Correspondence to: Richard W. Aldrich, Department of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Stanford University, Stanford, CA 94305. Fax:650-725-4463 E-mail:raldrich{at}leland.stanford.edu.
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Abstract |
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We irradiated cyclic nucleotidegated ion channels in situ with ultraviolet light to probe the role of aromatic residues in ion channel function. UV light reduced the current through excised membrane patches from Xenopus oocytes expressing the subunit of bovine retinal cyclic nucleotidegated channels irreversibly, a result consistent with permanent covalent modification of channel amino acids by UV light. The magnitude of the current reduction depended only on the total photon dose delivered to the patches, and not on the intensity of the exciting light, indicating that the functionally important photochemical modification(s) occurred from an excited state reached by a one-photon absorption process. The wavelength dependence of the channels' UV light sensitivity (the action spectrum) was quantitatively consistent with the absorption spectrum of tryptophan, with a small component at long wavelengths, possibly due to cystine absorption. This spectral analysis suggests that UV light reduced the currents at most wavelengths studied by modifying one or more "target" tryptophans in the channels. Comparison of the channels' action spectrum to the absorption spectrum of tryptophan in various solvents suggests that the UV light targets are in a water-like chemical environment. Experiments on mutant channels indicated that the UV light sensitivity of wild-type channels was not conferred exclusively by any one of the 10 tryptophan residues in a subunit. The similarity in the dose dependences of channel current reduction and tryptophan photolysis in solution suggests that photochemical modification of a small number of tryptophan targets in the channels is sufficient to decrease the currents.
Key Words: cyclic guanine monophosphate, electrophysiology, ligand-gated, ion channel gating, ultraviolet light
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INTRODUCTION |
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Electrical signals in visual and olfactory transduction arise from changes in the activity of cyclic nucleotidegated (CNG)1 ion channels (
Measurements of the cyclic nucleotide dependence of currents in photoreceptors (
Do other regions of CNG channels participate in channel gating? What are their energetic contributions to channel activation? It may be difficult to answer these questions using conventional mutagenesis techniques because of an inherent limitation of such approaches: functional assays may yield no detectable signal if the mutation disrupts a residue that is critical to the correct targeting, folding, or activity of the protein. These problems have been partially overcome by the use of chemical reagents to modify the reactive groups (generally the thiol function of cysteine residues) of expressed channels (
Photochemical reactivity is a property of many organic compounds (
330 nm), UV light also modifies cysteine and cystine residues (
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We used UV modification to assess the role of aromatic residues in CNG channel function. UV dramatically reduced the cyclic nucleotidedependent current through Xenopus oocyte membrane patches containing functional bovine retinal (RET) CNG channels. The dose dependence of the current reduction varied with the intensity and wavelength of the exciting light in a manner consistent with the absorption of single photons by tryptophan residues in an aqueous environment. The following paper (
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MATERIALS AND METHODS |
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Heterologous Channel Expression
The bovine rod photoreceptor channel cDNA was cloned from a bovine retinal cDNA library by PCR amplification using oligonucleotide primers that were designed based on the published sequence (
Channel proteins were expressed in Xenopus oocytes by microinjection of 4080 nl of mRNA at a concentration of 0.11 mg/ml. Injected oocytes were incubated at 18°C in ND96 pseudoextracellular solution. This solution contained (mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, 2.5 Na pyruvate, and 1% penicillin/streptomycin. For some experiments, oocytes were incubated at room temperature after injection to increase the rate of channel expression.
Electrical Recordings/Solutions
Macroscopic currents through excised, inside-out membrane patches from Xenopus oocytes expressing RET CNG channels were recorded 221 d after injection. The initial resistances of the borosilicate patch pipets were 0.65 M. To minimize attenuation of the UV light reaching the patches, the tips of the pipets were not coated with sylgard or other dielectrics. Membrane currents were recorded by an Axopatch 200 patch clamp amplifier (Axon Instruments, Inc.), filtered at 2 kHz, and acquired at 10 kHz by an analog-to-digital converter (Instrutech) interfaced with a Macintosh computer (Apple Computer), using Pulse software (HEKA Corp.).
Electrical recordings were made in a perfusion chamber whose top and bottom surfaces were of optical quality quartz for high UV light transmission. Unless otherwise indicated, macroscopic ionic currents were measured in symmetrical sodium solutions. The standard sodium solution contained (mM) 130 NaCl, 3 HEPES, 0.1 EDTA, pH 7.6 (NaOH). Cyclic GMP (Sigma-Aldrich) was present in the bath (intracellular) solution at concentrations ranging from 0 to 20 mM. Solution changes were made by a gravity-driven perfusion system, and required ~30 s. The patches were held at 0 mV and outward currents were recorded at +50 mV. Current amplitudes were taken as the average value of the current during the 76-ms depolarizing pulse, omitting points within 0.5 ms of the step onset. In some experiments, there was a time-dependent droop in the currents upon depolarization, presumably due to ion accumulation/depletion effects (
Relative monovalent cation permeabilities were estimated from currents recorded under biionic conditions. The pipet contained the standard sodium solution described above, while the bath solution substituted 130 mM of NH4Cl, KCl, LiCl, RbCl, or CsCl for NaCl, and also contained 500 µM cGMP (a nearly saturating concentration). Leak currents were recorded in otherwise identical solutions lacking cGMP.
UV Irradiation
Two different UV light sources were used. High-intensity UV was obtained from a pulsed laser. The fundamental infrared emission (1,064 nm) of a Q-switched Nd+3:YAG laser (DCR-I; Quanta Ray) was frequency doubled in a second harmonic generator (SHG) crystal. The 532-nm output of this crystal was frequency doubled in a second SHG crystal, yielding 266-nm pulses of 3-ns duration with typical peak intensities of 1.7 x 1016 photons · µm-2 · s-1 (= 12.7 mW · µm-2). Residual light at 1,064 and 532 nm was eliminated by dichroic mirrors. The laser linewidth was ~0.01 nm full width at half-maximum (FWHM).
Low-intensity UV light was obtained from a continuous-wave xenon arc lamp system (Oriel Corp.) using a stabilized power supply (#68806; Oriel Corp.). The broad-band output from the lamp was collected and collimated with an F/1 fused silica lens. The spectral composition of the UV light reaching the preparation was controlled using a combination of calibrated interference and long-pass filters (Oriel Corp. and Omega Optical). The spectral width of the excitation light was typically 10 nm FWHM. A bulb with a coated envelope that attenuated the UV output below 230 nm was used to minimize the formation of reactive ozone in the perfusion solution. No attempt was made to control the polarization of the light.
The collimated excitation light from either source was directed onto the preparation by reflection off an aluminum first-surface mirror (Newport Corp.) mounted in place of the condenser of an inverted microscope (Axiovert 35M; Carl Zeiss, Inc.). Excitation light from the arc lamp was refocused onto the preparation with a 2.5-cm focal length quartz lens, giving a typical unattenuated light intensity at the patch of 3 x 108 photons · µm-2 · s-1 (= 0.21 nW · µm-2) at 280 nm. The final focusing lens was omitted when the pulsed laser was used. Exposure times were controlled to the nearest millisecond by an electromechanical shutter (Uniblitz Model; Vincent Associates).
