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Light-dependent changes in outer segment free-Ca2+ concentration in salamander cone photoreceptors.

Sampath AP, Matthews HR, Cornwall MC, Bandarchi J, Fain GL - J. Gen. Physiol. (1999)

Bottom Line: Photopigment bleaching by the laser resulted in a considerable reduction in light sensitivity and a maintained decrease in outer segment Ca2+ concentration.Regeneration of the photopigment allowed repeated measurements of fluo-3 fluorescence to be made from a single red-sensitive cone during adaptation to steady light over a range of intensities.These measurements demonstrated that the outer segment Ca2+ concentration declines in a graded manner during adaptation to background light, varying linearly with the magnitude of the circulating current.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Science, University of California, Los Angeles, Los Angeles, California 90095, USA. apsampat@ucla.edu

ABSTRACT
Simultaneous measurements of photocurrent and outer segment Ca2+ were made from isolated salamander cone photoreceptors. While recording the photocurrent from the inner segment, which was drawn into a suction pipette, a laser spot confocal technique was employed to evoke fluorescence from the outer segment of a cone loaded with the Ca2+ indicator fluo-3. When a dark-adapted cone was exposed to the intense illumination of the laser, the circulating current was completely suppressed and fluo-3 fluorescence rapidly declined. In the more numerous red-sensitive cones this light-induced decay in fluo-3 fluorescence was best fitted as the sum of two decaying exponentials with time constants of 43 +/- 2.4 and 640 +/- 55 ms (mean +/- SEM, n = 25) and unequal amplitudes: the faster component was 1.7-fold larger than the slower. In blue-sensitive cones, the decay in fluorescence was slower, with time constants of 140 +/- 30 and 1,400 +/- 300 ms, and nearly equal amplitudes. Calibration of fluo-3 fluorescence in situ from red-sensitive cones allowed the calculation of the free-Ca2+ concentration, yielding values of 410 +/- 37 nM in the dark-adapted outer segment and 5.5 +/- 2.4 nM after saturating illumination (mean +/- SEM, n = 8). Photopigment bleaching by the laser resulted in a considerable reduction in light sensitivity and a maintained decrease in outer segment Ca2+ concentration. When the photopigment was regenerated by applying exogenous 11-cis-retinal, both the light sensitivity and fluo-3 fluorescence recovered rapidly to near dark-adapted levels. Regeneration of the photopigment allowed repeated measurements of fluo-3 fluorescence to be made from a single red-sensitive cone during adaptation to steady light over a range of intensities. These measurements demonstrated that the outer segment Ca2+ concentration declines in a graded manner during adaptation to background light, varying linearly with the magnitude of the circulating current.

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Simultaneous measurements of fluo-3 fluorescence  and circulating current from a red-sensitive cone during a 3-s laser  exposure. (A) Fluo-3 fluorescence signal excited by the laser spot  and recorded by the photodiode. (B) Circulating current measured by the suction pipette in response to the laser exposure. In  each case, 1 represents the first exposure of the dark-adapted cone  to the laser spot and 2 shows the response to a second laser exposure 10 s later. The light-induced decline in fluo-3 fluorescence (A,  1) could be fitted with the sum of two exponentials with time constants of 27 and 430 ms using a least-squares algorithm. The residual decline in fluo-3 fluorescence seen at the onset of the second  laser exposure (A, 2) is likely to correspond to the small recovery  in circulating current that had taken place at that time (B, 2). The  initial peak in the suction pipette recording (B) is likely to represent contamination of the photoresponse by voltage-sensitive conductances in the cone inner segment (Barnes and Hille, 1989).  The top trace is the laser light monitor. (Inset) The fall in fluo-3  fluorescence during the first 200 ms with the same exponential fit  as described above.
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Figure 1: Simultaneous measurements of fluo-3 fluorescence and circulating current from a red-sensitive cone during a 3-s laser exposure. (A) Fluo-3 fluorescence signal excited by the laser spot and recorded by the photodiode. (B) Circulating current measured by the suction pipette in response to the laser exposure. In each case, 1 represents the first exposure of the dark-adapted cone to the laser spot and 2 shows the response to a second laser exposure 10 s later. The light-induced decline in fluo-3 fluorescence (A, 1) could be fitted with the sum of two exponentials with time constants of 27 and 430 ms using a least-squares algorithm. The residual decline in fluo-3 fluorescence seen at the onset of the second laser exposure (A, 2) is likely to correspond to the small recovery in circulating current that had taken place at that time (B, 2). The initial peak in the suction pipette recording (B) is likely to represent contamination of the photoresponse by voltage-sensitive conductances in the cone inner segment (Barnes and Hille, 1989). The top trace is the laser light monitor. (Inset) The fall in fluo-3 fluorescence during the first 200 ms with the same exponential fit as described above.

