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(Investigative Ophthalmology and Visual Science. 2006;47:4956-4961.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-0524
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The Bestrophin Mutation A243V, Linked to Adult-Onset Vitelliform Macular Dystrophy, Impairs Its Chloride Channel Function

Kuai Yu, Yuanyuan Cui, and H. Criss Hartzell

From the Department of Cell Biology, The Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, Georgia.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. It has been proposed that Best vitelliform macular dystrophy (BVMD) is caused by dysfunction in the Cl– channel function of human bestrophin-1 (hBest1), but some patients with BVMD who have the hBest1 A243V mutation have normal electro-oculograms, suggesting that this mutation may not affect Cl– channel function. The purpose of this study was to determine whether the A243V mutation affects the Cl– channel function of hBest1.

METHODS. Wild-type alanine at position 243 was changed to valine by PCR-based mutagenesis. Wild-type (WT) and A243V hBest1 were transfected into HEK-293 cells, and Cl– currents were measured with the whole-cell patch-clamp technique. The trafficking of proteins to the plasma membrane was tested by cell-surface biotinylation.

RESULTS. WT hBest1 induced Ca2+-activated Cl– currents in HEK cells that were >1 nA in amplitude. The currents produced by the A243V mutant, however, were only approximately 10% as large as WT. This was not due to the inability of the A243V mutant to reach the plasma membrane, as shown by cell-surface biotinylation. The A243V mutation changed channel anion selectivity. The WT current exhibited a relative permeability PX/PCl order of SCN– ≥ I– ≥ NO3– > Br– > Cl– > HCO3– and a relative conductance GX/GCl order of NO3– > SCN– > I– ≥ Br– ≥ Cl– > HCO3–. However, the A243V current exhibited different sequences: PX/PCl was SCN– > NO3– > I– > Br– > Cl– > HCO3– and GX/GCl was SCN– > NO3– ≥ I– ≥ Br– > Cl– > HCO3–. Unlike several other hBest1 mutations that have dominant-negative effects on wild-type channels, the A243V-mutation did not influence the wild-type current when A243V and WT hBest1 were transfected together.

CONCLUSIONS. The disease-causing A243V mutation is associated with altered hBest1 Cl– channel activity. The absence of a dominant negative effect of A243V is consistent with the more mild symptoms associated with this mutation. These results are interpreted in terms of the hypotheses that bestrophins are Cl– channels and regulators of Ca signaling.

Mutations in the VMD2 gene have been found in patients with Best vitelliform macular dystrophy (BVMD, OMIM 153700; Online Mendelian Inheritance in Man; http://www.ncbi.nlm.nih.gov/Omim/ provided in the public domain by the National Center for Biotechnology Information, Bethesda, MD),1 2 adult-onset vitelliform macular dystrophy (AVMD, OMIM 608161),3 4 and autosomal dominant vitreoretinochoroidopathy (ADVIRC OMIM 193220).5

The link between VMD2 mutations and BVMD is extensive. BVMD is classically described as a juvenile-onset disease characterized by large deposits of a yellow-pigmented material in the subretinal space.6 In its initial stages, the deposits resemble an egg yolk (vitelliform), but later the vitelliform lesion disperses ("scrambles") and the affected area becomes deeply and irregularly pigmented. The disorder is progressive, and loss of vision often occurs. The gene was identified by positional cloning in several families with extensive pedigrees to be VMD2.1 2 Now approximately 100 mutations have been described in BVMD patients.7 8 Sun et al.9 and Tsunenari et al.10 proposed that VMD2 encodes a Cl– channel and that BVMD is a caused by hBest1 Cl– channel dysfunction. This proposal is supported by several lines of evidence: (1) The hallmark diagnostic feature of BVMD is a decrease in the light peak (LP) of the electro-oculogram (EOG). The LP in several species has been shown to be caused by a Cl– conductance in the basolateral membrane of RPE,11 12 13 and porcine Best-1 has been shown to be localized to the basolateral membrane of the RPE.14 (2) Overexpression of hBest1 in cell lines induces a novel Ca2+-activated Cl– current9 10 15 16 and different bestrophins produce currents that have different properties.9 10 (3) WT hBest1 currents are inhibited by membrane-impermeant, cysteine-reactive reagents, and this effect is eliminated when cysteine-69 is mutated to alanine.10 (4) Mutation of amino acids in the second transmembrane domain of several bestrophins results in altered anion permeability and conductance.10 15 17 18 This shows that bestrophin participates in forming the pore and does not simply upregulate endogenous Cl– channels.19 (5) Of the ~15 different BVMD-causing mutations that have been examined, all produce Cl currents that are much smaller than normal (<25%) when expressed in HEK cells.9

