and intracellular domains, and the presence of a leucine zipper motif in an extracellular domain set the stage for interaction with self or other proteins (e.g., dimerization) in organized membrane domains. Clearly, the process of new disk synthesis must involve the insertion of new membranous material, directed expansion along a particular axis (here orthogonal to the photoreceptor long axis), recognition of appropriate membrane expansion along that axis (size control), and finally, in the rod photoreceptor closure at the tips of the adjacent disks to place new topologically closed disks formally inside the plasma membrane. The interaction of prominin with protocadherin 21, which is also specifically expressed to the photoreceptor nascent disk microenvironment, and actin, facilitate this complex process that will likely be determined to have many more interacting partners and modulators.

11.2.3.4  BEST-1

The RPE is a polarized secretory epithelium [100, 101] (Fig. 11.13). Cells maintain a resting membrane potential between −40 and −45 mV and are subject neither to extreme shifts in membrane voltage nor action potential excitability [101, 102]. At the apical surface of the RPE are voltage-dependent Cl− channels that are found in association with bumetanide-sensitive Na+/K+/2Cl− exchangers, a Ba2+-sensitive inward rectifying K+ conductance, and aquaporins [179]. Cl− enters the RPE cell from the subretinal space and through the apical membrane channels. Na+ transport moves across the epithelium as a counterion to the Cl− transport. Water follows in response to the osmotic flux. The buildup of intracellular Cl− promotes an outwardly directed Cl− gradient across the basolateral surface of the RPE cell. A DIDS-sensitive Cl− conductance is found in the basolateral membrane of the RPE and is responsible for Cl− egress from the RPE cell and fluid transport.

BEST-1 is a member of a recently discovered family of Cl− channels of which there are four members in human [103]. BEST-1 is expressed exclusively to the basolateral membrane surface of the RPE adjacent to Bruch’s membrane and the choriocapillaris and also appears to be present, in part, in the intracellular ­membranes [22, 104]. Recent studies of the impact of BEST-1 mutations have mostly been conducted after expressing the WT and mutant proteins in HEK293

cells, a convenient system for stable electrophysiological analysis of channels [196, 201–202]. The WT BEST- 1 cDNA and many mutant cDNAs clearly promote the expression of a normal or altered Cl− conductance in HEK293 cells that is not present before transfection. All the mutants of BEST-1 that are expected to be pathogenic (not allelic variants) promote changes in the electrophysiological behavior of expressed Cl− channels, indicating that the altered function of basolateral membrane Cl− channels is the underlying basis of the disease. Mutations in the BEST-1 channels have promoted a loss of conductance to varying degrees (absolute to partial) and alterations in anion permeability, the latter of which is hard to relate to disease phenotype. Some mutations lead to protein misfolding that result in poor to absent trafficking to the plasma membrane surface, where functionality is assessed. Thus, there is a range of functions found in expressed mutant BEST-1 channels that is likely responsible for the range of phenotypic outcomes seen clinically. Most electrophysiological studies have been conducted with expressed WT and mutant BEST-1 channels in whole cells (whole-cell recording), where the entire populations of expressed channels are analyzed [103, 105, 106]. More recent studies have achieved single channel recording, which is critical to understanding the underlying kinetics and the modulation of the channels [107]. Whole-cell electrophysiological studies and more recent single channel studies have clearly shown that BEST-1 is a Cl− channel with low conductance (2 pS) and fast gating behavior (2 ms). Curiously, there is a minimal voltage sensitivity to the gating of these small WT BEST-1 Cl− channels, and only at extremes of potential that are unlikely to be physiologically meaningful. Moreover, both the wholecell and single-channel recording data show a linear current-voltage relationship indicating an ohmic channel conductance. These outcomes suggest that BEST-1, while being a basolateral Cl− conductance in the RPE, is unlikely to be modulated by whole-cell RPE membrane voltage. Its behavior must be modulated by other sources of energy. Prior studies have clearly shown that this channel is modulated by intracellular Ca2+ and also perhaps by ATP [103, 105, 108, 180]. Metabolic and likely receptor-mediated control of intracellular Ca2+ appear to act to modulate the BEST-1 Cl− conductance. Upon binding of ATP (or UTP) to a metabotropic P2Y purinergic receptor on the apical RPE surface membrane, there are large activations of conductances on

both the apical and basolateral membranes, which results in the stimulation of apical to basolateral fluid transport [109].