Mentions: Fig. 1 shows an example of a simultaneous measurement of fluo-3 fluorescence and suction pipette current from an isolated red-sensitive cone previously loaded with fluo-3 acetoxymethyl ester. First, the dark-adapted cone was exposed to the laser for 3 s, an exposure that will have bleached a considerable fraction of the photopigment (see methods), and which resulted in complete suppression of the circulating current (Fig. 1 B, 1). This was accompanied by a rapid decline in fluo-3 fluorescence (Fig. 1 A, 1), corresponding to a fall in the intracellular free-Ca2+ concentration. The measured fluorescence originated in the cone outer segment, since movement of the cone off the laser spot produced no detectable fluorescence when the laser shutter was opened. A second laser exposure 10-s later showed that at that time the circulating current remained almost completely suppressed (Fig. 1 B, 2) and that fluo-3 fluorescence had reached a minimum level (Fig. 1 A, 2). It can be seen from Fig. 1 A that fluo-3 fluorescence did not fall to zero during saturating illumination, but instead declined to a maintained pedestal level that was insensitive to further stimulation of the cone by laser light, much as is the case for rod photoreceptors. This light-insensitive component of fluorescence is unlikely to be due to compartmentalization of fluo-3 within the outer segment during loading, since the cone outer segment contains no closed compartments. Instead, this pedestal, which is greater than the minimum fluorescence evoked from fluo-3 under Ca2+-free conditions in vitro, may be attributable to a nonspecific interaction of fluo-3 with other molecules in the cone outer segment (Sampath et al., 1998). The decline in fluo-3 fluorescence during the first 2 s of exposure to saturating laser light could be fitted with the sum of two decaying exponentials, the shorter of which was of greater amplitude than the longer. Measurements from 25 red-sensitive cones yielded mean values for the two time constants of 43 ± 2.4 and 640 ± 55 ms (mean ± SEM), while the ratio of their amplitudes was 1.7 ± 0.11.


Light-dependent changes in outer segment free-Ca2+ concentration in salamander cone photoreceptors.

Sampath AP, Matthews HR, Cornwall MC, Bandarchi J, Fain GL - J. Gen. Physiol. (1999)

Simultaneous measurements of fluo-3 fluorescence  and circulating current from a red-sensitive cone during a 3-s laser  exposure. (A) Fluo-3 fluorescence signal excited by the laser spot  and recorded by the photodiode. (B) Circulating current measured by the suction pipette in response to the laser exposure. In  each case, 1 represents the first exposure of the dark-adapted cone  to the laser spot and 2 shows the response to a second laser exposure 10 s later. The light-induced decline in fluo-3 fluorescence (A,  1) could be fitted with the sum of two exponentials with time constants of 27 and 430 ms using a least-squares algorithm. The residual decline in fluo-3 fluorescence seen at the onset of the second  laser exposure (A, 2) is likely to correspond to the small recovery  in circulating current that had taken place at that time (B, 2). The  initial peak in the suction pipette recording (B) is likely to represent contamination of the photoresponse by voltage-sensitive conductances in the cone inner segment (Barnes and Hille, 1989).  The top trace is the laser light monitor. (Inset) The fall in fluo-3  fluorescence during the first 200 ms with the same exponential fit  as described above.
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Figure 1: Simultaneous measurements of fluo-3 fluorescence and circulating current from a red-sensitive cone during a 3-s laser exposure. (A) Fluo-3 fluorescence signal excited by the laser spot and recorded by the photodiode. (B) Circulating current measured by the suction pipette in response to the laser exposure. In each case, 1 represents the first exposure of the dark-adapted cone to the laser spot and 2 shows the response to a second laser exposure 10 s later. The light-induced decline in fluo-3 fluorescence (A, 1) could be fitted with the sum of two exponentials with time constants of 27 and 430 ms using a least-squares algorithm. The residual decline in fluo-3 fluorescence seen at the onset of the second laser exposure (A, 2) is likely to correspond to the small recovery in circulating current that had taken place at that time (B, 2). The initial peak in the suction pipette recording (B) is likely to represent contamination of the photoresponse by voltage-sensitive conductances in the cone inner segment (Barnes and Hille, 1989). The top trace is the laser light monitor. (Inset) The fall in fluo-3 fluorescence during the first 200 ms with the same exponential fit as described above.
Mentions: Fig. 1 shows an example of a simultaneous measurement of fluo-3 fluorescence and suction pipette current from an isolated red-sensitive cone previously loaded with fluo-3 acetoxymethyl ester. First, the dark-adapted cone was exposed to the laser for 3 s, an exposure that will have bleached a considerable fraction of the photopigment (see methods), and which resulted in complete suppression of the circulating current (Fig. 1 B, 1). This was accompanied by a rapid decline in fluo-3 fluorescence (Fig. 1 A, 1), corresponding to a fall in the intracellular free-Ca2+ concentration. The measured fluorescence originated in the cone outer segment, since movement of the cone off the laser spot produced no detectable fluorescence when the laser shutter was opened. A second laser exposure 10-s later showed that at that time the circulating current remained almost completely suppressed (Fig. 1 B, 2) and that fluo-3 fluorescence had reached a minimum level (Fig. 1 A, 2). It can be seen from Fig. 1 A that fluo-3 fluorescence did not fall to zero during saturating illumination, but instead declined to a maintained pedestal level that was insensitive to further stimulation of the cone by laser light, much as is the case for rod photoreceptors. This light-insensitive component of fluorescence is unlikely to be due to compartmentalization of fluo-3 within the outer segment during loading, since the cone outer segment contains no closed compartments. Instead, this pedestal, which is greater than the minimum fluorescence evoked from fluo-3 under Ca2+-free conditions in vitro, may be attributable to a nonspecific interaction of fluo-3 with other molecules in the cone outer segment (Sampath et al., 1998). The decline in fluo-3 fluorescence during the first 2 s of exposure to saturating laser light could be fitted with the sum of two decaying exponentials, the shorter of which was of greater amplitude than the longer. Measurements from 25 red-sensitive cones yielded mean values for the two time constants of 43 ± 2.4 and 640 ± 55 ms (mean ± SEM), while the ratio of their amplitudes was 1.7 ± 0.11.