Despite extensive evidence that bestrophins are Cl channels, it remains unclear whether BVMD is actually caused by Cl– channel dysfunction. Recent evidence from Marmorstein et al.20 21 has shown that the LP of the EOG in rodents is unlikely to be generated by bestrophin-1, because knockout of mBest1 in mice does not decrease the LP. Furthermore, Cl– currents in RPE cells isolated from the knockout21 appear normal. Other experiments show that bestrophin expression regulates Ca2+ currents and Ca2+ signaling.21 22 These observations have lead to the suggestion that although bestrophin may play a role in regulating the light sensitivity of the LP, bestrophin itself is not responsible for generating the LP (see the Discussion section).

Five VMD2 mutations have been reported in individuals with AVMD. There is no family history for three of these mutations (T6P, R47H, and A146K), but the A243V and D312N mutations have been described in families with a dominant inheritance pattern. Because AVMD is very similar to BVMD but has a later onset, smaller lesions, and slower progression, the possibility that some AVMD cases are mild cases of BVMD has been considered.3 4 Although BVMD is classically described as a disease with juvenile onset, penetrance of disease symptoms is variable.23 Some individuals never develop symptoms and the median age of onset has been reported to be 42 years, with a range of 5 to 58 years.23 Late-onset BVMD has also been reported in individuals with the Y227N24 and V89A25 mutations.

Yardley et al.5 described three VMD2 mutations (V86M, V239M, and Y236C) in five families with ADVIRC. Bioinformatic analysis suggests that exonic splicing may be altered. That these mutations alter exonic splicing was confirmed by inserting short hBest1 sequences into a fibronectin minigene, but the exact sequences of the transcripts that are produced by these mutations remain unknown. ADVIRC differs from BVMD in that the ERG is depressed in ADVIRC, but the ERG is often normal in BVMD.

Although a common feature of BVMD and ADVIRC is a decreased EOG, AVMD patients typically have normal or only slightly subnormal EOGs. This EOG pattern is particularly true of the A243V mutation in six patients with AVMD, five of whom had a normal EOG.3 The A243V mutation has also been reported in one family of patients with BVMD1 26 who had normal EOGs.26 A normal EOG in patients with the A243V mutation raises the question of whether the A243V mutation affects the Cl– channel function of hBest1. To examine this question, we measured the Cl– currents induced by expression of A243V hBest1 in HEK cells.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Heterologous Expression of hBest1 in HEK-293 Cells
hBest1 in pRK5 with six myc epitope tags at the C terminus was obtained from Jeremy Nathans (Johns Hopkins University, Baltimore, MD) The A243V mutation was made by using a PCR-based site-directed mutagenesis kit (Quickchanger; Stratagene, La Jolla, CA). hBest1 and an EGFP plasmid (pEGFP; Invitrogen, Carlsbad, CA) were transfected into HEK-293 cells (5:1 ratio, 2 µg total DNA per 3.5-cm plate; Fugene-6 transfection reagent; Roche, Indianapolis, IN). The EGFP plasmid alone (0.2 µg) was used as a transfection control. One day after transfection, cells were dissociated and replated at lower density for electrophysiological recording. Single cells identified by EGFP fluorescence were used for patch clamp experiments within 3 days.

Electrophysiological Methods
Recordings were performed with the whole-cell patch clamp. Borosilicate glass (Sutter Instrument Co., San Raphael, CA) patch pipettes (3–5 M{Omega}) were pulled (P-2000 puller; Sutter Instrument Co.) and fire polished. The bath was grounded via a 3-M KCl agar bridge connected to an Ag/AgCl wire. Solution changes were performed by perfusing the 1-mL chamber at a speed of 4 mL/min. To measure the steady state current-voltage relationship, we voltage clamped the cells from a holding potential of 0 mV with 750-ms duration pulses from –100 to +100 mV in 20-mV increments. Data were acquired by an amplifier (Axonpatch 200A) controlled by a data acquisition system (Clampex 8.1 via a Digidata 1322A; Axon Instruments, Inc., Foster City, CA). Experiments were conducted at room temperature (20°C–24°C). Because the liquid junction potentials were small (<2 mV), no correction has been made. Rectification ratio (RR) was calculated by dividing the current at +100 mV by the current at –100 mV.