The BEST-1 Cl− channel (bestrophin) must clearly modulate Cl− exit from the basolateral surface of the cell in response to electrochemical forces even if the membrane voltage is not involved in its opening and closing kinetics (probability) (Fig. 11.13). In RPE, the absorption of Cl− across the epithelium is mediated by an apical membrane Na+–K+–2Cl− cotransporter and the basolateral Cl− conductance [101]. Water follows Cl− efflux by osmotic forces toward Bruch’s membrane and the choriocapillaris. This net trans-RPE Cl− flux, through the cooperation of ionic conductances on both

the apical and basolateral surfaces of the RPE in this polarized epithelium, plays a major role in establishing and maintaining water flux from the subretinal space and keeping the retina opposed to the RPE. Breakdowns in this fluid transport lead to diseases such as BMD, central serous choroidopathy, and retinal detachment. The BEST-1 Cl− channel also regulates the voltage on the basal surface of the RPE cells and aids in setting up the trans-RPE voltage that is measured in the EOG. The conductance is activated slowly by intracellular Ca2+ and appears to be influenced by intracellular ATP levels. Recent studies have found that bestrophin may also be expressed to the intracellular membranes, such as lysosomes, where they could provide Cl− counterion

Fig. 11.13  BEST-1 cellular localization and function. (a) BEST-1 is localized to the

basolateral plasma membrane surface of the RPE cell. It appears to be a Ca2+-dependent Cl− channel that, under natural physiological conditions, promotes passive Cl− degress from the cytoplasm to the extracellular space down its electrochemical gradient. Loss of Cl− from the cell leads to the depolarization of the basolateral membrane surface, which appears to be a component of the c-wave of the ERG and the light peak of the EOG. (b) Molecular schematic of BEST-1. Hypothetic channel structure for BEST-1 is indicated. Ca2+-dependent

Cl− channels have both a voltage sensitive and Ca2+- sensitive “gate” which opens and closes conduction through the pore, and a Ca2+ binding site on the cytoplasmic surface which binds free cytoplasmic Ca2+ according to a titration isotherm in response to changes in intracellular Ca2+ concentration that occur during a variety of types of intracellular signaling

a

= apical

membrane Cl-

channel

Photoreceptors

= BEST-1

=pigment granule

=lipofuscin containing phagolysosome

=lysosome

containing phagocytized outer segments

Cl-

tight junctions

RPE

N

C

extracellular

+

+GATE

membrane

flux in response to a strong proton pumping by H+- ATPase into the lysosomal vesicle. The strong acid conditions of the lysosome microenvironment are essential for the degradation of phagocytized materials in the RPE, such as the rod and cone outer segment membranes. The proof of at least partial localization of bestrophin into the lysosomal membrane fraction would make logical sense to the occurrence of increased LF and A2E accumulation in Best’s disease due to a lack of pH-dependent enzymatic capacity and the accumulation of undigested materials in phagolysosomes [22, 108].

While the BEST-1 gene is thought to code for Cl− channels localized in the basolateral membrane of the RPE, what is less clear is their role in RPE-based clinical retinal electrophysiology. The EOG reflects the

standing potential that exists across the polarized RPE monolayer under conditions of dark and light adaptation. The ratio of the amplitude of the LP to the DT is used as the “Arden” ratio to evaluate the functionality of the RPE [110]. Arden ratio values less than 1.5 are thought to be pathological for RPE disease, and the EOG is thought to be the critical or pathognomonic test for BMD. Curiously, there is much less of a correlation between the EOG measures of patients with BEST-1 mutations and the dysfunction represented in expressed mutant Cl− channels. This has lead to the suggestion that while BEST-1 mutations may explain Cl− dysfunction, they do not necessarily explain the underlying nature of the cellular and clinical disease process of BMD and related syndromes [111, 112]. A first challenge­ to the thinking that BEST-1 proteins were Cl− channels came

in the identification that the expression of BEST-1 in rat RPE-J cells leads to alterations in the kinetics of volt- age-dependent L-type Ca2+ channel kinetics [113]. Next, was the finding that a knockout of BEST-1 in mouse did not alter the whole-cell Cl− channel measures, but in fact made the LP more sensitive to light at high and low extremes of intensity [112]. In addition, this study showed that the inhibition of ­voltage-dependent Ca2+ channels substantially reduced the LP, and raised the question as to whether BEST-1 proteins were actually Cl− channels. On the other hand, human BEST-1 mutations, when overexpressed in rats promoted changes in the LP [114].