Bottom Line: Photopigment bleaching by the laser resulted in a considerable reduction in light sensitivity and a maintained decrease in outer segment Ca2+ concentration.Regeneration of the photopigment allowed repeated measurements of fluo-3 fluorescence to be made from a single red-sensitive cone during adaptation to steady light over a range of intensities.These measurements demonstrated that the outer segment Ca2+ concentration declines in a graded manner during adaptation to background light, varying linearly with the magnitude of the circulating current.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Science, University of California, Los Angeles, Los Angeles, California 90095, USA. apsampat@ucla.edu

ABSTRACT
Simultaneous measurements of photocurrent and outer segment Ca2+ were made from isolated salamander cone photoreceptors. While recording the photocurrent from the inner segment, which was drawn into a suction pipette, a laser spot confocal technique was employed to evoke fluorescence from the outer segment of a cone loaded with the Ca2+ indicator fluo-3. When a dark-adapted cone was exposed to the intense illumination of the laser, the circulating current was completely suppressed and fluo-3 fluorescence rapidly declined. In the more numerous red-sensitive cones this light-induced decay in fluo-3 fluorescence was best fitted as the sum of two decaying exponentials with time constants of 43 +/- 2.4 and 640 +/- 55 ms (mean +/- SEM, n = 25) and unequal amplitudes: the faster component was 1.7-fold larger than the slower. In blue-sensitive cones, the decay in fluorescence was slower, with time constants of 140 +/- 30 and 1,400 +/- 300 ms, and nearly equal amplitudes. Calibration of fluo-3 fluorescence in situ from red-sensitive cones allowed the calculation of the free-Ca2+ concentration, yielding values of 410 +/- 37 nM in the dark-adapted outer segment and 5.5 +/- 2.4 nM after saturating illumination (mean +/- SEM, n = 8). Photopigment bleaching by the laser resulted in a considerable reduction in light sensitivity and a maintained decrease in outer segment Ca2+ concentration. When the photopigment was regenerated by applying exogenous 11-cis-retinal, both the light sensitivity and fluo-3 fluorescence recovered rapidly to near dark-adapted levels. Regeneration of the photopigment allowed repeated measurements of fluo-3 fluorescence to be made from a single red-sensitive cone during adaptation to steady light over a range of intensities. These measurements demonstrated that the outer segment Ca2+ concentration declines in a graded manner during adaptation to background light, varying linearly with the magnitude of the circulating current.

Show MeSH
Related in: MedlinePlus