Solutions
The standard pipette solution contained (mM): 146 CsCl, 2 MgCl2, 5 (Ca2+)-EGTA, 8 HEPES, 10 sucrose (pH 7.3), adjusted with NMDG. The zero-Ca2+ pipette solution contained 5 mM EGTA without added Ca2+. In the text, high-Ca2+ solution refers to a free Ca2+ concentration of ~4.5 µM. The standard extracellular solution contained (mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 15 glucose, and 10 HEPES (pH 7.4) with NaOH. This combination of intracellular and extracellular solutions set the reverse potential [Erev] for Cl– currents to zero, whereas cation currents carried by Na+ or Cs+ had very positive or negative Erev, respectively. When Cl– was replaced with another anion, NaCl was replaced on an equimolar basis with Na salt of the substitute anion. Osmolarity was adjusted with sucrose to 303 mOsM for all solutions.

Analysis of Data
Commercial software (Origin 6.0; Microcal, Northampton, MA) was used for calculations and graphical presentation. Data are expressed as the mean ± SEM. Relative permeability was determined by measuring the shift in Erev on changing the bath solution from 151 mM Cl– to 140 mM substitute anion plus 11 mM Cl–. The permeability ratio was calculated with the Goldman-Hodgkin-Katz equation:

Formula
where {Delta}Erev is the difference between Erev with the test anion X– and that observed with symmetrical Cl–, and the thermodynamic quantities are represented by their usual terms: F(Faraday constant), R(gas constant), and T(absolute temperature).

Cell Surface Biotinylation
Reactions were performed at 4°C. Nontransfected HEK-293 cells and HEK-293 cells transfected with WT and A243V hBest1 were placed on ice, washed three times with PBS, and biotinylated with 0.5 mg/mL biotin (Sulfo-NHS-LC; Pierce Chemical Co., Rockford, IL) in PBS for 30 minutes. The cells were washed with PBS, incubated in 100 mM glycine in PBS to quench unreacted biotin, washed three times with PBS, and then scraped from the dish and collected by centrifugation. The cells were suspended in lysis buffer (250 µL 150 mM NaCl, 5 mM EDTA, 50 mM HEPES [pH 7.4], 1% Triton X-100, 0.5% protease inhibitor cocktail III [Calbiochem, La Jolla, CA], and 10 µM phenylmethylsulfonyl fluoride [PMSF] per 100-mm dish). The extract was clarified at 10,000g, 15 minutes. Biotinylated proteins were isolated by incubation of 200 µL of extract with 100 µL of streptavidin beads (Pierce Chemical Co.) overnight with gentle agitation. The beads were collected by centrifugation (10,000g, 10 minutes) and washed four times with 0.6 mL lysis buffer+200 mM NaCl. The bound biotinylated proteins were eluted with 200 µL 2x Laemmli buffer. Protein samples were loaded onto an SDS-PAGE gel with ~10 µg of protein per well. Immunoblots were probed with anti-myc and anti-GAPDH antibodies, followed by secondary anti-mouse IgG. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL kit; GE Healthcare, Piscataway, NJ).


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effect of Disease-Associated A243V Mutation on hBest1 Cl– Channel Function
As reported previously,9 10 27 WT hBest1 expressed in HEK-293 cells produces a current that is activated by intracellular [Ca2+] (Fig. 1A 1B 1C) . The Ca2+-activated currents were time- and voltage- independent (Fig. 1B) . The I–V relationships reversed near 0 mV as expected for a Cl–-selective current (Fig. 1C) . In contrast, expression of the A243V mutant of hBest1 produced very little current (Figs. 1D 1E) . Typically, A243V currents were ~10% of the amplitude of the WT currents (Fig. 1E) .


