Gating of light-sensitive ion channels by cyclic GMP in rod photoreceptors

Neuroscience R~earch, Suppl. 6 (1987) $55-$66 0168-0102/87/$03.30 © 1987 Else~er Scientific Publishers Ireland Ltd.

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GATING OF LIGHT-SENSITIVE lON CHANNELS BY CYCLIC GMP IN ROD PHOTORECEPTOR8 GARY MATTHEWS AND SHU-ICHI WATANABE Department of Neurobiology and Behavior, The State University of New York, Stony Brook, NY 11794-5230, USA INTRODUCTION It has long been recognized that phototransduction in vertebrate photoreeeptors requires an internal transmitter that controls the sodium conductance of the outer segment plasma membrane.

The recent demonstration by Fesenko et al.

(i) of

a cationic conductance regulated by cyclic GMP (cGMP) in rod photoreceptor membrane has centered attention on the possibility that cGMP is that internal transmitter.

In this paper, we will present a brief account of some of our work com-

paring single ion channels closed by light in intact rods with single channels opened by cGMP in excised membrane patches.

These experiments are described in

more detail elsewhere (2, 3). MATERIAL AND METHODS Experiments were performed on rod photoreceptors isolated from the retina of the toad, Bu~o marinus, by mechanical dissociation (4).

Patch-clamp recordings

were made from cell-attached and excised patches using standard techniques (5). Patch pipettes were filled with modified Ringer solution (0 Ca/0 Mg Ringer) consisting of (in mM): Mg; pH = 7.8.

NaCI, 118; KCI, 2.5; HEPES, i0; EDTA, 0.15; no added Ca or

cGMP was dissolved in 0 Ca/0 Mg Ringer at i ~M to i mM and applied

to excised patches by local perfusion (6). Recordings were made under infrared illumination on the stage of a compound microscope equipped with an infrared-sensitive TV system.

Optical stimuli were

provided by a dual-beam stimulator similar in design to that of Baylor & Hodgkin (7).

Data were recorded on magnetic tape at 0-5000 Hz and later replayed through

an antialiasing filter for digitization and computer analysis. RESULTS The rationale leading to single-channel recording from the light-sensitive conductance was suggested by the action of divalent cations on the cGMP-activated conductance of excised outer segmentpatches.

Thus, it will be useful to begin

the discussion of the light-sensitive channel by briefly exploring those observations on the cGMP-sensitive conductance. Action of divalent cations on cGMP-activated conductance The size and rectification properties of the cGMP-activated conductance are affected by the concentration of divalent cations bathing the membrane

(8-11).

An example of this effect of divalent cations is shown in Fig. i, which shows

Presented at the 9th Taniguchi International Symposium on Visual Science, November 24-28, 1986.

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current-voltage relations from an excised, outside-out patch of rod plasma membrane.

In this experiment, the cGMP-sensitive conductance was activated by

including a saturating concentration (1.25 mM) of cGMP in the 0 Ca/0 Mg pipette solution bathing the intracellular face of the membrane.

With normal Ringer

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-100 -120 Fig. i. Effect of external divalent cations on current-voltage relation of cGMPactivated conductance of an outside-out patch of membrane excised from a rod outer segment. Intracellular face (inside pipette) bathed in 0 Ca, 0 Mg Ringer containing 1.25 mM cGMP. Extracellular face bathed either in normal Ringer (circles; 1.0 mM Ca, 1.6 mM Mg) or in 0 Ca, 0 Mg Ringer (triangles). Open and filled circles show relation in normal Ringer before and after 0 Ca, 0 Mg Ringer. The seal resistance of the cell-attached recording before rupturing the patch and forming the outside-out patch was i0 G~.

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bathing the extracellular face of the patch (circles), the current-voltage relation exhibited pronounced outward rectification similar to that of the lightsensitive conductance in intact rods (12).

When 0 Ca/0 Mg Ringer was substituted

on the external face, so that both faces of the membrane were bathed in 0 Ca/0 Mg solution, the conductance increased and the outward rectification vanished (triangles).

This effect was readily reversible.

In control experiments on

outside-out patches using patch pipettes filled with 0 Ca/0 Mg Ringer without cGMP, perfusion of the external face with 0 Ca/0 Mg Ringer reversibly increased patch conductance no more than 50% (average = 20%; N=4).