The UV power densities for the arc lamp were estimated by mounting a calibrated 50-µm diameter stainless-steel pinhole (Melles Griot) in the experimental chamber at the position of the tip of the patch pipet. The power of the beam passing through the pinhole was measured with a calibrated radiometer (#222; Graseby Optronics). The spatial homogeneity of the beam was estimated by mounting a 10-µm diameter pinhole in the light path; the transmitted power at various locations within a 250 x 250-µm area centered on the preparation was constant to within 11%. At a fixed location, the beam power drifted by <3% over 30 min. The laser power density was determined by directing the collimated beam through a hole of defined area in an opaque block outside the sample chamber and measuring the power of the transmitted beam with a calibrated calorimetric power meter (MC 2500; Scientech, Inc.).
Attenuation of UV light by absorption in the glass walls of the patch pipets was <6%, as calculated by scaling the UV transmittance of thin glass coverslips to the pathlength of the pipet walls (<2 µm). Consistent with the very small calculated attenuation, there was no correlation between a patch's apparent UV sensitivity and the pipet wall thickness. Power densities at the patches were also corrected for reflective losses (~4.5%) at the air-quartz and quartz-water interfaces in the chamber.
Absorption Spectra
Absorption spectra of the near-UV absorbing amino acids were obtained using compounds of the highest commercially available purity: L-tryptophan (99% Sigma-Aldrich); L-tyrosine, L-phenylalanine, L-histidine, L-cysteine hydrochloride monohydrate, and L-cystine (>99%; Sigma-Aldrich); N-acetyl L-tryptophanamide and N-acetyl L-tyrosineamide (Bachem Bioscience); Ala-Tyr, and Tyr-Ala (Sigma-Aldrich).
Absorption spectra were recorded at room temperature in a single-beam UV/visible spectrophotometer (#DU 640; Beckman Instruments) and were obtained as the difference between the spectrum of the chromophore in the indicated solvent and that of the solvent alone. Samples were contained in a 1-cm pathlength fused quartz cuvette (Spectrocell) or a specially made 11-cm pathlength cell. Possible distortions in the spectra were avoided by scanning the wavelength at progressively slower speeds until there were no discernible changes in lineshape or peak position. Spectra were recorded within 1 h of sample preparation to minimize oxidation or other degradation of the sample. Extinction coefficients were obtained by dividing the absorbance by the product of the sample pathlength and concentration, and were similar to published values where available. In some cases, spectra were averaged up to 10x to enhance the signal-to-noise ratio at wavelengths with very low absorbances.
Accurate determination of absorption spectra over the full range of wavelengths tested (250330 nm) was complicated on the long wavelength side of the spectra by a pronounced tail that was presumably caused by light scattering. The onset of the tail corresponds to the abrupt change in the curvature of the tryptophan absorption spectrum at > 320 nm (Fig 2 A).
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A clue to the physical origin of the tail came from the observation that it occurred at different wavelengths for the various compounds studied, but always appeared at wavelengths of low sample absorption. In a spectrophotometer, light scattering by a solute decreases the transmitted light intensity in a manner that is indistinguishable from true light absorption. Because of the exponential relation between transmitted light intensity and absorbance, the relative absorbance error introduced by this artifact is greatest when the true sample absorbance is very low. It is likely in Fig 2 A that the true tryptophan absorbance beyond 320 nm is masked by a much larger apparent absorbance due to light scattering.
The hypothesis that the tail arose from scattering was further tested by measuring tryptophan's fluorescence excitation spectrum, which is not subject to light scattering artifacts. For a molecule possessing a single emitting level whose fluorescence quantum yield is independent of the exciting wavelength, the fluorescence intensity at a fixed detection wavelength is expected to vary in direct proportion to the absorption cross-section at the excitation wavelength. The intensity of tryptophan fluorescence in a 10-nm interval centered at the peak emission wavelength (360 nm) was measured in a specially made fluorimeter. Tryptophan's fluorescence excitation spectrum, scaled to match its absorption spectrum at 320 nm, is shown by the solid circles in Fig 2 A. The absorption estimated from the fluorescence excitation spectrum was smaller than the measured absorbance by a factor of two at 330 nm and by a factor of 30 at 340 nm. These results confirm the idea that light scattering caused the tail in the absorption spectrum at > 320 nm. A corrected tryptophan absorption spectrum (Fig 3) was obtained by fitting a smooth curve through the fluorescence excitation spectrum and splicing this curve from 320 to 340 nm onto the original absorption spectrum from 220 to 320 nm.
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A long-wavelength tail was also present in the absorption spectrum of tyrosine in aqueous buffer at pH 7.6 (Fig 2 B, thin solid trace). The tyrosine tail is more complex than that of tryptophan, and includes two humps centered at ~295 and 340 nm. The extinction coefficients were over 100x smaller at these wavelengths than for the main peak at 275 nm, suggesting that one or both peaks may be due to an impurity. To test this idea, we measured the spectra of the dipeptides Tyr-Ala and Ala-Tyr in aqueous buffer at pH 7.6. We reasoned that the dipeptides are unlikely to contain the same impurities in the same amounts as tyrosine, yet should have a similar absorption spectrum to tyrosine, since they contain the same phenol chromophore as free tyrosine, and the alanine and peptide bond moieties have negligible absorption at wavelengths longer than 220 nm. The spectra of Tyr-Ala and tyrosine (Fig 2 B) at pH 7.6 nearly superimposed at wavelengths shorter than 290 nm, indicating that incorporation into a dipeptide did not perturb the phenol chromophore significantly. No 340-nm peak was observed in Tyr-Ala, suggesting that this feature in the tyrosine spectrum was likely due to an impurity. The absorption at 295 nm, on the other hand, was only two times smaller in Tyr-Ala than in tyrosine, and most of this effect could be accounted for by the loss of the broad 340-nm feature, suggesting that the 295-nm feature is a real component of the tyrosine spectrum.
A small fraction of tyrosine should be present as the tyrosinate anion at pH 7.6, since the pKa of the phenolic hydroxyl group of tyrosine is 10.1 ( > 300 nm in the pH 5.6 spectrum is probably due to light scattering. The spectra of Ala-Tyr (not shown) and Tyr-Ala were indistinguishable at the same pH.
Fig 3 shows the absorption spectra of tryptophan and Tyr-Ala from Fig 2 as well as the spectra of the other near UV-absorbing protein chromophores phenylalanine, cystine, tyrosinate, and the thiolate anion of cysteine. The 293-nm absorption maximum of tyrosinate in Fig 3 confirms the assignment of the 295-nm hump in the pH 7.6 spectra of tyrosine and Tyr-Ala (Fig 2 B) to this species.
Action Spectra of UV-absorbing Amino Acids
This section derives a theoretical expression for computing the wavelength-dependent probability of photochemical modification (the action spectrum) of a UV-absorbing amino acid from the molecule's absorption spectrum and the spectrum of the exciting light. Fig 9 (below) compares the action spectra estimated in this way for various UV-absorbing amino acids to the action spectrum of the UV effect on CNG channel currents.
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The attenuation of light traveling through a system of homogeneous, noninteracting absorbers is described by Beer's Law (
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(1) |
where Jt and J are the intensities of the transmitted and incident light, respectively, (quanta · cm-2 · s-1), is the absorption cross section of the absorbing molecules (cm2 · molecule-1), b is the pathlength of the sample (cm), and c is the absorber concentration (molecules · cm-3).