Figure 1
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  		FIGURE 1. Expression of WT and A243V mutant hBest1 in HEK-293 cells. The voltage protocol used is shown above (A). (A, B), Representative WT-hBest1–induced current traces with <20 nM (low) free [Ca2+]i (A) and 4.5 µM (high) free [Ca2+]i (B). (C) Mean current–voltage (I-V) relationships obtained from WT-hBest1–transfected cells with low and high [Ca2+]i. (D, E) Representative A243V-hBest1–induced current traces with 4.5 µM (high) free [Ca2+]i shown at low (D) and high (E) amplification. (F) Mean I-V curves from A243V-hBest1–transfected cells compared with WT. Each point represents data from at least seven cells.

 
Both BVMD and AVMD are inherited in an autosomal dominant manner. Furthermore, Sun et al.9 showed that several of the disease-causing mutations in hBest1 had dominant negative effects on Cl– currents produced by WT hBest1. Therefore, we tested whether the A243V mutant also had dominant negative effects on the WT current. Equal amounts of WT and A243V plasmids were transfected into HEK-293 cells. Cotransfection of the A243V plasmid had no effect on the currents induced by WT hBest1 (Fig. 2) .


Figure 2
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  		FIGURE 2. Coexpression of WT and A243V hBest1. Whole-cell recording was performed as in Figure 1 with high [Ca2+]i. (A) Representative current traces from cells transfected with equal amounts of WT and A243V hBest1. (B) Mean I-V relations for WT (n= 13) and cotransfection of WT and A243V (n= 12). A243V mutant has no significant influence on the WT current (P > 0.05, two-tailed t-test).

 
To be sure that differences in current amplitudes in Figures 1 and 2 were not partly due to differences in cell size, which could occur in response to Cl– channel expression, current densities were normalized to cell capacitance (Fig. 3) . The A243V mutation produces currents that are much smaller than those produced by WT and coexpressing the A243V mutant with WT has no effect on the WT current.


Figure 3
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  		FIGURE 3. Ca dependence of WT and A243V Cl– current density. Cl– current density was calculated by normalizing amplitudes of the steady state currents at the end of the –100 mV (Figure 3, inward currents) and +100 mV ({square}, outward currents) pulses by cell capacitance for HEK-293 cells transfected with GFP alone and GFP plus WT hBest1, A243V-hBest1, or A243V plus WT hBest1. Each data point was from at least seven cells.

 
Bestrophin Localization on the Plasma Membrane
The small whole-cell currents produced by the A243V mutation could be caused by altered trafficking of the mutant hBest1 to the plasma membrane. To test this, plasma membrane bestrophin was labeled in intact cells 4°C with an membrane-impermeant biotinylation reagent (NHS-LC-Biotin; Pierce Chemical Co.). Biotinylated proteins were then solubilized and purified from the lysates with streptavidin beads. The presence of hBest1 in the plasma membrane was determined in immunoblots by using antibody directed against the myc tag on the hBest1 (Fig. 4A) . There was no band in nontransfected cells (control), but both wild-type (WT) and A243V hBest1 had 68-kDa bands corresponding to hBest1 in the biotinylated fractions from both WT and mutant hBest1-transfected cells (Fig. 4A) . The data confirmed that hBest1 is located on the cell surface and excluded the possibility that the A243V mutation eliminated Cl– current by altering trafficking of bestrophin to the surface.


Figure 4
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  		FIGURE 4. Localization of WT and A243V mutant hBest1 in the plasma membrane of HEK-293 cells. (A) Biotinylated proteins from nontransfected (control), WT, and A243V mutant hBest1-transfected cells were probed with antibody to the myc tag on hBest1 (68-kDa band). (B) Total extracts or (C) biotinylated proteins were probed with antibody to GAPDH (37-kDa band) to show that the biotinylation reaction labeled only cell surface proteins.

 
To be certain that the biotinylation reaction did not label intracellular proteins, biotinylated proteins that were recovered from an aliquot of the lysate using streptavidin beads were immunoblotted with an antibody against GAPDH, a cytosolic protein. As expected, if the biotinylation labeled only cell surface proteins, the 37-kDa GAPDH band was found only in the total lysate (Fig. 4B) and was not present in the biotinylated fraction (Fig. 4C) .

