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Address correspondence to Criss Hartzell, Department of Cell Biology, Emory University School of Medicine, 615 Michael St., Whitehead Building 535, Atlanta, GA 30322-3030. Fax: (404) 727-6256; email: criss.hartzell{at}emory.edu
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ABSTRACT |
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= 16.6 s), suggesting that SCN binding to the channel altered channel gating as well as conductance. The data in this paper and other data support a model in which TMD2 plays an important role in forming the bestrophin pore. We suggest that the major determinant in anion permeation involves partitioning of the permeant anion into an aqueous pore whose structural features are rather flexible. Furthermore, anion permeation and gating may be linked.
Key Words: chloride channels • ion permeation • electrophysiology • mutagenesis • ion channel
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; Qu and Hartzell, 2003; Tsunenari et al., 2003; Qu et al., 2004; Pusch, 2004). The suggestion that bestrophins are Cl channels is supported by several lines of evidence. (a) Mutations in human best-1 produce an early-onset macular degeneration known as Best vitelliform macular dystrophy (Petrukhin et al., 1998). This disease is characterized by decreased amplitude of the slow light peak component of the electro-retinogram, suggesting that an ion channel defect may be responsible for the pathology (Francois et al., 1967; Deutman, 1969). Because the slow light peak of the electro-retinogram has been linked to a basolateral Cl conductance in retinal pigment epithelial cells (Gallemore et al., 1998), it has been suspected for some time that hBest1 is a Cl channel. hBest1 may play a role in ion transport in the retina and in regulating the ionic environment around photoreceptor cells. hBest1 has been shown to be localized in or near the basolateral membrane of retinal pigment epithelial cells (Marmorstein et al., 2000). (b) Vertebrates have four genes that code for bestrophins and C. elegans has >25 bestrophin genes. All bestrophins that have been tested from various species from human to worms induce Ca-activated Cl currents when overexpressed in several cell lines (Sun et al., 2002; Hartzell and Qu, 2003; Qu and Hartzell, 2003; Qu et al., 2004). The currents differ somewhat in their rectification and kinetic properties depending on the subtype of bestrophin expressed (Sun et al., 2002; Hartzell and Qu, 2003; Tsunenari et al., 2003). The dependence of the current on the type of bestrophin provides a dose of confidence that the Cl currents are not artifacts of heterologous expression. (c) Overexpressed bestrophin appears at the cell surface as assessed by cell-surface biotinylation or immunocytochemical criteria (Hartzell and Qu, 2003; Qu et al., 2004). (d) Substitution of certain amino acids in hBest1 and mBest2 with cysteine results in Cl currents that can be modified with sulfhydryl-reactive MTS reagents (Tsunenari et al., 2003; Qu et al., 2004). (e) Mutation of serine-79 to cysteine results in Cl currents with altered permeability and conductance, particularly to SCN (Qu et al., 2004). Because ion permeability and conduction are controlled by the partitioning of the ion into the channel pore and the binding of the ion to sites in the permeation pathway, the mutational data provide prima facie evidence that bestrophins contribute to the pore of the Cl channel (Pusch, 2004).
Because serine-79, which we mutated in our previous paper, is located in the putative second transmembrane domain (TMD2), in this paper we have investigated the role of other residues in TMD2 in anion permeability in mBest2. The question that we sought to address was whether S79 was a key residue in determining the biophysical properties of the channel or whether other residues contributed to anion selectivity. To date, structure–function relationships have been studied extensively only in three families of cloned Cl channels: the ligand-gated anion channels like the GABAA and glycine receptors (Keramidas et al., 2002b), the cystic fibrosis transmembrane conductance regulator CFTR (Dawson et al., 1999), and the ClC channel family (Fahlke, 2001). Ligand-gated anion channels select among permeant ions based mainly on charge. Mutations in one or a few key amino acids can convert the channel from anion to cation selective. In contrast, CFTR and the ClC channels have selectivity filters composed of clusters of a number of amino acids located in more than one TMD (Dawson et al., 1999; Linsdell et al., 2000; Dutzler et al., 2002). In these channels, changes in selectivity can be introduced by mutations in an abundance of amino acid residues, but the changes in selectivity are seldom as dramatic as one observes in K channels where mutations can cause >100-fold changes in relative permeability (Armstrong, 2003). The modest changes (<10-fold) in selectivity in CFTR and ClC channels are probably related to the fact that these channels have low intrinsic selectivity and that permeation does not depend as precisely on the details of the pore as it does in K channels.
Here we show that mutations in seven different amino acids in mBest2 produce changes in anion permeability and conduction consistent with a model of the bestrophin channel pore in which the details of the structure of the pore are not as important in determining selectivity as the lyotrophic nature of the permeant anions and their partitioning into an aqueous pore.
