From the Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom
To study the actions of Ca2+ on "early" stages of the transduction cascade, changes in cytoplasmic calcium concentration (Ca2+i) were opposed by manipulating Ca2+ fluxes across the rod outer segment membrane immediately following a bright flash. If the outer segment was exposed to 0 Ca2+/0 Na+ solution for a brief period immediately after the flash, then the period of response saturation was prolonged in comparison with that in Ringer solution. But if the exposure to 0 Ca2+/0 Na+ solution instead came before or was delayed until 1 s after the flash then it had little effect. The degree of response prolongation increased with the duration of the exposure to 0 Ca2+/0 Na+ solution, revealing a time constant of 0.49 ± 0.03 s. By the time the response begins to recover from saturation, Ca2+i seems likely to have fallen to a similar level in each case. Therefore the prolongation of the response when Ca2+i was prevented from changing immediately after the flash seems likely to reflect the abolition of actions of the usual dynamic fall in Ca2+i on an early stage in the transduction cascade at a site which is available for only a brief period after the flash. One possibility is that the observed time constant corresponds to the phosphorylation of photoisomerized rhodopsin.
Key words: photoreceptor; retinal rod; light adaptation; calcium; rhodopsinPhotoreceptor light adaptation is believed to be largely
if not exclusively controlled by cytoplasmic calcium
concentration (Ca2+i) (Matthews et al., 1988; Nakatani
and Yau, 1988
; Fain et al., 1989
; Koutalos et al., 1995b
;
Matthews, 1995
). Both biochemical and electrophysiological evidence indicates that Ca2+ acts at a number of
different sites in the transduction mechanism. These
can broadly be subdivided into those associated with
"early" stages in the transduction cascade, which are
likely only to be accessible for a relatively brief period
after the flash, and those "late" in transduction which
will be available throughout the flash response (Koutalos et al., 1995b
; Matthews, 1996
). The former are believed to involve actions of Ca2+i on the production
(Lagnado and Baylor, 1994
; Jones, 1995
) or the subsequent quenching via phosphorylation (Kawamura and
Murakami, 1991
; Kawamura, 1993
; Chen et al., 1995
)
of photoisomerized rhodopsin (Rh*),1 whereas the latter include the inhibition of guanylyl cyclase (Lolley
and Racz, 1982
; Koch and Stryer, 1988
; Koutalos et al., 1995a
) and modulation of the cGMP affinity of the
outer segment conductance (Hsu and Molday, 1993
;
Nakatani et al., 1995
) by Ca2+i.
Photoreceptor Ca2+i is governed by the balance between Ca2+ influx through the outer segment conductance (Yau and Nakatani, 1984a; Hodgkin et al., 1985
)
and Ca2+ efflux via an Na+:Ca2+,K+ exchanger (Yau
and Nakatani, 1984b
; Hodgkin et al., 1987
; Cervetto et
al., 1989
). When the outer segment conductance is suppressed during the response to a bright flash, this balance is upset and Ca2+i falls (Yau and Nakatani, 1985
;
McNaughton et al., 1986
; Ratto et al., 1987
; Gray-Keller
and Detwiler, 1994
; McCarthy et al., 1994
) with a dominant time constant in salamander rods of 0.5-1 s (Yau
and Nakatani, 1985
; Hodgkin et al., 1987
). If the flash is sufficiently bright to hold the response in saturation
for several seconds, Ca2+i is likely to fall sufficiently low
that its actions late in transduction will be essentially
complete by the time that recovery commences. However, this dynamic fall in Ca2+i may also have a more
modest effect on stages early in the transduction cascade during the period for which they remain accessible after the flash (Matthews, 1996
).
The light-induced fall in Ca2+i can be opposed by superfusing the outer segment with a 0 Ca2+/0 Na+ solution designed to minimize simultaneously Ca2+ influx
and efflux (Matthews et al., 1988; Nakatani and Yau,
1988
; Fain et al., 1989
). The removal of external Ca2+
minimizes Ca2+ influx through the outer segment conductance, while the removal of external Na+ prevents
Ca2+ efflux through the Na+:Ca2+,K+ exchanger. I have
investigated the actions of Ca2+ on early stages in transduction by briefly exposing the outer segment to this
solution just after a bright flash, thereby delaying the
onset of the usual dynamic fall in Ca2+i.