If the only transmission loss through the sample is due to light absorption, then:
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(2) |
where Ja is the intensity of light absorbed by the sample. Combining Equation 1 and Equation 2, the absorbed intensity is given by Equation 3:
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(3) |
For an optically dilute sample (such as channel proteins in a membrane patch), bc is much less than one, so that:
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(4) |
In this limit, the probability of absorbing a photon, Pa, is the same for all of the absorbers in the sample, and is given by the ratio:
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(5) |
where Qa is the total number of quanta absorbed by the sample and Na is the number of absorbers in the sample. In a brief time interval dt, the number of absorbed quanta, dQa, is given by:
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(6) |
where A is the cross-sectional area of the light beam (cm2). After irradiation for a time (s), the total number of absorbed quanta is obtained by integrating Equation 6:
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(7) |
where use was made of Equation 4. The number of absorbers in the illuminated volume is the absorber concentration multiplied by that volume:
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(8) |
where the illuminated volume is A · b. The probability of photon absorption by an individual chromophore is obtained by combining Equation 5, Equation 7, and Equation 8:
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(9) |
Let denote the (unitless) quantum yield, which is the fraction of absorptions that are followed by a photochemical transformation (Fig 1). The probability that a chromophore will undergo photochemical modification, Pm, is then (Equation 10):
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(10) |
Since the absorption cross sections of the amino acids (Fig 3) are constant to within 1% over the narrow bandwidth of the UV laser (0.01 nm FWHM), the excitation light in the experiments using the pulsed laser may be considered monochromatic. This approximation is not valid, however, for the much broader bandwidth light (10 nm FWHM) from the arc lamp. For those experiments, the probability of photochemical modification of an individual absorber is obtained by integrating Equation 10 over all wavelengths:
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(11) |
where the wavelength dependence of J and is now shown explicitly. For simplicity, the quantum yield was assumed to be independent of wavelength and therefore removed from the integrand in Equation 11. This is a reasonable approximation if absorption at different wavelengths populates the same excited electronic state. The intensity distribution of the exciting light reaching the preparation, J(
), is determined by the spectral output of the arc lamp, L(
), and the combined transmission spectrum F(
) of all optical filter(s) in the beam path:
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(12) |
The total intensity of the excitation beam, Jtot, is the sum of these contributions at all wavelengths:
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(13) |
Defining Sn() as the normalized spectral distribution of the excitation light,
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(14) |
Pm is obtained by substituting Equation 12Equation 13Equation 14 into Equation 11:
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(15) |
All of the factors on the right-hand side of Equation 15 except the quantum yield were measured directly. The total light intensity at the sample, Jtot, was obtained by the calibration procedure described above. The factor Sn(
) was obtained using Equation 14 as follows. For a given set of optical filters, the combined transmission spectrum, F(
), was computed from the product of the transmission spectra of the filters in the beam, which were measured individually in a spectrophotometer. The spectral output of the xenon arc lamp, L(
), was measured using calibrated interference filters and a calibrated radiometer, and agreed with the manufacturer's published spectrum. The product F(
) · L(
) was then divided by its integral over all wavelengths, ensuring that Sn(
) obeyed the normalization condition (Equation 16):
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(16) |
The wavelength-dependent absorption cross sections, (
) (µm2) were obtained from the corresponding extinction coefficients,
(
), in molar-1 · cm-1 (Fig 2 and Fig 3) using the relation
(
) =
(
) · 3.83 x 10-13. The wavelength-dependent photochemical modification probability for each of the UV-absorbing amino acids in Fig 3 was then computed using Equation 15 (assuming a wavelength-independent quantum yield for each compound).
One- and Multi-Photon Photochemistry
The wavelength dependence of the absorption probability (Equation 9) was derived in the previous section starting with the Lambert-Beer law (Equation 1). The latter applies only to so-called one-photon absorption processes, in which a chromophore is excited by the absorption of a single photon. A generalized form of Equation 9 that accounts for absorption by higher-order mechanisms is (
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(17) |
In Equation 17, Pa(r) denotes the probability of photoexcitation by absorption of r photons simultaneously, and r denotes the r -photon absorption cross section [cm(2r) · s(r - 1)]. The steep power-law dependence of the absorption probability on the incident light intensity may be used to determine the mechanism of light absorption in a photochemical modification experiment. For an r -photon absorption process, the probabilities of exciting a chromophore using lights of different intensities (denoted J hi and J lo), are equal if the corresponding exposure durations (denoted
hi and
lo) are related by:
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(18) |
For the special case of a one-photon absorption mechanism, the absorption probability depends only on the total photon dose D, since D = J · .
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UV DoseResponse Relation
This section develops a quantitative theory for the dose dependence of CNG channel macroscopic currents. Several models for the effects of photochemical modification on channel function are considered, and equations for the UV doseresponse relation are derived for each model.
Assume that the channels contain n identical target residues per channel subunit. (Target residues are defined as the amino acids whose UV modification changes the channel current.) Since photochemical modification of the targets occurs randomly, irradiation transforms a patch of N initially identical channels into a complex mixture containing channel subpopulations with different sets of modified targets. The macroscopic current I after exposure to a photon dose D is the sum of the currents through all channel subpopulations. Assume that the effect of UV on the current through any channel depends only on k, the total number of modified targets in that channel. The dose dependence of the patch current is then:
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(19) |
where N(k,D) is the number of channels with k modified targets after a UV dose D [0 N(k,D)
N], and isc(k) and Po(k) are the single channel current and open probability of channels with k modified targets, respectively. The summation index in Equation 19 ranges from 0 to 4n because the channels are assumed to assemble as tetramers (
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(20) |
for all values of D, reflecting the assumption that UV does not "destroy" channels. (As discussed below, functional destruction of a channel is accounted for by setting the product of its unitary current and open probability to zero).
If the photochemical modifications occur independently, then the fraction of channels f containing a total of k modified targets after a dose D is given by the Bernoulli distribution (see, for example,
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(21) |
where 0 f(k,D)
1. The first term on the right side of Equation 21 is the appropriate binomial coefficient, while the exponential terms in brackets represent the probabilities that an individual target is or is not modified, respectively. Before irradiation (Equation 22),
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(22) |
The number of modified targets increases with UV dose until, after an arbitrarily large dose (Equation 23),
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(23) |
Using Equation 19 and the identity (Equation 24)
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(24) |
the fraction of current remaining as a function of dose is
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(25) |
In this formalism, the dose dependence of the UV effect is contained entirely in the f(k,D) factors.
The following sections describe three possible physical models for the effect of UV on CNG channels activated by saturating concentrations of ligand. "Rules" are given for each model that specify the effect of each target modification on the channels' unitary current, isc(k), and open probability, Po(k). The dose dependence of the channel current is obtained by combining these rules with Equation 25.
Independent, "All-or-None" Model
Suppose that photochemical modification of a channel's UV targets has no effect on the current until alteration of a critical number of targets (denoted n*) renders the channel nonconducting. This model does not identify whether UV alters the channels' unitary current or open probability. However, their product is given by:
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(26) |
The UV doseresponse relation obtained by combining Equation 25 and Equation 26 is:
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(27) |
In the limit that all of the targets must be modified to produce the all-or-none effect (i.e., n* = 4n), Equation 27 simplifies to:
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(28) |
In the opposite limit that modification of a single target abolishes the channel current (i.e., n* = 1), Equation 27 reduces to single exponential form (Equation 29):
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(29) |
Proportional Conductance Model
Suppose that each target modification reduces a channel's unitary conductance by an equivalent amount without affecting its open probability, and that the current through a single open channel is reduced to zero after all of the targets are modified. Then (Equation 30 and Equation 31):
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(30) |
and
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(31) |
The UV doseresponse relation (Equation 25) in this case reduces to
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(32) |
Evaluation of Equation 32 yields the unexpected result:
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(33) |
i.e., the UV doseresponse relation is a single exponential function and does not depend on the total number of targets.