Biophysical Properties of the A243V Current
The WT and A243V hBest1 Cl– currents exhibited different rectification properties. The WT current slightly outwardly rectified (RR = 1.46 ± 0.05, n = 25), whereas the A243V current strongly outwardly rectified (RR = 1.73 ± 0.11, n = 30; Fig. 5 ). The current recorded when the cells were cotransfected with WT and A243V hBest1 was the same as WT current alone (RR = 1.37 ± 0.14, n = 7; Fig. 5B ).


Figure 5
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  		FIGURE 5. The A243V mutation alters the rectification of hBest1 currents. (A) Normalized I-V curves for WT and A243V hBest1. Currents were normalized to the current at –100 mV. (B) Mean RR of WT hBest1 (n = 25), A243V hBest1 (n = 30), and WT plus A243V (n= 7). The difference between WT and A243V was significant (P > 0.05, two-tailed t-test).

 
The finding that the A243V current outwardly rectified suggests that the biophysical properties of the current may be altered. Therefore, we compared the relative anion permeability and conductance of WT and A243V channels by measuring I-V curves in the presence of various extracellular anions. Figures 6A 6B show typical I–V curves obtained from voltage ramps for WT and A243V hBest1-transfected cells with different extracellular anions. The relative permeability (PX/PCl; Fig. 6C ) was calculated from the shift in Erev under bi-ionic conditions, and the relative conductance (GX/GCl; Fig. 6D ) was measured as the slope of the I–V curve between ±25 mV of Erev. The WT current exhibited a PX/PCl order of SCN– ≥ I– ≥ NO3– > Br– > Cl– > HCO3–, and a GX/GCl order of NO3– > SCN– > I– ≥ Br– ≥ Cl– > HCO3–. However, A243V current exhibited different sequences: for PX/PCl the sequence was SCN– > NO3– > I– > Br– > Cl– > HCO3– and for GX/GCl the sequence was SCN– > NO3– ≥ I– ≥ Br– > Cl– > HCO3–.


Figure 6
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  		FIGURE 6. Ionic selectivity of WT and A243V hBest1 currents. The I-V curves were obtained with a ramp voltage protocol from –100 to +100 mV and high intracellular [Ca2+]i Representative current traces were normalized to the peak amplitude of the current with symmetrical Cl– at +100 mV for each cell. Typical traces from WT (A) and A243V (B) currents were recorded with extracellular Cl–, Br–, I–, NO3–, SCN– and HCO3–. (C) The relative permeability ratios for each anion were calculated by using the Goldman-Hodgkin-Katz equation. (D) Mean relative conductance ratios (GX/GCl) for WT and A243V hBest1. Each data point represents at least seven cells.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
We have shown that the A243V mutation alters the Cl– channel function of hBest1 expressed in HEK cells. The mutation results in currents that are ~10% as large as WT. A243V hBest1 is expressed on the cell surface at approximately the same level as WT hBest1, which suggests that the A243V mutation alters channel gating or permeability. This view is supported by the observation that the A243V mutation alters the rectification and anion selectivity of the channel.

Bestrophin Topology and Location of A243
Tsunenari et al.10 showed that positions 212, 218, 223, 227, 261, 264, and 267 are likely to be extracellular. However, because hydropathy analysis predicts that residues 228 to 260 are very hydrophobic,10 A243 may be located in a re-entrant membrane loop. Another recent model28 suggests that A243 is located in a transmembrane domain. A243 is identical in all vertebrate bestrophins and is located at the N-terminal end of a highly conserved cluster, 233PLVYTQVVT[VIL]A243. In hBest1, there are 12 disease-causing mutations between I201 and A243. One of these mutations, Y227N, is associated with late-onset BVMD and may disrupt targeting of hBest1 to the RPE basolateral membrane.24 Although the A243V mutant is trafficked to the membrane, we do not know whether it is targeted basolaterally.