Preparation, Recording, and Light Stimuli
Rod photoreceptors were isolated mechanically under infrared illumination from the dark-adapted retina of the larval tiger salamander Ambystoma tigrinum after decapitation and pithing in dim red light. The circulating current was recorded by drawing the inner segment of the rod into a suction pipette, leaving the outer segment exposed to the superfusing solution. All experiments were carried out at room temperature (maintained at 20°C). The suction pipette current signal was low-pass filtered at 20 Hz (6-pole Bessel filter) and digitized continuously for subsequent analysis at a sampling rate of 100 Hz. Light stimuli were unpolarized 20-ms flashes of wavelength 500 nm. Light intensities were adjusted with neutral density filters and measured with a calibrated silicon photodiode (Graseby Optronics, Orlando, FL); they can be converted to isomerizations using a collecting area of 20 µm2.
Solutions and Solution Changes
Ringer solution contained 111 mM NaCl, 2.5 mM KCl, 1.0 mM
CaCl2, 1.6 mM MgCl2, and 3.0 mM HEPES, adjusted to pH 7.7 with NaOH, and 10 µM EDTA to chelate impurity heavy metals.
The Ringer solution continuously perfusing the recording chamber also included 10 mM glucose. 0 Ca2+/0 Na+ solution was
modified from Ringer solution by the equimolar substitution of
choline chloride for NaCl, the omission of CaCl2 and MgCl2, the
removal of EDTA, and the inclusion of 2 mM EGTA (Matthews, 1996). The normally inward dark current is inverted in this choline-substituted solution due to the efflux of K+ (Hodgkin et al.,
1985
; Matthews, 1995
; Lyubarsky et al., 1996
; Matthews, 1996
).
The removal of Mg2+ served to prevent the substantial Mg2+ influx which would otherwise have occurred under these conditions (Hodgkin et al., 1985
) and which has been shown to retard
response recovery (Matthews, 1995
). Rapid external solution
changes were effected by translating the boundary between two
flowing streams of solution across the exposed outer segment
(Hodgkin et al., 1985
) using a piezo-electric actuator (Matthews,
1994
). Recordings were corrected by subtraction of the junction
current measured when the same solution changes were carried
out during intense steady illumination at the end of the experiment, scaled for coincidence of saturating level before and after
the solution change. The junction current, shown in Figs. 1 and 2
as the solution monitor, normally rose to 90% of its final value
within 50 ms, suggesting a similar time course for the solution
change. Times of solution changes in Figs. 2 and 3 are given as
the half-rise times of the junction current.
Fig. 1 illustrates the effect of briefly exposing the outer
segment to 0 Ca2+/0 Na+ solution either shortly before
or shortly after a bright flash. If the outer segment was
stepped into 0 Ca2+/0 Na+ solution 1 s before the flash
and returned to Ringer solution 1 s thereafter (Fig. 1 A,
Pre & Post), then the flash response recorded by the
suction pipette recovered from saturation slightly later
than when the outer segment remained in Ringer solution throughout the response. A similar prolongation
of the response was also induced by a 1 s exposure to 0 Ca2+/0 Na+ solution immediately after the flash (Fig. 1
B, Post). Both of these procedures will have delayed the
dynamic fall in Ca2+i, which normally ensues when the
circulating current is suppressed by the flash, until after the outer segment was returned to Ringer solution.
These data were quantified by measuring the time for the response to recover to a criterion level (Pepperberg
et al., 1992; Matthews, 1995
, 1996
). Since the shape of
the initial recovery was virtually unaffected by these manipulations, measurements of the delay in recovery will
be essentially independent of the criterion selected,
which was taken here as 25% of the original dark current. 0 Ca2+/0 Na+ exposures as in Fig. 1, A and B, prolonged the bright flash response by 0.81 ± 0.15 s (11 cells) and 0.75 ± 0.10 s (10 cells) respectively (mean ± SEM; flash delivered 1,320 or 5,560 photons µm
2),
which do not differ significantly at the 5% level (Student's t test, t = 0.38). The earlier recovery of the flash
response in Ringer solution when Ca2+i was allowed to
fall can be contrasted with the much greater shortening of the response induced by nearly saturating steady light (5.0 ± 0.5 s, 8 cells) which should produce a static
reduction in Ca2+i of similar magnitude.