Energy Additive Model
Assume that modification of the UV targets does not affect the single channel current,
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(34) |
but instead reduces the macroscopic current by lowering the channels' open probability. The dependence of Po on k is complex and is derived as follows. First, assume that each subpopulation of fully liganded channels with k modified targets has a single closed state (denoted Ck) in equilibrium with a single open state (denoted Ok):
where the equilibrium constant K(k) = [Ok]/[Ck]. Many cyclic allosteric models that are used to describe CNG channel activation reduce to a two-state system when ligand is present at saturating concentration (
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(35) |
and to the standard free energy difference between Ok and Ck, G0(k), by the relation:
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(36) |
In Equation 36, the equilibrium constant is expressed in RT units, where R is the gas constant and T is the absolute temperature. The UV effect on channel current is modeled by assuming that the modification of k targets alters the free energy difference between the channels' open and closed states by a factor G0(k). The reduction in channel current by UV (see below) implies that
G0(k) is unfavorable (i.e., positive) and thus represents the free energy "cost" of modifying k targets. If
G0(k) is assumed to be independent of
G0(0), the free energy difference between the channels' open and closed states before irradiation, then
G0(k) is equal to the sum:
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(37) |
If the targets are assumed to be identical and independent, then the free energy costs associated with their modification will be equal and additive, so that:
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(38) |
Combining Equation 35Equation 36Equation 37Equation 38 yields the desired expression for Po(k):
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(39) |
The UV doseresponse relation is obtained by substituting Equation 34 and Equation 39 into Equation 25:
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(40) |
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RESULTS |
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Effect of UV Light on Uninjected Oocytes
Because UV light changed the conductance of patches from oocytes lacking exogenously expressed channels, it was necessary to characterize this effect before studying the effect of UV on CNG channels. For example, Fig 4 A shows that the small initial leak current (23 pA at +50 mV) through a patch excised from an uninjected oocyte increased slightly after three equivalent doses of 1.08 x 1010 photons · µm-2 at 280 nm, but then increased rapidly after additional exposures, reaching 1,824 pA after a cumulative dose of 5.41 x 1010 photons · µm-2.
Two properties of this current suggested that it represented a UV-activated membrane conductance rather than an increased leakage current due to deterioration of the seal between the pipet wall and the oocyte membrane. First, as shown in Fig 4 B, the current in symmetrical sodium solutions was 15% smaller at +70 than at -70 mV. The currentvoltage relation for this patch (Fig 4 C) demonstrates the inward rectification more clearly. Although the extent of the inward rectification varied between patches, it was observed in all experiments. The leakage current through the seal resistance is not expected to rectify in this manner. The UV-activated conductance also exhibited ion selectivity. The voltage ramps in Fig 4 D were applied in symmetrical 130-mM NaCl solutions (*) or with 130 and 26 mM NaCl in the pipet and bath solutions, respectively. In the latter configuration, the Nernst potentials for Na+ and Cl- are +40 and -40 mV, respectively. The patch current reversed sign at 42.1 mV, indicating that the conductance was strongly selective for cations over anions. Before irradiation, the seal leakage conductance did not exhibit ion selectivity. Taken together, these results suggest that the large currents induced by irradiation of uninjected oocyte patches passed through UV-activated conductance sites.
The markedly nonlinear currentdose (I-D) relations for the UV-activated conductances from nine separate patches are illustrated in Fig 5 A. The slopes of the curves and their positions on the dose axis varied as expected if the patches contained different numbers of UV-activated conductance sites. It should be possible to correct for this difference in scaling by normalizing the results for different patches. However, all attempts to measure the maximal UV-activated current directly were unsuccessful because the current amplitudes invariably exceeded the saturation limit of the patch-clamp amplifier. Moreover, the leak currents after large UV doses were often unstable and sometimes drifted in a time-dependent manner. Lacking a direct estimate for the limiting current amplitude, we analyzed the unnormalized I-D relations using a variant of the all-or-none model described above. Assume that there are N identical, initially dormant UV-activated conductances in a patch, and that activation of a site occurs in an all-or-none fashion upon modification of a critical number (n*) of UV targets in the conductance. The product of the unitary current and open probability of a site containing k modified targets is then (Equation 41):
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(41) |
The current through a patch containing many sites is given by:
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(42) |
The form of Equation 42 suggests that the I-D curves for patches containing different numbers of UV-activated conductances should be the same, save for a vertical displacement, when plotted on double logarithmic coordinates. Indeed, the shapes of the relations were similar when displayed in this manner (Fig 5 B). The invariant shape of the relations is more apparent when the results in Fig 5 B are shifted vertically to correct for differences in the number of UV-activated conductance sites per patch (Fig 5 C). The target number obtained from the fit to the combined results using Equation 42 (solid curve) indicates that each UV-activated conductance site is activated after UV modification of about four or five targets. The quantum yield for modification of the targets in the UV-activated conductance was estimated from a second fitting parameter, which is equal to the product ·
. Since the nature of the target residues is unknown, multiple estimates for
were made using the known cross sections (
) of various UV-absorbing amino acids (Fig 3). The quantum yields were 0.0028 ± 0.0007 for tryptophan targets and 0.014 ± 0.003 for tyrosine targets. These values are comparable with the photochemical quantum yields of these amino acids obtained from UV photolysis studies of various globular proteins (
We were unable to identify pharmacological blockers of the UV-activated conductance. However, other experiments (not shown) indicated that these sites are not affected by cGMP at concentrations up to 1 mM. Since the currents through CNG channels were obtained as the difference between currents in the presence and absence of cGMP, the UV-activated conductance did not interfere with our measurements of UV effects on CNG channel currents.
Possible interference from the UV-activated conductance might also be avoided by carrying out the UV-modification experiments in cells other than Xenopus oocytes. However, possibly related UV-activated conductances have been reported in a variety of mammalian cell lines (
Effect of UV Light on CNG Channel Currents
We assessed the functional effects of UV light on CNG channels by comparing the amplitudes of currents through inside-out membrane patches expressing RET channels before and after irradiation. Patches were rejected for further use if the cGMP-dependent current amplitude varied more than ±5% before UV. Unless otherwise noted, due to the strong absorption of cGMP in the near UV (max = 14,167 M-1 · cm-1 at
max = 253.5 nm, this work), patches were irradiated in the absence of ligand to prevent photochemical modification of cGMP or its cross linking to the channels.
Fig 6 A shows results from a typical experiment. Each UV exposure decreased the cGMP-dependent outward current, as shown by the solid traces with successively smaller amplitudes. The amplitude of the cGMP-activated current, I, normalized by its value before irradiation, I0, is shown as a function of time for this patch in Fig 6 B. and , respectively, indicate trials that were or were not immediately preceded by a UV exposure. The current amplitude decreased in a dose-dependent manner, reaching 34% of its initial value after a cumulative dose of 5.1 x 109 photons · µm-2 (corresponding to the 11th
). The amplitude of this residual current remained constant for the next 48 min in the absence of additional UV, ruling out light-independent mechanisms for the rundown. The irreversibility of the current is consistent with a mechanism in which UV transforms channel residues into stable, covalently modified products (Fig 1).
