ATP Consumption by Mammalian Rod Photoreceptors in Darkness and in Light

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Current Biology 18, 1917–1921, December 23, 2008 ª2008 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2008.10.029 Report ATP Consumption by Mammalian Rod Photoreceptors in Darkness and in Light Haruhisa Okawa,1 Alapakkam P. Sampath,2 transduction. To calculate the energy required to pump out Simon B. Laughlin,3 and Gordon L. Fain4,* Na+ entering through the channels, which must be removed 1Neuroscience Graduate Program to keep the cell at steady state, we assumed a normal dark 2Department of Physiology and Biophysics resting current of 25 pA; mouse rod responses in excess of Zilkha Neurogenetic Institute 20 pA are routinely observed in our laboratories. Approxi- USC Keck School of Medicine mately 7% of the current is from Na+/Ca2+ exchange, so we Los Angeles, CA 90089 could directly estimate the Na+ influx in darkness (see Supple- USA mental Data available online). We then divided by 3 to calculate 3Department of Zoology ATP consumption by the Na+/K+ pump, because three Na+ University of Cambridge ions are pumped out of the rod for every ATP. The dependence Downing Street of ATP consumption on light intensity over the physiological Cambridge CB2 3EJ range was evaluated from measurements of mouse rod UK current responses to steady illumination [10] and are shown 4Departments of Physiological Science and Ophthalmology in Figure 1A. ATP utilization falls by an amount equivalent to University of California, Los Angeles 2.3 3 106 ATP s21 per pA decrease in inward current, from Los Angeles, CA 90095-7000 about 5.7 3 107 ATP in darkness to zero at an intensity of about USA 104 Rh* s21, which closes all the cGMP-gated channels. For enzymatic components, photoexcited rhodopsin (Rh*) produces the exchange of GTP for GDP on the a subunit of Summary transducin (Ta); the GTP is hydrolyzed by the GTPase activity of Ta in conjunction with the proteins of the GAP complex Why do vertebrates use rods and cones that hyperpolarize, (see [11]). Although it was once thought that a single Rh* could when in insect eyes a single depolarizing photoreceptor produce as many as 500 Ta-GTP molecules during its lifetime, can function at all light levels [1, 2]? We answer this question more recent measurements indicate that, in vivo, this number at least in part with a comprehensive assessment of ATP is closer to 20 in a dark-adapted mouse rod [12]. ATP is also consumption for mammalian rods from voltages and required to phosphorylate rhodopsin. Though under certain currents and recently published physiological and biochem- conditions as many as 6–7 phosphate groups can be attached ical data. In darkness, rods consume 108 ATP s21, about the to the rhodopsin molecule [13], under most conditions many same as Drosophila photoreceptors [3]. Ion fluxes associ- fewer sites appear to be phosphorylated, probably no more ated with phototransduction and synaptic transmission than three [14, 15]. Reduction of all-trans retinal to all-trans dominate; as in CNS [4], the contribution of enzymes of the retinol and regeneration of 11-cis retinal in the retinal pigment second-messenger cascade is surprisingly small. Suppres- epithelium would require another 2–3 ATP molecules. Alto- sion of rod responses in daylight closes light-gated chan- gether, we therefore estimated the total number of ATPs re- nels and reduces total energy consumption by >75%, but quired for transducin activation and response termination by in Drosophila light opens channels and increases consump- multiplying the number of Rh* by 25 (Figure 1A). These are prob- tion 5-fold [5]. Rods therefore provide an energy-efficient ably overestimates in bright illumination, because the rate of mechanism not present in rhabdomeric photoreceptors. rhodopsin kinase is apparently accelerated in light [16],and Rods are metabolically less ‘‘costly’’ than cones, because this would reduce the number of TaGTPs formed per Rh*. cones do not saturate in bright light [6, 7] and use more The ATP required for cGMP synthesis as a function of light ATP s21 for transducin activation [8] and rhodopsin phos- intensity was calculated (see Supplemental Data) from the phorylation [9]. This helps to explain why the vertebrate ret- dependence of the cGMP cyclase on Ca2+ concentration ina is duplex, and why some diurnal animals like primates [17], the maximal cyclase activity of mouse retina [18], and have a small number of cones, concentrated in a region of the free-Ca2+ concentration in the mouse rod outer segment high acuity. [19]. We assumed that in steady illumination, the rod free- Ca2+ concentration scaled with the value of the outer segment Results and Discussion current [20]. Figure 1A gives the ATP required for cGMP synthesis as a function of light intensity, as well as the total ATP Outer Segment consumption of transduction in the outer segment, which + Vertebrate rods and cones have