Thus, the large revers-

ible increase in conductance shown in Fig. 1 occurred only when cGMP was present at the intracellular face of the membrane.

This suggests that the effect of

removing external divalent cations was a specific action on the cGMP-dependent conductance, rather than a nonspecific effect on membrane leak or on seal resistance.

In other experiments on inside-out patches, this was demonstrated

directly; perfusing the intracellular face with 0 Ca/0 Mg Ringer had little e f f e ~ on patch conductance (13, 14), whereas 0 Ca/0 Mg Ringer + cGMP induced a large ohmic conductance (e.g., Fig. 2). As shown in Fig. 2, the rectification properties of the cGMP-activated conductance were also affected by varying the concentration of divalent cations on the intracellular face of the membrane.

The current-voltage relations in Fig. 2 were

obtained from an inside-out patch of outer segment membrane with normal Ringer (open and filled circles), normal Ringer + 1 mM cGMP (triangles), or 0 Ca/0 Mg Ringer + 1 mM cGMP (squares) bathing the intracellular face of the patch. pipette solution bathing the extracellular face was 0 Ca/0 Mg Ringer. cribed previously

The

As des-

(Matthews, 1986a), when the divalent cation gradient was re-

versed so that normal Ringer bathed the internal rather than the external face, the current-voltage curve showed inward rectification Fig. i).

(the reverse of that in

Once again, when 0 Ca/0 Mg Ringer bathed both faces, the cGMP-activated

conductance was large and ohmic. Single light-sensitive channels Under normal physiological conditions, the current through a single lightsensitive channel in an intact rod is too small to be resolved in patch-clamp recordings, and noise analysis is required to study its characteristics 15-17).

(ii,

However, if the light-sensitive conductance of intact rods is the same

as the cGMP-activated conductance of excised patches, the results shown in Figs. 1 & 2 suggest that inward current through single channels might be sufficiently. large to allow single-channel recording when external divalent cations are removed.

To examine this, cell-attached patch-clamp recordings were made from

outer segments of dark-adapted rods using patch pipettes filled with 0 Ca, 0 Mg Ringer.

In such recordings, brief pulses of inward current were observed in

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-60 Fig. 2. Effect of divalent cations on current-voltage relation of cGMP-activated conductance of an inside-out patch from rod outer segment. The pipette solution bathing the extracellular face of the membrane was 0 Ca, 0 Mg Ringer. Circles show current-voltage curves with normal Ringer on the intracellular face of the membrane at the beginning (open circles) and end (filled circles) of the experiment, corresponding to ohmic seal resistances of 10.3 and 9.5 G~ respectively. The cGMP-sensitive conductance was then activated by perfusing the intracellular face with 1.25 m M cGMP in normal Ringer (triangles) or in 0 Ca, 0 Mg Ringer (squares). With the divalent cation gradient reversed from that in Fig. i, the direction of rectification was also reversed, so that the cGMP-activated conductance passed inward current better than outward current (triangles). As in Fig. i, with 0 Ca, 0 Mg Ringer on both sides of the membrane (squares), the conductance was large and approximately ohmic.

darkness; these events were suppressed by illumination in the normal response range of the dark-adapted rod. Fig. 3.

Examples of this channel activity are shown in

In the experiment of Fig. 3, light was restricted to a narrow, trans-

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versely oriented slit, which was centered on the recorded patch of membrane or displaced longitudinally along the outer segment.

When the slit was positioned

37 ~m distant from the recorded patch, sensitivity was 1.3 to 1.6 log units lower than when illumination fell directly on the recorded region.

The effect of

illumination on the rod dark current is known to be spatially restricted, spreading only a few ~m or less along the outer segment from an illuminated region (18, 19).

Thus, the result shown in Fig. 3 confirms that the light-sensitive channels

behaved in the manner expected of the channels responsible for the dark current of the rod.

Because the reduction in sensitivity with displaced illumination was

similar to the measured fall in light intensity at 30-40 ~m from the slit (20), the response obtained with the longitudinally displaced slit was likely due to light scatter.