The last two UV doses for the experiment shown in Fig 6 did not reduce the cGMP-activated current significantly; however, the leak current at this point was much larger than the cGMP-activated current (probably due to the contribution of the UV-activated conductance, see above). Such large, unstable leak currents were observed in most patches before rupture, and prevented an accurate determination of the limiting current amplitude. The leak current for some patches was smaller in proportion to the cGMP-activated currents (perhaps due to a relatively small ratio of UV-activated conductance sites to CNG channels). These results provided an upper bound of ~5% for the limiting value of I/I0.
Strategy for Identifying Target Residues
What are the nature, number, and location of the target amino acid(s) in CNG channels? In principle, modification of any channel residue might alter the current amplitude. We used UV wavelengths longer than 240 nm to restrict the possible UV targets to a small subset of the channel residues. The only amino acids that absorb in this region of the spectrum are tryptophan, tyrosine, phenylalanine, cysteine, and cystine (Fig 3). The identity of the target(s) was narrowed further by comparing the wavelength dependence of the UV effect (the action spectrum) to the wavelength dependence of the absorption probability (the absorption spectrum) of each candidate target amino acid (see Fig 8 Fig 9 Fig 10). Mutagenesis was also used in an effort to identify individual target residues in the channel sequence (see Fig 12).
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Intensity Dependence
The strategy outlined above uses the distinct absorption spectra of the amino acids (Fig 3) as "fingerprints" to identify the UV targets in CNG channels. The light used in the channel photochemical modification experiments (Fig 6) was much more intense than the weak light used to probe the absorption of the amino acids (Fig 2 and Fig 3), raising the possibility that some of the channel targets were modified after simultaneous absorption of multiple photons. Our absorption spectra, however, measure single-photon absorptions exclusively. Since the one-photon and higher-order absorption spectra of a given chromophore may differ radically (
Fig 7A and Fig B, compares the currents through RET channels irradiated with UV from a continuous arc lamp source ( = 280 nm) or from a much more intense pulsed UV laser (
= 266 nm). Each panel displays collected results from many experiments as UV doseresponse relations, which plot the cGMP-activated current, normalized by its pre-UV amplitude, against the logarithm of the cumulative photon dose. Results from independent experiments at each light intensity were pooled by a two-part procedure. The results from each experiment were fit with the simplified version of the "all-or-none" model (Equation 28), in order to estimate D1/2, the UV dose that reduced the current to half of its initial amplitude. Each set of points was then shifted along the abscissa so that the D1/2 for the shifted results was equal to the average D1/2 of the unshifted results for all experiments at similar light intensities. The shifted results allowed a better comparison of the shapes of the UV doseresponse relations than the unshifted points. Since the fits were used only to characterize the slopes of the curves and their positions on the abscissa, the validity of conclusions drawn from comparisons of these parameters are independent of the correctness of the all-or-none model.
The UV doseresponse relations for channels irradiated with low- and high-intensity UV were strikingly similar, despite the eight order of magnitude difference in light intensities. The similarity in the D1/2 values indicates that the UV effect depended only on the total photon dose, and not on the rate at which photons were delivered to the patches. This finding is consistent with absorption by a one-photon absorption mechanism, but not two-photon or higher-order mechanisms. The small difference between the wavelengths of the two light sources has only a slight (<7%) effect on the channels' UV sensitivity (see Fig 8 and Fig 9, below). The maximal slopes of the relations were also similar for low- and high-intensity irradiation, suggesting that the nature and relative amounts of the various photoproducts formed were independent of light intensity.
The results for low-intensity UV were obtained by irradiating the channels in the absence of cGMP, while those for high-intensity UV were obtained by irradiating the channels in the presence of a half-saturating concentration of cGMP (100 µM). However, similar results were obtained for channels irradiated with high-intensity UV in the absence of cGMP, indicating that the effect of UV was not dependent on the activation state of the channels. For high-intensity UV, we confined the analysis to results on channels irradiated in the presence of cGMP, due to the much larger number of experiments performed under those conditions.
The D1/2 values for the results in Fig 7A and Fig B, were converted into 1/2 values (the exposure times required for 50% current reduction) using the relation (Equation 43):
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(43) |
and the ratio of 1/2 values is shown in Fig 7 C ("data"). This ratio is compared with the predicted
1/2 ratios computed from Equation 18 for one- and two-photon absorption mechanisms. The Jlo and Jhi factors in Equation 18 were obtained from the average light intensities used in the low-intensity (Fig 7 A) and high-intensity (B) experiments, respectively. The experimental
1/2 ratio was similar to the predicted ratio for a one-photon absorption mechanism, but was ~8 log units smaller than the ratio for absorption by a two-photon mechanism. (The
1/2 ratios predicted for three-photon and higher-order absorption mechanisms were even larger.) This analysis indicates conclusively that UV modification of the target residue(s) in the channels followed absorption by a one-photon mechanism, and validates the strategy outlined above for identifying the channel targets.
Wavelength Dependence
Fig 8 shows the wavelength dependence of the UV effect on currents through RET channels activated by saturating cGMP. The spectral width of the exciting light in each experiment was ~10 nm FWHM, and the range of peak wavelengths employed (250330 nm) spanned the lowest energy one-photon absorption bands of all possible target amino acids (Fig 3). UV decreased the channel currents in a dose-dependent manner at all wavelengths studied. The efficacy of photons of different wavelengths varied greatly, however, as evidenced by the large variations in the horizontal positions of the corresponding UV doseresponse relations.
Slope factors and D1/2 values were obtained by fitting the relations with Equation 28. On a quantal basis, 280-nm photons were most effective, with D1/2 = (4.6 ± 0.3) x 109 photons · µm-2 (mean ± SEM). Photon efficiencies fell off asymmetrically about the maximum at 280 nm: D1/2 was 74-fold larger at 310 nm compared with 280 nm, but only 1.5-fold larger at 250 nm than at 280 nm. Photons of 330-nm wavelength, the longest tested, were 500x less effective than 280-nm photons.
Fig 9 A plots the channels' UV sensitivity (defined as the reciprocal of D1/2) as a function of wavelength. Also shown are the predicted action spectra for tryptophan and tyrosine targets, computed from the corresponding absorption spectra using Equation 15. The predicted action spectra differed from the corresponding absorption spectra due to corrections for the finite bandwidth of the exciting light and for wavelength-dependent variations in the lamp intensity. The magnitude of these corrections (see MATERIALS AND METHODS) were largest at the wavelengths with the steepest absorption slopes. For tryptophan, the correction procedure increased the absorption probability at 280 nm relative to that at 300 nm by ~45% compared with the uncorrected values. No other corrections or adjustments were made to any of the spectra in Fig 9 A aside from the normalization at 270 nm.
The channels' action spectrum (Fig 9, ) matched the calculated tryptophan action spectrum (solid line) quantitatively over most of its extent (260310 nm). This close agreement indicates that the main photochemical effects on the channels were initiated by tryptophan absorption. The tyrosine spectrum declined more steeply than the channels' action spectrum, particularly on the long-wavelength side of the peak, while the spectra of phenylalanine, cystine, and cysteine targets (not shown) bore little resemblance to the action spectrum and are omitted for clarity (see Fig 3).
The channels' UV sensitivity was 4.5x larger at 320 nm and 7x larger at 330 nm than expected for absorption by tryptophan. At 250 nm, the sensitivity was 1.3x larger than expected for tryptophan absorption. These discrepancies may result from wavelength-dependent variations in the nature of the photoproducts or their quantum yield of formation. Possible sources of these variations are considered in the DISCUSSION.