If, instead, the exposure to 0 Ca2+/0 Na+ solution took place immediately before the flash (Fig. 1 C, Pre) or was delayed until 1 s after the flash (Fig. 1 D, Late) then it had little effect on response recovery. These manipulations only prolonged the time for 25% recovery of the dark current by 0.02 ± 0.03 s (10 cells) and 0.05 ± 0.04 s (8 cells), respectively, neither of which differs significantly from zero. These observations indicate that the response was only prolonged when Ca2+i was prevented from falling during a brief period immediately after the flash and not as the result of exposure to 0 Ca2+/0 Na+ solution per se.
The period during which the dynamic fall in Ca2+i
could exert this effect was explored in Fig. 2 by varying
the time at which the outer segment was returned to
Ringer solution after the flash. In each case the outer
segment was stepped to 0 Ca2+/0 Na+ solution 1 s before the flash, and returned to Ringer solution at the
time (t) after the flash indicated beside each trace. As
the time spent in 0 Ca2+/0 Na+ solution after the flash
decreased, the duration of the response progressively
declined towards that in Ringer solution.
These data are quantified in Fig. 3 by once again
measuring the time for recovery of 25% of the dark current in each case. The longest exposures to 0 Ca2+/0
Na+ solution prolonged the response by nearly 1 s in
comparison with the response in Ringer, but this delay
in recovery progressively decreased as the time spent in
0 Ca2+/0 Na+ solution after the flash was reduced.
These data could be well fitted by a single exponential
of time constant 0.49 ± 0.03 s (8 cells). The simplest interpretation of this result is that it reflects the progressive removal of a Ca2+-sensitive stage early in the transduction cascade. When the dynamic fall in Ca2+i was
delayed in this way an ever-smaller proportion of these sites would remain to respond to it. The small displacement of the fitted exponential curve from the origin
probably arises largely from the delay between the flash
and the complete suppression of the circulating current (Cobbs and Pugh, 1987) which initiates the dynamic fall in Ca2+i. This delay will have been augmented
by the finite flash duration, a 20-ms group delay from
the Bessel low-pass filter, and the time taken for completion of the solution change.
Stepping the outer segment to 0 Ca2+/0 Na+ solution is
believed greatly to retard changes in Ca2+i, largely preventing the light-induced fall in Ca2+i for some 10-15 s
after the solution change (Fain et al., 1989; Lyubarsky
et al., 1996
). The short exposures used here therefore seem likely to have held Ca2+i near to its pre-existing
level before the solution change, and thereby to have
delayed the subsequent dynamic fall in Ca2+i until after
the return to Ringer solution.
After the return to Ringer solution the response to
the bright flash remained in saturation for a period sufficient to allow Ca2+i to fall substantially (Yau and Nakatani, 1985; Hodgkin et al., 1987
), thereby presumably
resulting in near maximal activation of guanylyl cyclase
before the circulating current began to recover (Koch
and Stryer, 1988
). Therefore the prolongation of the
response when the outer segment was exposed to 0 Ca2+/0 Na+ solution immediately after the flash seems
likely to reflect instead the abolition of actions of the
dynamic fall in Ca2+i on an early stage in the transduction cascade which was only accessible to Ca2+ for a relatively brief period after the flash (Koutalos et al., 1995b
; Matthews, 1996
). These changes in response duration are much smaller than those induced by near
saturating steady light (Fain et al., 1989
; Pepperberg et
al., 1992
; Matthews, 1995
), which presumably reduces
Ca2+i to a static level similar to that ultimately attained
after a bright flash. This observation implies that the
site for Ca2+ early in transduction is likely to become inaccessible at least as rapidly as the dynamic fall in Ca2+i
induced by the flash, thereby limiting the effect on response duration. The time constant of 0.49 ± 0.03 s obtained in Fig. 3 from the dependence of the response
prolongation on the duration of the exposure to 0 Ca2+/0 Na+ solution is most readily interpreted as reflecting the time course with which this early site for
Ca2+ is removed after the flash. Indeed, this interpretation follows directly, irrespective of the precise time
course of the stereotypical decline in Ca2+i once the
outer segment is returned to Ringer solution, if it is assumed that the early site decays stochastically and that reduced Ca2+i accelerates its removal independent of
time after the flash.
