ATP consumption by mammalian rod photoreceptors in darkness and in light

Abstract

Why do vertebrates use rods and cones that hyperpolarize, when in insect eyes a single depolarizing photoreceptor can function at all light levels? We answer this question at least in part with a comprehensive assessment of ATP consumption for mammalian rods from voltages and currents and recently published physiological and biochemical data. In darkness, rods consume 10(8) ATP s(-1), about the same as Drosophila photoreceptors. Ion fluxes associated with phototransduction and synaptic transmission dominate; as in CNS, the contribution of enzymes of the second-messenger cascade is surprisingly small. Suppression of rod responses in daylight closes light-gated channels and reduces total energy consumption by >75%, but in Drosophila light opens channels and increases consumption 5-fold. Rods therefore provide an energy-efficient mechanism not present in rhabdomeric photoreceptors. Rods are metabolically less "costly" than cones, because cones do not saturate in bright light and use more ATP s(-1) for transducin activation and rhodopsin phosphorylation. This helps to explain why the vertebrate retina is duplex, and why some diurnal animals like primates have a small number of cones, concentrated in a region of high acuity.

Figure 1

Principal contributors to ATP consumption in mammalian rod over the physiological range of steady light intensities. (A) Outer segment. ATP required for extrusion of Na⁺ entering cGMP-gated channels (■), transducin GTP hydrolysis and rhodopsin phosphorylation (□), synthesis of cGMP (○), and sum of all of these processes (X). (B) Inner segment and total rod ATP consumption. ATP required for extrusion of Na⁺ entering through i_h channels (□), extrusion of Ca²⁺ entering voltage-gated channels at synaptic terminal (○), sum of ATP for Na⁺ and Ca²⁺ extrusion (△), and sum of ATP turnover in whole rod (●). For the global sum, we used light intensities at which voltage responses had been recorded (Fig. 2B) and estimated photocurrents by interpolation from step response-intensity data in Woodruff et al. [10]. See text and Supplemental Material for details of calculations.

Figure 2

Perforated-patch current-clamp recordings of membrane potential from mouse rod photoreceptors in dark-adapted retinal slices. (A) Response waveforms to 5 s light steps of intensities 33, 83, 210, 520, 1300, 3200, and 8100 Rh* rod⁻¹ s⁻¹. Data from 8 cells were averaged individually for each background light intensity and were corrected for a measured liquid junction potential of ~10 mV. Average input resistance was 5 GΩ and average access resistance, 300 MΩ. Inset: Same responses at higher temporal resolution showing rapid relaxation or “nose” in voltage waveform at high light intensities caused by activation of i_h. (B) Response-intensity curve of voltage response, averaged during the interval 4.5–5 s from the responses in A, as function of steady light intensity. Methods: Experiments were conducted in accordance with protocols approved by institutional IACUC committees. Light-evoked membrane potential changes during current-clamp recordings were measured in response to background light steps of 5 sec duration delivered from an LED (λ_max ~ 470 nm). To estimate the number of rhodopsin molecules activated per flash, we measured the light intensity of a 520 μm spot focused on the slice preparation by the 20X 0.75NA (Nikon) condenser objective using a calibrated photodiode (United Detector Technologies, San Diego, CA). Light intensities were converted to equivalent 501 nm photons by convolving the power-scaled spectral output of the LED with the normalized spectral sensitivity curve for mouse rhodopsin. These were then converted to Rh* s⁻¹ by estimating the collecting area of rod photoreceptors in the experimental setup. Dim flashes were delivered during suction electrode recordings from rod outer segments in clusters [42], and the mean Rh* per rod was determined from the scaling of the time-dependent variance to the mean response [43]. Based on these factors we estimated the rod collecting area in the experimental setup to be 0.18 μm² (n = 6 rods).

Figure 3

Maximal and minimal ATP expenditure required for the biochemistry of the transduction cascade and by ion extrusion in a mouse rod in darkness and in bright illumination. The letters D and L indicate darkness and bright light. See text.