The slope factors (n* in Equation 28) for the UV doseresponse relations clustered between 1.12 and 1.60 (indicated by the dashed lines in Fig 9 B) for excitation wavelengths between 260 and 310 nm. The wavelength invariance of the slope over this region of the spectrum suggests that wavelength affects only the probability of absorption, and not the efficiency or nature of the subsequent photochemistry. The UV doseresponse relation was significantly steeper for excitation at 250 nm (n* = 1.92), and significantly shallower for 320 or 330 nm UV (n* = 0.69 and 0.87, respectively). Interestingly, the channel action spectrum and the tryptophan spectrum deviated at these same wavelengths (Fig 9 A). The simplest explanation for these results is that UV irradiation of the channels generated at least two different photoproducts with distinct effects on channel function. The slopes of the UV doseresponse relations at 250, 260310, and 320330 nm would likely vary if the photoproducts were formed in different relative proportions after irradiation at those wavelengths. However, the slopes of the curves are not expected to vary with wavelength if UV produced a single photoproduct with a wavelength-dependent quantum yield, or generated multiple photoproducts in the same relative proportions at different wavelengths.
Effect of Environment on Tryptophan Absorption
Previous studies indicated that inserting tryptophan into a protein matrix shifts max, the wavelength of maximal absorbance, by about +4 nm at most, and has negligible effect on the spectral lineshape (
max to environment is in marked contrast to the extremely large protein-induced shifts (up to -45 nm) in the wavelength of maximal tryptophan fluorescence (
What is the environment of the target tryptophans in the channel? We approached this question by measuring the spectrum of the tryptophan derivative N-acetyl L-tryptophan amide (NATrpA) in a variety of organic solvents and comparing it with the action spectrum of channel modification. NATrpA is a popular model for protein tryptophans because of the similarity of its acetyl and amide linkages to the peptide linkages of tryptophans in proteins (max by only +1.0 and +1.3 nm, respectively, relative to the value for tryptophan in aqueous buffer. Ethyl alcohol, which contains one more nonpolar methylene group than methyl alcohol, shifted the spectrum by +2.6 nm. Dimethylformamide and dimethylsulfoxide, which contain many of the functional groups present in nonpolar amino acids, shifted the spectra by +3.4 and +4.1 nm, respectively. These values are comparable with the largest shifts observed for buried tryptophans in proteins. The lineshape of the spectra were not altered appreciably by the solvent, as evidenced by their unchanged bandwidth and the preservation of distinct "shoulders" on the short and long wavelength sides of the peak in all solvents tested.
Because the tryptophan spectra are relatively flat near the peak at 280 nm, but very steep at 300 nm, shifts of only a few nanometers in max may alter the ratio of absorption at these two wavelengths by a large factor. The 280/300-nm absorption ratio varied between values of 15.7 for tryptophan in buffer and 4.3 for NATrpA in DMSO, with intermediate values in the other solvents (Fig 10 B). The 280/300-nm UV sensitivity ratio of CNG channels (17.1 ± 2.0) is consistent with the absorption of tryptophan in aqueous solution, but not with the absorption by NATrpA in any of the organic solvents tested, suggesting that the target tryptophans in the channels occupy a water-like chemical environment.
UV Photolysis of Tryptophan in Solution
Do tryptophan targets in CNG channels undergo the same photochemical reactions as free tryptophan in solution? The photochemistry of tryptophan in a protein may differ from that in solution because the photoexcited residues may react with surrounding protein residues (
We monitored the photolysis of tryptophan in aqueous solution spectrophotometrically. Irradiation at 280 nm converted the initial tryptophan absorption peak centered at 280 nm into a broad, diffuse band (Fig 11 A). The absorbance changes indicate that the tryptophan photoproducts absorb more strongly than tryptophan from 230270 nm and at wavelengths longer than 290 nm, but less strongly than tryptophan from 270 to 290 nm. The nature of the spectral changes in Fig 11 A are consistent with the formation of the photoproducts in Fig 1: kynurenine absorbs UV maximally at 230, 257, and 360 nm, while N-formyl kynurenine absorbs strongly at 235 and 318 nm (
Assuming that the components in the solution absorb light independently, the sample absorbance after a UV dose D is the sum of the absorbances of the solutes (
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(44) |
In Equation 44, i and ci(D) are the extinction coefficient and molar concentration of the ith component, respectively, and b is the sample pathlength. For the scheme in Fig 1, the sample absorbance is expected to vary with dose according to
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(45) |
where A(D) is the absorbance change as a function of dose,
A(
) is the maximal absorbance change,
is the absorption cross section of tryptophan at the exciting wavelength, and
i is the photochemical quantum yield for forming the ith photoproduct. Photochemical modification of tryptophan by 280 nm UV was monitored at 280 and 310 nm (Fig 11 B). The absorbance change at each probe wavelength depended exponentially on UV dose, as shown by the fits of Equation 45 (solid curves) to the results. The apparent quantum yield for the absorbance change at 310 nm (0.026 ± 0.002) was smaller than that at 280 nm (0.055 ± 0.005). A likely explanation for the difference in quantum yields at the two probe wavelengths is that the absorbance change at 280 nm reflects mainly the disappearance of tryptophan, which depends on the quantum yield for forming all primary photoproducts, while the absorbance change at 310 nm reflects both the formation of N-formyl kynurenine and its conversion to secondary photoproducts (see DISCUSSION).
The UV dose that caused a half-maximal change in tryptophan absorbance at 280 nm (5.8 x 109 photons · µm-2, Fig 11) was remarkably similar to the D1/2 value for current reduction in CNG channels activated by saturating cGMP at the same wavelength (4.6 x 109 photons · µm-2, Fig 8). The similarity of these values suggests that UV modifies tryptophan in solution and tryptophan targets in the channels with nearly equal efficiencies.
UV Sensitivity of Tryptophan Mutants
Which tryptophans in CNG channels are the UV targets? Fig 12 A is a schematic diagram showing the location of the 10 tryptophan residues in a CNG channel subunit. Many of the tryptophans are located in channel domains that are involved in activation gating, such as W9, W166, and W178 in the N-S2 domain, W353 in the channel pore, and W435 and W440 in the C-linker.
We attempted to locate the UV targets by examining the UV sensitivity of channels in which tryptophans were replaced by other amino acids. If the mutations have no effect on the channels other than to alter their tryptophan content, then the UV sensitivity of some of the mutant channels should be reduced or possibly abolished. Cyclic GMP-activated currents were not detected for a tryptophan-less channel, or for any of a number of mutant channels that lack more than one of the native tryptophans in each subunit. In contrast, all of the 10 possible mutants lacking a single tryptophan per subunit yielded functional channels. Exposure to 280 nm UV reduced the current through each of these mutants to a small fraction of its initial value (Fig 12 B), indicating that the channels must contain more than one target tryptophan residue per subunit.
The UV sensitivities of 9 of the 10 tryptophan mutants were similar to that of wild-type channels (Fig 12 C). In addition, the slopes of their UV doseresponse relations (average n* = 1.33 ± 0.04) were similar to that of wild-type channels (n* = 1.39 ± 0.05), suggesting that the mutations did not alter the mechanism of the current reduction by UV.