two distinct regions: an outer closely follows that required for the influx of Na except at segment containing the enzymes and channels of the photo- the brightest intensities. Thus in darkness [21, 22] and over transduction second-messenger cascade and an inner seg- the physiological operating range of the rod, the extrusion of + ment with mitochondria, ion pumps, nucleus, and presynaptic Na dominates the consumption of ATP required for the outer terminal. In the outer segment, the principal contributors to segment, whereas the contribution from the biochemistry of ATP consumption are the Na+ influx through cGMP-gated signal transduction is unexpectedly small. channels and enzymatic processes necessary for signal Inner Segment Because the major contributors to ATP consumption in the *Correspondence: [email protected] inner segment are voltage-gated influxes of Na+ and Ca2+, Current Biology Vol 18 No 24 1918 Figure 2. Perforated-Patch Current-Clamp Recordings of Membrane Po- tential from Mouse Rod Photoreceptors in Dark-Adapted Retinal Slices (A) Response waveforms to 5 s light steps of intensities 33, 83, 210, 520, 21 21 Figure 1. Principal Contributors to ATP Consumption in Mammalian Rod 1300, 3200, and 8100 Rh* rod s . Data from 8 cells were averaged over the Physiological Range of Steady Light Intensities individually for each background light intensity and were corrected for a measured liquid junction potential of w10 mV. Average input resistance (A) Outer segment. ATP required for extrusion of Na+ entering cGMP-gated was 5 GU and average access resistance, 300 MU. Inset: Same responses channels (filled square), transducin GTP hydrolysis and rhodopsin phos- at higher temporal resolution showing rapid relaxation or ‘‘nose’’ in voltage phorylation (open square), synthesis of cGMP (open circle), and sum of all waveform at high light intensities caused by activation of i . of these processes (X). h (B) Response-intensity curve of voltage response, averaged during the (B) Inner segment and total rod ATP consumption. ATP required for extru- interval 4.5–5 s from the responses in (A), as function of steady light inten- sion of Na+ entering through i channels (open square), extrusion of Ca2+ h sity. Errors are standard deviations. entering voltage-gated channels at synaptic terminal (open circle), sum of Methods: Experiments were conducted in accordance with protocols ATP for Na+ and Ca2+ extrusion (open triangle), and sum of ATP turnover approved by institutional IACUC committees. Light-evoked membrane in whole rod (closed circle). For the global sum, we used light intensities potential changes during current-clamp recordings were measured in re- at which voltage responses had been recorded (Figure 2B) and estimated sponse to background light steps of 5 s duration delivered from an LED photocurrents by interpolation from step response-intensity data in Wood- (l w470 nm). To estimate the number of rhodopsin molecules activated ruff et al. [10]. See text and Supplemental Data for details of calculations. max per flash, we measured the light intensity of a 520 mm spot focused on the slice preparation by the 203 0.75NA (Nikon) condenser objective with a which must be pumped out to achieve homeostasis, we calibrated photodiode (United Detector Technologies, San Diego, CA). Light needed first to know how steady light affects the mammalian intensities were converted to equivalent 501 nm photons by convolving the rod membrane potential. No measurements of this kind had power-scaled spectral output of the LED with the normalized spectral sen- 21 previously been made, so we performed perforated-patch sitivity curve for mouse rhodopsin. These were then converted to Rh* s by estimating the collecting area of rod photoreceptors in the experimental recordings to measure voltage responses to light steps from setup. Dim flashes were delivered during suction electrode recordings mouse rods in retinal slices (see legend to Figure 2). We from rod outer segments in clusters [42], and the mean Rh* per rod was used 5 s light steps, the longest we could employ and obtain determined from the scaling of the time-dependent variance to the mean reliable measurements, because total recording time lasted response [43]. Based on these factors, we estimated the rod collecting 2 only a few minutes before the pipette went whole cell. The volt- area in the experimental setup to be 0.18 mm (n = 6 rods). age hyperpolarized with increasing light intensity from a dark resting potential of 237.3 6 2.3 mV (Figure 2A), with the wave- We combined these measurements of voltage with previous form in bright light showing a characteristic rapid relaxation or determinations of mean conductance and voltage depen- + + ‘‘nose,’’ caused by activation of a Na /K current activated by dence of ih in guinea pig rods [24] to calculate the current hyperpolarization, usually referred to as ih (Figure 2A, insert).

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