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Fig. 3. Chart recorder traces showing effect of slit stimuli on light-sensitive channel activity in a cell-attached recording from a dark-adapted rod. A. The slit was centered on the patch electrode. The nominal width of the slit was 3.5 ~m, and the long axis of the slit was perpendicular to the long axis of the rod. Bottom traces of each pair show timing of illumination, and the numbers to the left give light intensity in photons ~m -2 s -l, measured full-field without the slit~ Bandwidth: 0-80 Hz. Patch electrode was clamped to bath potential, i.e., the patch was not hyperpolarized from the resting dark potential. B. The same as A, except that the slit was displaced longitudinally 37 ~m from the recorded patch. Illumination was less effective than in A.

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The effect of slit position on the response to illumination (Fig. 3) also provides indirect evidence that channel activity is abolished by light itself and not by the resulting hyperpolarization of the cell.

The hyperpolarization pro-

duced by the slit illumination of a particular intensity would be approximately constant regardless of slit position, but light displaced from the recorded patch was 1-2 log units less effective than light placed directly on the patch. experiments described elsewhere

In

(2, 3), it was demonstrated directly that hyper-

polarizing the patch has little effect on the rate of op~ning of the lightsensitive channel.

Thus, light-sensitive channel activity is reduced by light

per se rather than voltage changes. A higher resolution recording of light-sensitive channel activity is shown in Fig. 4.

In the wide-bandwidth recording (bottom trace), it can be seen that

single openings occur as a burst of brief openings and closings, i.e., the channel "flickers."

Restricting the recording bandwidth by low-pass filtering

(top trace) gives a more impressive signal-to-noise ratio and results in a more rectangular event waveform; however, the high-frequency behavior of the channel is obscured in the process.

For that reason, only wide-bandwidth records were

used for the analysis presented in this paper.

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Is there more than one conductance state? Light-sensitive channel events appeared to have more than one amplitude. Occasionally,

smaller events were observed that were about 1/3 the size of those

shown in Fig. 4.

In addition, the current sometimes fluctuated between the large

and small values within a single event.

Similar behavior has been described for

cGMP-activated channels in excised patches (i0).

The existence of two amplitudes

of channel current suggests that the light-sensitive channel has more than one conductance state.

However, the rapidity of the flicker prevented unequivocal

demonstration of two (or more) conducting states.

For

example, the probability

density function of patch membrane current in darkness gave no clear indication of two peaks that would correspond to two conductance states.

An example of the

probability density function of current amplitude is shown in Fig. 5.

There was

a large Gaussian peak centered at zero membrane current, corresponding to baseline noise in the absence of channel opening, and a tail of inward current due to channel activity.

In Fig. 5, there is an excess of probability density over that

expected from the baseline fluctuations at about 1 pA, and there is some indication of another peak at about 2-3 pA.

It might be argued that the smaller-

amplitude peak represents complete closures and openings that are sufficiently brief to appear incomplete, but because of the presence of relatively long smallamplitude events, such an explanation cannot account for all of the smalleramplitude peak.

It is possible, however, that the small maintained events

represent large events with particularly rapid flicker that is unresolved at the recording bandwidth. Comparison of light-sensitive and cGMP-activated channels in the same patch Haynes et al. (9) and Zimmerman & Baylor (i0) have recently shown that single cGMP-activated channels can be recorded from inside-out patches of outer segment membrane.

To make a direct comparison of the light-sensitive channel with the

cGMP-activated channel, we recorded cGMP-activated channels in excised patches after recording light-sensitive channel activity in the same patch in the intact rod.

Records from one experiment of this type are shown in Fig. 6.

cGMP-

activated channel events were indistinguishable from light-sensitive channel activity.

To compare the overall shape of the channel events, difference power

spectra of light-sensitive and cGMP-activated channel activity were obtained by subtracting the spectrum in bright light from the spectrum in darkness and by subtracting the spectrum without cGMP from the spectrum in the presence of cGMP. As shown by Matthews

(2), these power spectra of light-sensitive and cGMP-

activated channels were the same, demonstrating that the two channels were kinetically identical.

Spectra were fitted by a sum of two Lorentzian components

with corner frequencies differing i0- to 15-fold.

The parameters of the two

Lorentzian components of the power spectra for light-sensitive and cGMP-activated

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Current (pA) Fig. 5. Probability density function of membrane current of cell-attached patch of dark-adapted rod outer segment in darkness. Bin width = 0.005 pA. Patch hyperpolarized by 148 mV. Same cell as Fig. 4.

channels in 6 experiments are summarized in Table i. As discussed by Matthews & Watanabe

(3), the kinetics of light-sensitive and

cGMP-activated channels were also compared by measuring the distributions of burst duration (i.e., the duration of a burst of rapid openings and closings), open duration (duration of individual openings, including each individual opening during a burst), and closed duration within a burst.