One of the mutant channels was unusual. W353Y channels, in which tyrosine replaces a highly conserved tryptophan residue in the channel pore, were approximately seven times more UV sensitive than wild-type channels. This result was unexpected since W353Y channels contain fewer tryptophan residues than wild-type channels. Possible mechanisms for the anomalous behavior of W353Y channels are considered below and in the accompanying paper (
Modeling of UV DoseResponse Relation
Does UV reduce CNG channel currents mainly by lowering the channels' open probability or by decreasing their unitary conductance? The UV-activated conductance (see above) made direct single channel measurements before and after UV impractical. Therefore, we adopted the alternative approach of analyzing the 280-nm UV doseresponse relations of wild-type and W353Y mutant channels using the three physical models for UV modification described in the THEORY section (above). For each model, the quantum yield for target modification was estimated for various target numbers, using the fitting parameter ·
(see Equation 28, Equation 33, and Equation 40), and an absorption cross section of 2.17 x 10-17 cm2 for free tryptophan in aqueous solution at 280 nm (Fig 3). The total number of UV targets per channel was assumed to be 4n (where n is the number of targets per subunit), consistent with the tetrameric composition of the channels (
The all-or-none model assumes that UV has no effect on channel function until a critical number of target residues (n*) are modified, which renders the channel nonconducting. The model does not distinguish between UV effects on the channel's unitary conductance and open probability (see Equation 26). Fits of this model (Equation 28) to the UV doseresponse relation of wild-type channels for various values of n* are shown in Fig 13 A. The simulated curves were very sensitive to the values of n* and the quantum yield . Varying
shifted the curves horizontally without altering their shape, while changing n* affected both the position of the curves on the abscissa and their slope. The fits were not sensitive to the total number of targets, although increasing this parameter had the effect of increasing the total effective absorption cross section of the targets, which caused a proportional decrease in the estimated quantum yield (Equation 28). For all possible values of n (1
n
10), the relations for wild-type channels were fit equally well using critical target numbers of 1 or 2. The curves for n* = 1 were slightly shallower than the observed relations, while the curves for n* = 2 were slightly steeper than the experimental points. The simulated relations for n* > 2 were much steeper than the experimental points, establishing an upper limit of 2 for the critical target number. The quantum yields obtained from the fits depended on both n and n*, and varied from 0.0018 for n = 10, n* = 1 to 0.048 for n = 1, n* = 2.
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It is reasonable to assume that the "effective" quantum yield for modifying targets in W353Y mutant channels is less than or equal to that for wild-type channels, since the mutant channels contain one less tryptophan residue. (We define effective quantum yield as the quantum yield for modifying one target multiplied by the number of targets.) If the other model parameters (i.e., n and n*) were kept the same as for wild-type channels, decreasing the value of had the effect of shifting the simulated curves to the right along the abscissa. This shift is opposite the observed leftward shift of the UV doseresponse relation for W353Y channels compared with wild-type channels (Fig 12 B). The only way to shift the relation for W353Y channels to the left of the relation for wild-type channels was by decreasing n*. Since n*
2 for wild type channels (see above), this condition could only be satisfied if n* = 1 for W353Y channels. Simulated UV doseresponse relations were generated for all possible values of n for W353Y channels, assuming n* = 1 and using the quantum yields obtained for wild-type channels with n* = 2 and the same value of n. The simulated curves for W353Y were well to the right of the experimental points for all values of n. This failure of the all-or-none model to fit the UV doseresponse relations of the wild-type and mutant channels simultaneously suggests that UV reduced the channel currents in a graded fashion.
The proportional conductance model assumes that each UV target modification decreases the channels' unitary current by an equivalent amount. This model might apply if the channels' unitary conductance decreased stepwise as targets in each subunit were modified. In fact,
The fits to the UV doseresponse relation of wild-type channels were identical for all values of n (see Equation 33) and were somewhat shallower than the results (Fig 13 B). The proportional conductance model was ruled out because it could not reproduce the leftward-shift of the UV doseresponse relation of W353Y channels relative to that of wild-type channels without violating the constraint that the photochemical quantum yield for W353Y channels must be less than or equal to that for wild-type channels (see above).
The energy additive model assumes that UV lowers the channels' open probability by stabilizing the closed channel relative to the open channel. For simplicity, it was assumed that each target modification exerts an equal and additive effect (represented by the parameter G0(1), see Equation 40) on the free energy difference between the channels' open and closed states. The model fit the UV doseresponse relation of wild-type channels well for all possible target numbers (1
n
10, Fig 13 C). The free energy "cost" of modifying a single target,
G0(1), varied inversely with the value of n, and the total energetic cost of modifying all of the UV targets, 4n ·
G0(1), remained roughly constant for different values of n. In contrast to the all-or-none and proportional conductance models, the energy additive model also reproduced the observed leftward shift of the UV doseresponse relation of W353Y channels. The relations for wild-type and W353Y channels could be fit simultaneously using the same values of n,
, and
G0(1) for both channels, but only if
G0(0) was much more positive for W353Y channels. This finding suggests that the mutation lowered the channels' open probability substantially. Other observations are consistent with this idea. Cyclic GMP-dependent currents were not observed for this mutant initially, but were rescued by the application of low cytoplasmic concentrations of Ni+2 ions to the patches, which increases the open probability of RET channels (
The fitting parameters for all three models are compiled in Table 1.
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UV Effect on Ion Selectivity
We favor the idea that the main effect of UV is on the open probability of CNG channels, but cannot rule out some effect on the channels' unitary conductance. We tested this model-dependent conclusion in another way by measuring the ion selectivity of modified channels. Although CNG channels do not discriminate between monovalent cations as finely as related channels such as Shaker potassium channels, they do exhibit ion selectivity. For example, they are about nine times more permeable to ammonium ions than to cesium ions (
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(46) |
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In Equation 46, F is the Faraday constant and z is the charge on the ions (+1). The magnitude and rank order of the relative cation permeabilities were very similar before and after irradiation.
The relative effect of UV on the amplitudes of currents carried by the different monovalent cations provided a second measure of possible UV effects on permeation. The ratio of the outward current amplitudes before and after UV were computed for each ion after first correcting the currentvoltage relations for the small changes in reversal potential after irradiation. The fractional reduction in current was similar for all ions tested (Fig 15 B). The results in Fig 14 and Fig 15 thus provide no evidence for UV effects on ion permeation through CNG channels, consistent with the idea that the main effect of UV is on the channels' open probability.
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DISCUSSION |
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Interpretation of Action Spectrum
The irreversible reduction of current through CNG channels by UV light involves two distinct steps. Membrane particles that we call "initiators" absorb the UV photons. The energy of the absorbed photons drives subsequent photochemical transformations of membrane particles that we refer to as "targets." It is the latter process that reduces the amplitude of the channel currents. Since UV reduced the currents to 5% of their pre-UV amplitude, it is likely that the targets are intrinsic to the CNG channels. We cannot rule out the alternate possibility that the targets are located in other membrane components that interact strongly with the channels.
The close agreement between the channels' action spectrum and the absorption spectrum of tryptophan in aqueous solution (Fig 9 and Fig 10) indicates that the main initiators of target modification are tryptophan residues occupying a water-like environment. Three distinct photochemical mechanisms might be triggered by absorption in tryptophan. Mechanism I (depicted in Fig 1) assumes that the photochemical modifications that reduce the channel currents occur in the same tryptophan residues that initially absorb UV.
Mechanism II assumes that the initial light-absorbing tryptophans are not themselves modified by UV, but rather that these initiators sensitize the excitation of other amino acids (the target residues) by resonance energy transfer ( B or E
E. The solvent-exposed initiator tryptophans in CNG channels (Fig 10) thus are unlikely to act as efficient photosensitizers of other tryptophans, ruling out energy transfer as an important mechanism for target modification in the channels.
