighs these and other possible drawbacks. As the interest in VCF increases and other groups join in the excitement, we should see improved understanding of
the voltage-dependent activation and inactivation mechanisms, as novel approaches match the number of
technical challenges that lie ahead. There is a bright future ahead for the application of fluorescence techniques to voltage-dependent phenomena.
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REFERENCES |
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---|
1. | Armstrong, C.M., and F. Bezanilla. 1973. Currents related to movement of the gating particles of the sodium channels. Nature. 242: 459-461 [Medline]. |
2. | Baker, O.S., H.P. Larsson, L.M. Mannuzzu, and E.Y. Isacoff. 1998. Three transmembrane conformations and sequence-dependent displacement of the S4 domain in Shaker K+ channel gating. Neuron. 20: 1283-1294 [Medline]. |
3. | Cha, A., and F. Bezanilla. 1997. Characterizing voltage-dependent conformational changes in the Shaker K+ channel with fluorescence. Neuron. 19: 1127-1140 [Medline]. |
4. |
Cha, A., and
F. Bezanilla.
1998.
Structural implications of fluorescence quenching in the Shaker K+ channel.
J. Gen. Physiol.
112:
391-408
|
5. | Cohen, L.B., B. Hille, R.D. Keynes, D. Landowne, and E. Rojas. 1971. Analysis of the potential-dependent changes in optical retardation in the squid giant axon. J. Physiol. (Camb.). 218: 205-237 [Medline]. |
6. | Cohen, L.B., R.D. Keynes, and B. Hille. 1968. Light scattering and birefringence changes during nerve activity. Nature. 218: 438-441 [Medline]. |
7. | De Biasi, M., H.A. Hartmann, J.A. Drewe, M. Taglialatela, A.M. Brown, and G.E. Kirsch. 1993. Inactivation determined by a single site in K+ pores. Pflügers Arch 422: 354-363 [Medline]. |
8. |
Doyle, D.A.,
J.M. Cabral,
R.A. Pfuetzner,
A. Kuo,
J.M. Gulbis,
S.L. Cohen,
B.T. Chait, and
R. MacKinnon.
1998.
The structure of
the potassium channel: molecular basis of K+ conduction and selectivity.
Science.
280:
69-77
|
9. | Hille, B. 1992. Ion channels of excitable membranes. Sinauer Associates, Inc., Sunderland, MA. |
10. | Hubbell, W.L., and H.M. McConnell. 1968. Spin-label studies of the excitable membranes of nerve and muscle. Proc. Natl. Acad. Sci. USA. 61: 12-16 [Medline]. |
11. | Keynes, R.D., and E. Rojas. 1974. Kinetics and steady-state properties of the charged system controlling sodium conductance in the squid giant axon. J. Physiol. (Camb.). 239: 393-434 [Medline]. |
12. | Landowne, D.. 1985. Molecular motion underlying activation and inactivation of sodium channels in squid giant axons. J. Membr. Biol. 88: 173-185 [Medline]. |
13. | Larsson, H.P., O.S. Baker, D.S. Dhillon, and E.Y. Isacoff. 1996. Transmembrane movement of the Shaker K+ channel S4. Neuron. 16: 387-397 [Medline]. |
14. |
Loots, E., and
E.Y. Isacoff.
1998.
Protein rearrangements underlying slow inactivation of the Shaker K+ channel.
J. Gen. Physiol.
112:
377-389
|
15. | Mannuzzu, L.M., M.M. Moronne, and E.Y. Isacoff. 1996. Direct physical measure of conformational rearrangement underlying potassium channel gating. Science. 271: 213-216 [Abstract]. |
16. | Mendel, D., J.A. Ellman, Z. Chang, D.L. Veenstra, P.A. Kollman, and P.G. Schultz. 1992. Probing protein stability with unnatural amino acids. Science. 256: 1798-1802 [Medline]. |
17. | Nowak, M.W., P.C. Kearney, J.R. Sampson, M.E. Saks, C.G. Labarca, S.K. Silverman, W. Zhong, J. Thorson, J.N. Abelson, N. Davidson, et al . 1995. Nicotinic receptor binding site probed with unnatural amino acid incorporation in intact cells. Science. 268: 439-442 [Medline]. |
18. | Noren, C.J., S.J. Anthony-Cahill, M.C. Griffith, and P.G. Schultz. 1989. A general method for site-specific incorporation of unnatural amino acids into proteins. Science. 244: 182-188 [Medline]. |
19. |
Olcese, R.,
R. Latorre,
L. Toro,
F. Bezanilla, and
E. Stefani.
1997.
Correlation between charge movement and ionic current during
slow inactivation in Shaker K+ channels.
J. Gen. Physiol.
110:
579-589
|
20. | Perozo, E., R. MacKinnon, F. Bezanilla, and E. Stefani. 1993. Gating currents from a nonconducting mutant reveal open-closed conformations in Shaker K+ channels. Neuron 11: 353-358 [Medline]. |
21. |
Yang, Y.,
Y. Yan, and
F.J. Sigworth.
1997.
How does the W434F mutation block current in Shaker potassium channels?
J. Gen. Physiol.
109:
779-789
|
|
|