As expected from the fact

that the power spectra were the same, all of these temporal parameters were the same for light-sensitive and cGMP-activated channels in the same patch.

For the

light-sensitive channel (17 experiments), burst duration, open duration, and closed duration within a burst averaged 0.78 ÷ 0.03, 0.18 ± 0.008, and 0.37 ± 0.02 ms (mean ± s.e.m.), respectively; experiments),

for the cGMP-activated channel (ii

the comparable values were 0.85 ± 0.07, 0.23 + 0.01, and

0.38 + 0.03 ms.

In 7 experiments in which both channels were studied in the same

patch, the value for the cGMP-activated channel divided by the value for the light-sensitive channel averaged 1.09 + 0.08, 1.12 ± 0.08, and 1.01 ± 0.06 for

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Cell-Attached, Light-Sensitive

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20 ms Fig. 6. Comparison of light-sensitive and cGMP-sensitive channel activity within the same patch of outer segment membrane. Traces in the left column show lightsensitive channel activity in a cell-attached recording from an intact rod. The bottom trace is during saturating light, and all others are in darkness. The patch was hyperpolarized by 148 mV. Traces in the right column show cGMPsensitive channel activity in the same patch of membrane after detaching the patch from the rod. In the bottom trace, the intracellular face of the patch was perfused with 0 Ca, 0 Mg Ringer. In all other traces, the intracellular face was perfused with 0 Ca, 0 Mg Ringer + I0 ~M cGMP. The patch electrode was filled with 0 Ca, 0 Mg Ringer. The patch membrane potential was -148 mV. Bandwidth: 0-5000 Hz.

burst duration, open duration, and closed duration within a burst; none of these ratios is significantly different from the ratio of 1.0 expected if the two channels were kinetically identical. We also estimated single-channel conductance from the slope of the relation Uetween single-channel current and holding potential.

For the light-sensitive

channel, the average conductance was 20.5 ± 1.07 pS (n = 17); the conductance of the cGMP-activated channel was slightly larger, averaging 24.0 ± 1.19 pS ( n = l l ) . Over a i00 mV range, holding potential had no effect on the kinetics of either channel.

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TABLE I Parameters of two Lorentzian components fitted to power spectra of lightsensitive and cGMP-activated channel activity. The results are from six experiments in which both types of channel were recorded in the same patch. All numbers are means ± s.e.m. Abbreviations: SI(0) , zero-frequency asymptote of the low frequency component; fl, corner frequency of the low-frequency component; Sh(0), zero-frequency asymptote of the high-frequency component; fh' corner f~equency of the high-frequency component.

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3.3 _+ 1.3 x 10 -4

1038 ± 137

cGMP-activated

1.2 ± 0.3 x 10 -3

83.0 + 4.8

8.8 ± 1.8 x 10 -5

913 ± 122

DISCUSSION The light-sensitive channel and the cGMP-activated channel recorded in the same patch of outer segment membrane before and after excision from the cell were indistinguishable in a number of properties.

These include flicker in the open

state, power spectral density, burst duration, open duration, closed duration within a burst, and the absence of an effect of membrane potential on the temporal properties of the channel.

Channel conductance was also similar (24.0

pS for cGMP-activated channels and 20.5 pS for light-sensitive channels).

These

results indicate that the channel that is regulated by light in intact rods is the same as the channel opened by cGMP in excised outer segment patches.

As

reviewed recently by Stryer (21), biochemical experiments have established that there is a rapid fall in internal cGMP concentration in the outer segment in response to illumination and have detailed the molecular machinery coupling photoisomerization of rhodopsin to hydrolysis of cGMP.

Taken together with the

direct linkage between light-sensitive and cGMP-activated channels reviewed here, this leaves little doubt that cGMP js the internal transmitter that mediates the effect of light on plasma membrane conductance during phototransduction. ACKNOWLEDGEMENT Supported by grant EY03821 from the National Eye Institute, USPHS. REFERENCES I.

Fesenko EE, Kolesnikov SS,

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Matthews G (1986a) Proc. Natl. Acad. Sci. USA 84:299-302

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