The strongest candidate for such a site of action for
Ca2+ early in the transduction cascade is Rh* itself,
whose inactivation via phosphorylation is known to be
modulated by Ca2+i (Kawamura, 1993; Chen et al.,
1995
). Suppose that Rh* were phosphorylated more
rapidly when Ca2+i was reduced, or that its ability to activate transducin were decreased. Under control conditions the dynamic fall in Ca2+i would act on Rh* to
lower the activation of transducin resulting from the
flash. If instead the dynamic fall in Ca2+i were delayed
until long after the flash, by which time Rh* would have
been completely phosphorylated, then this effect would
be abolished, and the response to a bright flash prolonged. However, as the delay between the flash and the
dynamic fall in Ca2+i was reduced, the overlap between
the lifetime of unphosphorylated Rh* and the dynamic
fall in Ca2+i would increase, thereby progressively shortening the flash response. Thus the time constant derived
from these measurements may represent the effective lifetime of unphosphorylated Rh* in the dark-adapted rod.
Previous estimates of Rh* lifetime have been derived
from the time constant of around 2 s which dominates
the recovery of the flash response (Pepperberg et al.,
1992, 1994
). However, the insensitivity of this longer
time constant to Ca2+i (Lyubarsky et al., 1996
; Matthews, 1996
) and light (Pepperberg et al., 1992
, 1994
;
Murnick and Lamb, 1996
) is difficult to reconcile with
biochemical evidence for the Ca2+ dependence of Rh*
phosphorylation (Kawamura, 1993
; Chen et al., 1995
).
If the more rapid time constant of about 0.5 s obtained here for the removal of the Ca2+-sensitive site early in
transduction represents Rh* phosphorylation then this
problem would be resolved, suggesting that the longer time constant governing response recovery might originate instead from subsequent events in Rh* inactivation or from the inactivation of later stages in the transduction cascade (Lyubarsky et al., 1996
; Matthews, 1996
;
Murnick and Lamb, 1996
). This value is also in close
agreement with the shorter of the two time constants required to model the kinetics of the flash response
when the light-induced fall in Ca2+i is prevented (Lyubarsky et al., 1996
).
Such rapid phosphorylation of Rh* even in darkness
would have several functional implications for the light
response. First, to account for the magnitude of the
change in sensitivity induced by bright light (Pepperberg et al., 1994; Jones, 1995
; Matthews, 1996
) or
greatly reduced Ca2+i (Lagnado and Baylor, 1994
;
Koutalos et al., 1995b
), it seems possible that the time
constant for Rh* phosphorylation might shorten further in the fully light-adapted rod by at least three- to
fourfold from this already rapid value in darkness. Second, much Rh* phosphorylation appears to take place
during the rising phase of even the dark-adapted dim
flash response, before it has reached its peak. Such
rapid phosphorylation of Rh* might thereby contribute
to the ability of lowered Ca2+i to reduce the apparent
gain of the flash response rising phase (Lagnado and
Baylor, 1994
; Jones, 1995
).
Original version received 22 September 1996 and accepted version received 8 November 1996.
Address correspondence to Dr. H.R. Matthews, Physiological Laboratory, University of Cambridge, Downing Street, Cambridge CB2 3EG, United Kingdom. Fax: 01223-333840; E-mail: hrm1{at}cam.ac.uk
I am grateful to Dr. G.L. Fain for helpful comments on the manuscript.Supported by the Wellcome Trust.
Rh*, photoisomerized rhodopsin.