Model of Bestrophin Function
We propose that bestrophin participates in epithelial transport across the RPE/choroid, either directly as a Cl– ion channel and/or indirectly as a regulator of Ca2+ signaling. A precedent for the possibility that bestrophin is both a Cl– channel and a regulator of other ion channels is provided by CFTR, which is both a Cl– channel and a channel regulator.29 The evidence is persuasive that bestrophins can function as Ca2+-regulated Cl– channels9 10 15 16 17 18 30 31 and that Cl– channel dysfunction correlates with BVMD.9 Furthermore, an abnormal LP is a highly penetrant feature of BVMD. The LP is affected both by knockout and by overexpression of best1 and best1 mutants in rodents.20 21 Although the data in rodents are not consistent with a simple model in which best1 is the generator of the LP (discussed later), the data remain consistent with the idea that best1 is involved in ion and fluid transport across the RPE. We suggest, therefore, that the primary defect in BVMD is a disorder in RPE transport. Because the development of vitelliform lesions is considerably less penetrant than the reduced LP, vitelliform lesions are clearly not an obligatory consequence of disrupted epithelial transport but are governed by other environmental and/or genetic factors that confer susceptibility or protection.

How might disrupted epithelial transport be linked to development of vitelliform lesions? Major functions of RPE include regeneration of the visual pigment and phagocytosis of photoreceptor discs.32 If epithelial ion and fluid transport is disrupted, the composition and fluid volume of the subretinal space will be altered. This could affect both the transport of retinoids between photoreceptors and RPE and the ability of RPE cells to phagocytose photoreceptor outer segments. Both processes could result in abnormal accumulation of retina-derived pigments.

The development of BVMD, therefore, is likely to require two steps. Disruption of epithelial transport could result in a decrease in RPE-photoreceptor interaction which potentially results in accumulation of vitelliform pigment, depending on other factors. The mechanisms of disruption of epithelial transport could occur through dysfunction of Cl– transport through hBest1 channels9 or disruption of Ca2+ signaling.21

Abnormal LP as a Feature of BVMD
The development of vitelliform lesions is very variable in BVMD. BVMD is rarely symmetrical, with one eye typically being more affected than the other. Although, as mentioned, BVMD is usually a juvenile-onset disease, many patients do not manifest lesions until they are adults23 25 33 35 and some genetically affected individuals never have vitelliform lesions.34

In contrast to the incomplete penetrance of vitelliform lesions, an abnormal LP is tightly correlated with disease-causing VMD2 mutations. There are, however, exceptions to this generalization: the T6P,3 R47H,3 R105C,34 T216I,4 36 A243V,3 26 {Delta}I295,37 D312N,3 and L567F4 36 mutations have been described in patients with AMD, BVMD, or AVMD who have normal or slightly subnormal EOGs. The evidence that the R47H, R105C, T216I, and L567F mutations are actually disease-causing is not compelling, because they were found in sporadic cases with no family history and are not well conserved phylogenetically. The T6P, A243V, {Delta}I295, and D321N mutations, in contrast, are likely to be disease-causing mutations because they are associated with a family history of disease and are highly conserved residues. Although some patients with these mutations have normal LPs, others have subnormal LPs.

Normal LPs in some patients may be explained by the fact that the LP is an indirect measure of Cl– channel activity and may not be a sensitive measure of the basolateral Cl– conductance. The LP reflects the difference between the voltage decreases across the apical and basolateral RPE membranes produced by the increased Cl– conductance in the basolateral membrane (the LP conductance).12 Thus, decreases in LP amplitude caused by decreases in the LP conductance could be blunted by changes in other conductances that could come about secondarily to changes in membrane potential or ionic gradients. Variability between patients could be caused by environmental factors or polymorphisms in other channels. The ability of the EOG to detect a change in the LP conductance may also be compromised by the fact that the generation of the LP requires diffuse illumination of the entire retina: Even relatively large spots of light do not elicit a LP.12 Accordingly, if the LP conductance were affected in only a part of the retina, this may not be revealed as an LP reduction. In the case of the A243V mutation, which is not dominant negative, environmental, or genetic factors (perhaps including allele-specific transcriptional silencing) may result in geographic differences in expression of WT and A243V alleles.

That there are patients with VMD2 mutations and vitelliform lesions but normal EOGs could either be evidence that the defective LP mechanism is not causative of the disease or an indication that the EOG is not capable of detecting subtle changes in the underlying LP conductance. In the case of the A243V mutation, the fact that Cl– conductance is abnormal clearly supports the latter hypothesis, as it seems unlikely that each of the 16 disease-causing mutations that alter Cl– channel function also alter some other unrelated function of hBest1.

