THE ROLE OF CNG CHANNELS IN PHOTORECEPTOR PHYSIOLOGY

Proteins required to maintain structure and function of the outer segment, including CNG channels, are synthesized within the main cell body and, on maturation, are actively transported to their destination.

In rods, the outer segment consists of about 1,000 stacked membrane disks that are covered by the plasma membrane. The membrane constituting the disks harbors the proteins involved in photoelectrical transduction, except for the CNG channels. The CNG channels are located in the plasma membrane at high density. In cones, the outer segment is organized differently. Instead of disks, the plasma membrane forms deep invaginations and is the locale of all membrane-bound or integrated proteins involved in photoelectrical transduction.

The Activation Phase of the Light Response

In the dark, micromolar concentrations of free cGMP keep 1–10% of the CNG channels open in both rods and cones. This open fraction conducts an inward current of about 20 pA that is carried by sodium (Na+) and calcium (Ca2+) ions. This steady inward current (“dark current”) depolarizes photoreceptors to a resting voltage of about −35 mV. In rods, 8–12% of the dark current is carried by Ca2+, while in cones this fraction is 15–25%, twice as high as in rods. The Ca2+ entry is balanced by Ca2+ extrusion through a Na+/K+,Ca2+ exchanger, maintaining a resting Ca2+ concentration of 400–700 nM in both rods and cones.

The photoelectrical transduction triggered by light stimulation consists of a complex sequence of biochemical reactions, studied most extensively in rod photoreceptors. On the absorption of a photon, rhodopsin transforms into its active enzymatic form. Active rhodopsin allows the G protein transducin to exchange bound guanosine diphosphate (GDP) for guanosine triphosphate (GTP). GTP-bound transducin activates the effector enzyme phosphodiesterase (PDE), which efficiently hydrolyses cGMP (Fig. 1).

The degradation of cGMP and the subsequent closure of CNG channels has two main consequences. First, the photoreceptor hyperpolarizes and releases less of the neurotransmitter glutamate. Second, the interrupted Ca2+ influx into the outer segment leads to a drop in the intracellular Ca2+ concentration due to the continuing export of Ca2+ by the light-insensitive Na+/K+,Ca2+ exchanger. The drop in intracellular Ca2+ is sensed by several Ca2+-binding proteins that accelerate the recovery of the resting state in photoreceptors after light activation and mediate the adaptation of photoreceptors to continuous light exposure (see next section).

Photoelectrical transduction in cones is similar to that in rods, and the enzymes involved in light-triggered cGMP degradation display only modest functional differences compared to their rod cousins. Since rods and cones implement functionally similar enzymes in the photoelectrical cascade, which factors are responsible for the differences in light sensitivity, speed, and dynamic response range? It has been suggested that different Ca2+ dynamics in rods and cones partially account for the distinct response properties [2]. Compared with rods, cones display at least a tenfold faster Ca2+ clearance from the outer segment, a lower Ca2+ buffer capacity, and a twofold higher Ca2+ influx through the CNG channels. These differences suggest that the changes in the intracellular Ca2+ concentration are faster and larger in cone outer segments, allowing faster response times and a broader dynamic range of light adaptation.

Recovery After a Light Stimulus and Adaptation to Continuous Illumination

The drop in the intracellular Ca2+ concentration on CNG channel closure is sensed by Ca2+-binding proteins that allow photoreceptors to recover after a light stimulus and to adapt to continuous illumination. Specifically, the protein recoverin is involved in a process that increases the rate of rhodopsin inactivation and guanylate cyclase-activating protein (GCAP) promotes the synthesis of new cGMP. The process of reopening CNG channels and therefore the recovery of the dark current is also Ca2+ dependent. In vitro studies demonstrate that the rod CNG channel is exquisitely sensitive to physiologically relevant levels of Ca2+/calmodulin (e.g., [1, 3]). These data suggest that, in the dark, Ca2+/calmodulin binds to CNG channels and keeps them in a state of low cGMP sensitivity. On the light-triggered drop in intracellular Ca2+, calmodulin dissociates from the CNG channels, thus raising their cGMP sensitivity. As a consequence, less cGMP is required for the channel to reopen. In fact, the cGMP concentration required for half-maximal activation [EC50(cGMP)] of rod channels changes with calmodulin up to twofold from high to low intracellular Ca2+. In contrast, cone CNG channels either respond weakly or are insensitive to modulation by Ca2+/calmodulin. It is possible that another unidentified Ca2+-binding protein modulates the cGMP sensitivity of cone CNG channels over an even broader range [4]. For example, in electropermeabilized cones from the striped bass, the average EC50(cGMP) of CNG channels increases about fourfold from low to high intracellular Ca2+ concentrations.

In summary, the Ca2+ permeation and the regulation of activity by intracellular Ca2+ are profoundly different for CNG channels from rods and cones, supporting the notion that the CNG channel is a pivotal determinant of the dynamics of Ca2+ homeostasis in photoreceptors.

CNG Channels in the Synaptic Transmission of Cone Photoreceptors

In cones, CNG channels are present not only in the outer segment but also in the synaptic terminal, where they might serve an additional function absent in rods. At the photoreceptor dark resting potential of −35 mV, a voltage-activated Ca2+ channel in the synaptic terminal of rods and cones permits continuous Ca2+ entry that sustains a tonic release of glutamate [5]. The Ca2+ channel is characterized by an activation threshold of about −45 mV. When the graded light response reaches −45 mV, the Ca2+ channels close, and in rods, synaptic transmission ceases. In contrast to rods, cones continue synaptic transmission as the light-induced voltage response grows to −70 mV.

The discovery of CNG channel clusters in the synaptic terminal of cones offers an explanation for the extended operating range of synaptic transmission [5, 6]. If the clusters are located near release sites, the CNG channels could underlie Ca2+-dependent release of glutamate. In fact, experimental maneuvers that activate CNG channels also trigger exocytotic events and the release of glutamate from the cone terminal. The cGMP required to activate the synaptic CNG channels might be produced by a nitric oxide (NO)-stimulated soluble guanylate cyclase (GC) [7]. Consistent with this model, a NO synthase is present in cone photoreceptors, completing the set of enzymes required for the activation of synaptic CNG channels [7].