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Biophys J, September 1999, p. 1189-1191, Vol. 77, No. 3
Department of Molecular Biology and Biochemistry, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021 USA
In 1964, K. T. Brown and M. Murakami
reported a flash-induced electrical response with no detectable latency
in the retina. This early receptor potential (ERP) was characterized in
some detail over the next several years by R. A. Cone, W. L. Pak, T. G. Ebrey and others in a variety of systems. The ERP is a
biphasic response comprising an initial cornea-positive fast phase
(R1) followed by a slower cornea-negative (R2)
phase. The action spectra of both R1 and R2
matched that of rhodopsin, and the amplitudes of both phases were
linearly proportional to the fraction of rhodopsin bleached by the
stimulus flash. It was therefore proposed that the net displacement of
electric charge in rhodopsin molecules undergoing light-dependent
conformational changes generated the ERP. Since the charge displacement
was proportional to the number of pigment molecules that
photoisomerized, the ERP was a convenient method to obtain the
photosensitivities of rods and cones. A. L. Hodgkin and P. M. O'Bryan (1977) In an ambitious study in this issue of Biophysical Journal,
J. M. Sullivan and P. Shukla describe the measurement of the ERC generated by rhodopsin heterologously expressed in tissue culture. They
use a whole-cell gigohm-seal voltage clamp with millisecond-order time
resolution to demonstrate that ERCs can be obtained from single or
fused giant HEK293S cells expressing human opsin regenerated with
11-cis-retinal. The plasma membranes of these cells
apparently contain rhodopsin in densities similar to that found in the
plasma membranes of rod photoreceptor cells. The data show
conclusively that the flash-activated ERC results from
photoactivation of rhodopsin. This work has the potential to open a new
approach to the study of the important problem of vectorial information
transfer across cell membranes.
The ERC signal arises from charge displacement orthogonal to the plane
of the membrane bilayer and is a probe of electric changes directed
across the membrane dielectric. The ERC is kinetically complex,
suggesting transitions among several contributing electrical states.
The protein conformation changes underlying these transitions are
unknown. However, the electrical difference between any two conformational states could be due to the net displacement of a bound
charge, dipole reorientation, an induced structural dipole moment, or
an interfacial charge migration such as proton uptake or release (Honig
et al., 1986 Rhodopsin is an excellent model system for biophysical studies of the
superfamily of G protein-coupled receptors (Sakmar, 1998 The primary photoproduct of rhodopsin, bathorhodopsin, relaxes
through a series of spectrally defined intermediates, including lumirhodopsin, Meta I, and Meta II. There is good evidence that a
protein conformational change occurs in the rhodopsin to Meta I
transition, and that steric rather than electrostatic interactions are
responsible for the structural changes leading to Meta I. Electrostatic
interactions are more clearly involved in the subsequent conversion of
Meta I to the active receptor, R*. Transfer of the Schiff base proton
in the formation of Meta II is a key electrostatic trigger that
precedes receptor activation. At least five concerted events are known
to occur in forming the active R* conformation: retinal isomerization;
Schiff base deprotonation; protonation of the Schiff base counterion;
transmembrane helix movement, primarily of helical segments 3 and 6;
and proton uptake at the cytoplasmic surface of the
receptor. Schiff base deprotonation and subsequent protein
conformational changes are likely to be driven by electrostatic interactions. These conformational changes should all contribute to the
ERC waveform. In particular, it is likely that the R2 phase of the ERC represents in part the conversion of Meta I to Meta II.
The mechanistic details of electrically active conformational changes
in membrane proteins are of great interest. Since it is not unusual for
biological membranes to have significant transmembrane potentials,
membrane proteins may be exposed to intense electric fields. Their
structural conformations may be functionally coupled to the electric
field for the purpose of switching, transport, or energy transduction
(Honig et al., 1986 The work by J. M. Sullivan and P. Shukla provides an exciting new
method to study the relationship between structure and electrostatics in membrane protein receptors. It is reasonable to expect that this
method will bring a new dimension to structure-function studies of
rhodopsin and other visual pigments. In particular, it should be
possible to correlate specific features of the ERC to specific electrostatically based conformational changes in rhodopsin. It is
reasonable to expect that site-specific mutants of rhodopsin that are
expressed at high levels in the plasma membrane will be amenable to
study. This approach would allow the identification of the molecular
components responsible for R2. A major challenge will be to
design experiments to identify the mechanistic events that underlie the
ERC. These could include chromophore dipole reorientation, charge
separation, dipole movement, intramolecular proton transfer, and
vectorial proton uptake or release at the aqueous interface.
ARTICLE
used the ERP to obtain an apparent photosensitivity of
turtle cone cells, and more recently C. L. Makino, W. R. Taylor, and D. A. Baylor (1991) determined photosensitivites by
measuring the early receptor current (ERC) in voltage-clamped rod and
cone photoreceptors from larval salamanders. The ERC is the charge
motion underlying the ERP. They calculated the magnitude of the outward
component (R2) to correspond to the movement of about 0.18 e across the membrane per photoisomerization.
). Rhodopsin might well display all of these mechanisms as
it undergoes a well-documented series of conformational changes
initiated by the absorption of a photon of visible light.
; Gether and
Kobilka, 1998). Molecular biological approaches in combination with
various spectroscopic and biophysical methods have allowed the study of
recombinant mutant pigments to address the physical basis of spectral
tuning and the molecular mechanism of receptor activation
(Kochendoerfer et al., 1999; Farrens et al., 1996). It has been known
since the pioneering work of G. Wald and T. Yoshizawa that rhodopsin
contains an 11-cis retinylidene chromophore, which
isomerizes to the all-trans conformation upon photon
absorption. But how is chromophore isomerization coupled to receptor
activation? How do the conformational changes in the chromophore-binding pocket of rhodopsin propagate to the cytoplasmic surface of the receptor? What are the potential electrostatic changes
associated with these conformational changes? The dominant interaction
between chromophore and opsin is the electrostatic interaction between
the retinylidene protonated Schiff base and its protein counterion, a
glutamic acid residue. However, it is likely that both steric and
electrostatic factors contribute to receptor activation (Shieh et al.,
1997).
). For example, ion channels undergo conformational
changes that open and close an ion-permeable pore across the membrane.
In voltage-dependent channels this gating process is influenced by
transmembrane voltage. Charge motions in rhodopsin that result in the
ERC are conformation-dependent currents, as are ion channel gating
currents. Perhaps not surprisingly, the ERC is remarkably similar in
waveform and kinetics to an ion channel gating current. In
voltage-dependent ion channels, conformational states cannot be
measured independently of gating current. However, in rhodopsin a flash
of light can change the conformational state, and the conformational
changes can be probed independently of the ERC by a variety of
spectroscopic techniques including time-resolved absorption
spectroscopy. Several investigators including C. F. Stevens and R. Ranganathan have previously noted that the ERC is akin to a rhodopsin
gating current. J. M. Sullivan and P. Shukla also address this notion.
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FOOTNOTES |
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Received for publication 14 July 1999 and in final form 22 July 1999.
Address reprint requests to Thomas P. Sakmar, Dept. of Molecular Biology and Biochemistry, Rockefeller University, Howard Hughes Medical Institute, Box 284, RRB 515, 1230 York Ave., New York, NY 10021.
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Biophys J, September 1999, p. 1189-1191, Vol. 77, No. 3
© 1999 by the Biophysical Society 0006-3495/99/09/1189/03 $2.00
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