Mechanism III proposes that the photo-excited initiator tryptophan residues react with nearby residues in the channel protein, and that the covalent modification of these neighboring amino acids (the target residues) reduces the channel current. The target residues need not be tryptophans. For example, in some proteins, photoexcited initiator tryptophans may undergo redox reactions with nearby disulfide targets (
Although we cannot rule out Mechanism III, we note that the half-maximal doses for tryptophan photolysis in solution (Fig 11 B) and channel current reduction (Fig 8 A) by 280 nm UV are very similar. This similarity suggests that the photochemistry of the targets in the channels is like that of isolated tryptophans. Therefore, we favor the idea that UV absorption by channel tryptophans, and the subsequent photochemical modification of those same residues, causes the current reduction in CNG channels (Mechanism I).
As noted above, the CNG channel action spectrum and the tryptophan absorption spectrum differed at 250 and 320330 nm (Fig 9 A). There are several possible reasons for these discrepancies. RET channels contain a large number of residues other than tryptophan that absorb in the 250330-nm range, including 30 tyrosines, 33 phenylalanines, and 7 cysteines per subunit. These other amino acids may contribute additional spectral components to the channels' action spectrum if they initiate photochemical reactions that alter the channel currents. We tested for such components by fitting the action spectrum to linear combinations of the absorption spectra of tryptophan and the other UV-absorbing amino acids. As shown in Fig 16, adding together tryptophan and cystine absorption spectra in roughly equal proportions (based on the molar extinction coefficients of the two compounds, see Fig 3) provided an excellent fit to the channel action spectrum over the entire range of wavelengths studied. The action spectrum is a weighted average that depends on: (a) the number of each type of initiator residue, (b) the absorption spectra of the individual initiators, (c) the relative nature and quantum yields of the photoproducts formed from modification of the target residues, and (d) the magnitude of the current loss associated with formation of each of these photoproducts. These complications prevent us from estimating the relative effects on channel function of modifying a single tryptophan target compared with a single cystine target. However, the fit to the action spectrum in Fig 16 suggests that tryptophan photolysis dominates the UV effect at excitation wavelengths shorter than 315 nm, while cystine photolysis is the more important effect at longer wavelengths.
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Experiments using the cross-linking reagent copper phenanthroline (CuPhe) indicate that disulfide formation between cysteines in the amino- and carboxy-terminal regions of CNG channels stabilize the channels' open state (
Adding tyrosine or phenylalanine spectral components to the tryptophan absorption spectrum did not improve the fit to the channels' action spectrum, suggesting that these residues are not modified by UV, or that their photochemical modification does not alter channel function significantly.
A second mechanism may also contribute to the channels' anomalously high UV sensitivity (relative to tryptophan absorption) at wavelengths longer than 320 nm. The absorption cross section of the tryptophan photoproduct N-formyl kynurenine (NFK; Fig 1) is so much larger than that of tryptophan at 320 nm (
3,180 vs. 3 M-1 cm-1, respectively, see
320 nm) excitation wavelengths (Fig 9 B).
Target Number
How many tryptophan targets (denoted neff) must be modified to reduce the channel current by 50% or more? The UV doseresponse relations of wild-type and W353Y mutant channels were inconsistent with an all-or-none model for the UV effect (Fig 13 A), indicating that the current reduction required modification of more than one target. Although the relations were consistent with the energy-additive model, the analysis did not provide an upper limit for neff.
Our results also did not provide an exact estimate for the total number of tryptophan targets (denoted ntot) in CNG channels. However, two lines of evidence suggest that only a subset of the 10 tryptophans per channel subunit are targets. First, the channels' action spectrum is similar to the spectrum of free tryptophan in solution, but inconsistent with the spectrum of fully or partially buried tryptophans (Fig 10). Many of the channel tryptophans are in transmembrane segments of the channel (Fig 12 A), where they are probably not exposed fully to solvent. Second, the UV dose required to photolyze tryptophan in aqueous solution (Fig 11) was very similar to the dose that reduced the currents through CNG channels (Fig 8). The simplest explanation for this similarity is that the channels contain a small number of targets, and that their photochemical quantum yields are similar to the quantum yield for free tryptophan in solution. Since all 10 tryptophan point mutants remained sensitive to UV, the channels must contain more than one target per subunit. This observation is consistent with the conclusion (see above) that neff > 1, since ntot neff.
We were unable to locate the tryptophan targets within the channels' amino acid sequence, since all mutant channels that replaced individual tryptophans by other amino acids remained sensitive to UV. Since the channels contain more than one target residue per subunit (see above), it may be necessary to remove several tryptophans by mutagenesis to identify the UV targets. Results in the following paper (
Our results provide no information on the total or effective number of cystine targets in the channels.
Mechanism of Current Reduction by UV
Our analysis of the UV doseresponse relations of wild-type and W353Y mutant RET channels (Fig 13) suggests that UV reduces CNG channel currents primarily by lowering the channels' open probability rather than by decreasing their unitary conductance. This notion is supported by the lack of a UV effect on the channels' ion selectivity (Fig 14 and Fig 15). It is unlikely that changes in the channels' unitary conductance sufficient to produce the large current reduction by UV (95%) could have occurred without significant changes in the structure of the channel pore. However, the channels' ion selectivity was essentially unchanged by UV (Fig 15 A). It might be argued that UV effects on the channel pore were missed in those experiments because the current after UV was carried exclusively by unmodified channels. However, this idea implies an all-or-none mechanism for the UV effect, which was ruled out previously (Fig 13 A). The following paper (
We attempted to assess directly the magnitude of the UV effects on the channels' open probability and unitary conductance using single-channel methods. This approach was unsuccessful, however, because the currents through patches containing single CNG channels were overwhelmed by currents through UV-activated conductance sites (results not shown).
State Dependence of UV Effect
The ability of many reagents to block or chemically modify CNG channels depends on the channels' conformational state. For example, the pore blocker tetracaine binds much more tightly to closed than to open channels (
In contrast to the results for blockers and chemical modifying agents, we found that UV reduced CNG channel currents with equal effectiveness when the channels were exposed to half-saturating cGMP or no cGMP (Fig 7A and Fig B). A significant difference between the chemical and photochemical modification experiments is that the channels' conformation does not affect the "access" of photons to their targets appreciably: the absorption cross section of tryptophan is relatively insensitive to its environment (
A more subtle form of state dependence that we did not investigate is the possibility that the channels' action spectrum may be sensitive to the conformational state of the channels during irradiation.
Relation to Previous Work
Before the advent of modern molecular biological techniques, UV light was a preferred method for altering the sidechains of protein amino acids (
The UV sensitivity of gramicidin channels is conferred by their tryptophan residues (
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Footnotes |
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1 Abbreviations used in this paper: CNG, cyclic nucleotidegated; RET channel, bovine retinal CNG channel; FWHM, full-width at half-maximum; I-D, currentdose; NATrpA, N-acetyl L-tryptophanamide.
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Acknowledgements |
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The authors thank Cynthia Adams and Christine Warren for experimental assistance in the early stages of this work, Steven Boxer for the generous loan of the Nd+3:YAG laser, Laura Mazzola for critical reading of the manuscript, Steve Baylor, Dan Cox, and Alan Miller for helpful discussions, and Robert Schneeveis for excellent technical assistance.
This work was supported by grants from the National Institutes of Health (NS23294), the National Eye Institute (EY01543), and the McKnight Foundation. Richard W. Aldrich is an investigator with the Howard Hughes Medical Institute.
Submitted: 27 March 2000
Revised: 1 June 2000
Accepted: 5 June 2000
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