Is there a way to combine these dissociate opinions into a cohesive hypothesis? The LP of the EOG is a slow electrophysiological signal that occurs over ­minutes after light exposure and is mediated by a depolarization of the basolateral membrane of the RPE that is due to the activation of a Cl− conductance [100, 115, 116, 192]. In order to manifest a depolarization of the basolateral membrane, the equilibrium potential for Cl− must rest at more depolarized potentials than the resting membrane potential of the RPE cell. Then, when these channels open, Cl− leaves the cell to result in a local membrane depolarization. How is control over such channels manifested? The LP continues to occur even when both ON and OFF bipolar pathway inhibitors of the ERG are present. This outcome indicates that the LP must arise from a photoreceptor-RPE interaction. Photoreceptors have long been thought to release a diffusible “light peak substance” upon photoactivation that promotes physiological changes in adjacent RPE cells [117]. Current knowledge suggests that this mediator is likely to be ATP [109]. There are purine receptors (P2Y receptors) on the apical surface of the RPE which are able to respond to ATP in the subretinal space [118]. Purine receptors are A-class G-protein coupled receptors that bind small ligands in the mem- brane-bound core of the integral membrane protein. Activation of P2Y or other purine receptors is known to modulate intracellular Ca2+ through the modulation of intracellular stores [118]. Activation of such P2Y2 receptors on the apical surface of the RPE is expected to lead to the mobilization of intracellular Ca2+ stores through G-protein (i.e., Gq) activation and the activation of phospholipase C with the liberation of inositol triphosphate (IP3). Direct addition of IP3 into the cytoplasm of RPE cells promotes the activation of Cl− currents [119]. Moreover, increase in IP3 is also known to promote the stimulation of L-type VDCC in RPE cells

[102]. The increase in intracellular Ca2+, due to both, the release from intracellular stores and IP3-mediated stimulation of VDCCs, is expected to promote the opening of BEST-1 Cl− channels in the basolateral membrane of the RPE. DIDS, a proven inhibitor of BEST-1 Cl− channels is known to suppress the LP [40, 115]. As a result of Cl− exit from the cell, the basal membrane of the RPE would depolarize into a range of potentials over which VDCCs activate. Curiously, these L-type VDCC are also activated by IP3, which suggests that they too are modulated by P2Y receptors. The influx of Ca2+ into the cytoplasm would be expected to promote Ca2+-induced Ca2+ release from the intracellular stores thus further prolonging the temporal span of the basal membrane depolarization in a positive feedback loop, to account for the timescale of the LP which is on the order of minutes. The persistence of a Cl− channel conductance in RPE cells of VMD2−/− mice is readily explained by the known expression in mouse RPE of BEST-2 which has similar but not identical properties to BEST-1 and may even be able to form heterodimers with BEST-1 [103, 108]. The presence of BEST-2 in RPE cells may also be able to explain the persistent LP in VMD2−/− mice and the enhanced light sensitivity at low and high fluence rates in this line, which prompted the suggestion that BEST-1 was an antagonist of the LP [112]. The LP signal is clearly not the result of a single protein (e.g., BEST-1). Rather, it is the response of a system of functionalities (ligands, receptors, intracellular Ca2+ stores, surface membrane channels) that are distributed across at least two cell types. A change in any component of the system (e.g., switch of BEST-2 for BEST-1 in the system) would be expected to alter the system properties (e.g., the relationship between calcium-activated Cl− channels, which drive membrane voltage changes, and VDCCs which respond to those voltage changes to influence Ca2+ inside the cell). What is challenging to explain with this model is the alteration of VDCC gating in VMD2−/− RPE cells, if one assumes that an adequate voltage clamp was acquired, because there is no prior knowledge to support the hypothesis that Cl− channels form a close spatial relationship with VDCCs which influences gating kinetics. On the other hand, intracellular [Cl−] is known to affect the function of other cellular proteins [120]. Clearly, more research work will be needed to discern between these alternatives. Other voltage-dependent Cl− channels (e.g., ClC-2) are expressed in RPE and knockout of ClC-2 promotes an early onset form of severe retinal degeneration [121].