Cl– Channel Dysfunction Correlates with BVMD Disease-Causing Mutations
All the disease-causing VMD2 mutations that have been examined have altered Cl– channel function. These include the T6P, A10V, Y85H, R92C, W93C, N99K, D104E, R218S, Y227N, A243T, Q293K, G299E, E300D, D301E, T307I,3 and A243V mutations, all of which produce currents with amplitudes <20% as large as WT.

Our data are consistent with the hypothesis that BVMD is caused by a defect in Cl– channel function, because the A243V mutation alters the Cl– channel function of hBest1. That symptomatic patients with the A243V mutation have normal or only slightly subnormal EOGs can be explained by the observation that the A243V mutation does not have a dominant negative effect on WT current. Because both BVMD and AVMD are dominantly inherited, we expected that the A243V mutation, like other dominant VMD2 mutations that have been examined,9 would suppress the WT current; but, because the A243V mutant does not have a dominant negative effect, the level of expression of the WT allele in some heterozygous individuals may be sufficient to produce a normal EOG. Other factors may determine either the level of expression of the WT allele or the impact of haploinsufficiency on the EOG. The Arden ratio is considered normal within a large range, from 1.5 to >2.5, but most A243V AVMD patients have Arden ratios at the lower end of this range. Haploinsufficiency, although inadequate in some patients to produce a decrease of the Arden ratio below the 1.5 threshold, may still diminish the Cl– conductance enough to cause disease.

Another possibility is that the altered biophysical characteristics (rectification, gating, or permeation) of the current produced by the A243V allele, even in the presence of functional WT protein, upsets ionic homeostasis. In this regard, hBest1 may physiologically conduct other anions besides Cl–, such as HCO3 or organic anions. Although the A243V mutation does not apparently affect HCO3 permeability, it may affect the permeability of other anions.

hBest1 and Regulation of Ca2+ Channels
Although the hypothesis that hBest1 is responsible for generating the LP is attractive, evidence has recently been presented against this idea. The most compelling evidence is that mice with the mBest1 gene disrupted (mBest1–/– null mutant) have the same maximum LP amplitudes as do WT animals.21

Marmorstein et al. and others20 21 22 have proposed that hBest1 regulates the LP indirectly via its effects on voltage-gated Ca2+ channels. The LP is dependent on a Ca2+ channel, because the LP is reduced by the Ca2+ channel blocker nimodipine.21 This Ca2+ channel apparently contains the CACNB4 subunit, because mice with CACNB4 deleted have a greatly diminished LP.21 The observation that hBest1 overexpression alters the voltage dependence of Ca2+ currents in RPE-J cells,22 therefore suggests that hBest1 may modulate the LP via its effects on voltage-gated Ca2+ channels.

These experiments question whether a diminished LP is causative of BVMD, because mice lacking CACNB4 and the LP do not exhibit retinal degeneration. Humans with CACNB4 mutations (OMIM 601949) have not been reported to exhibit vision problems, although it is not known whether they have normal EOGs.

Although these results support a role for hBest1 in regulating voltage-gated Ca2+ currents and that Ca2+ currents are important in generating the LP, the Ca2+ channel modulator hypothesis is faced with the same questions as the Cl– channel hypothesis. In both cases, the mechanistic link between channel dysfunction (dysfunction of a Cl– channel or dysregulation of a Ca2+ channel) and vitelliform lesions remains to be established. However, all the presently available data support the conclusion that hBest1 mutations disrupts epithelial transport, regardless of whether hBest1 functions as a Cl– channel, a Ca2+ regulator, or both.


   Footnotes
 
Supported by a grant from the American Health Assistance Foundation, National Eye Institute Grant EY014852, and National Institute of General Medical Sciences Grant GM060448.

Submitted for publication May 11, 2006; revised June 12 and June 29, 2006; accepted August 21, 2006.

Disclosure: K. Yu, None; Y. Cui, None; H.C. Hartzell, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: H. Criss Hartzell, Emory University School of Medicine, Cell Biology/Whitehead 535, 615 Michael Street, Atlanta, GA 30322; criss.hartzell{at}emory.edu.


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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