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MATERIALS AND METHODS |
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). mBest2 cDNA (ATCC, IMAGE clone ID: 4989959, GENBANK/EMBL/DDBJ accession no. BC031186 and NM_145388) and its mutants in pCMV-SPORT6 vector were cotransfected into HEK293 cells (ATCC) using Fugene-6 transfection reagent (Roche). pEGFP (Invitrogen) was also transfected at a 10:1 ratio to identify transfected cells. To obtain modest amplitude of Ca2+-activated Cl– currents (1–2 nA per cell), 0.05 
0.1 µg mBest2 wild type or 0.025 0.3 µg mBest2 mutant DNA was used to transfect one 35-mm culture dish. 1 d after transfection, cells were dissociated and replated on glass coverslips for electrophysiological recording. Transfected cells were identified by EGFP fluorescence and used for patch clamp experiments within 3 d after transfection.
With the F80R and F80E mutants, the behavior of the currents seemed to depend on the level of expression. When the usual amounts of cDNA were used for transfection, 50% of EGFP-expressing cells had very small currents when cotransfected with the F80E mBest2, whereas 50% of the cells cotransfected with F80R had huge currents that were not well voltage clamped. When the amount of cDNA was adjusted to give moderately sized currents, this heterogeneity disappeared.
Electrophysiology
Recordings were performed using the whole-cell recording configuration of the patch clamp technique. Patch pipettes were made of borosilicate glass (Sutter Instrument Co.), pulled by a Sutter P-2000 puller (Sutter Instrument Co.), and fire polished. Patch pipettes had resistances of 2–3.5 M
filled with the standard intracellular solution (see below). The bath was grounded via a 3 M KCl agar bridge connected to a Ag/AgCl reference electrode. Solution changes were performed by perfusing the 1-ml chamber at a speed of 4 ml/min. Experiments were performed with 100 or 200-ms duration voltage ramps from –100 to +100 mV. The start-to-start interval was 2 or 10 s. Data were acquired by an Axopatch 200A amplifier controlled by Clampex 8.1 via a Digidata 1322A data acquisition system (Axon Instruments). Experiments were conducted at room temperature (20–24°C). Liquid junction potentials were measured using the liquid junction potential calculator in Clampex 8.1 to correct Erev of various bionic conditions. Rectification ratios (RR) were determined from the quotient of the slope conductance measured at +50 mV from the reversal potential divided by the slope conductance measured at –50 mV from the reversal potential.
The standard pipette solution contained (in mM) 146 CsCl, 2 MgCl2, 5 (Ca2+)-EGTA, 8 HEPES, 10 sucrose, pH 7.3, adjusted with NMDG. The free [Ca2+] in the solution was determined as described by Tsien and Pozzan (1989) (Kuruma and Hartzell, 2000). The calculated Ca2+ concentration in the "high Ca intracellular solution" was confirmed as 4.5 µM by fura-2 (Molecular Probes) measurements using an LS-50B luminescence spectrophotometer (Perkin Elmer). The standard extracellular solution contained (in mM) 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 15 glucose,10 HEPES, pH 7.3 with NaOH. This combination of solutions set Erev for Cl– currents to zero, while cation currents carried by Na or Cs would have very positive or negative Erev, respectively. When Cl– was substituted with another anion, NaCl was replaced on an equimolar basis with NaX, where X is the substitute anion. Solution osmolarity was 303 mOsm for both intra- and extracellular solutions (Micro Osmometer, Model 3300; Advanced Instrument). Small differences in osmolarity were adjusted by addition of glucose. DIDS (Molecular Probes) was suspended in water at 50 mM as a stock before working solutions were made.
Sulfhydryl Modification
MTSET (2-trimethylammonioethylmethanethiosulfonate, bromide), MTSES (sodium [2-sulfonatoethyl] methanethiosulfonate) (Toronto Research Chemicals), and NEM (N-ethylmaleimide) (Pierce Chemical Co.) were prepared in water, stored on ice, and used within 90 min. The standard extracellular solution with the reagents diluted to the indicated working concentration was made immediately before use.
Analysis of Data
For the calculations and graphical presentation, we used OriginPro 7.0 software (Microcal). Data are expressed as mean ± SEM. Relative permeability of the channels was determined by measuring the shift in Erev upon changing the bath solution from one containing 151 mM Cl– to another with 140 mM X and 11 mM Cl–, where X is the substitute anion (Qu and Hartzell, 2000). The permeability ratio was estimated using the Goldman-Hodgkin-Katz equation: Px/PCl = [Cl–]i/([X]o exp(
ErevF/RT)) – [Cl–]o/[X]o, where Erev is the difference between the reversal potential with the test anion X and that observed with symmetrical Cl–, and F, R, and T have their normal thermodynamic meanings.
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RESULTS |
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). The SCN block was virtually abolished by mutation of serine-79 to cysteine. These data suggested that S79 was located near the anion binding site of the channel. To explore further the role of S79 in anion binding, we mutated S79 to other amino acids and determined the effects of these mutations on Cl and SCN conductances. Each of these substitutions produced measurable currents. I–V curves for representative mutations S79A and S79Y are shown in Fig. 1 (A and B), and the mean relative permeabilities and conductances for all the mutations are shown in Fig. 1 (C and D). With each of the mutations, the relative SCN permeability was less than for wild type (Fig. 1 C), and the SCN conductance was similar to or greater than the Cl conductance (Fig. 1 D), suggesting that SCN binding was reduced in these mutants in the same way as we have described for S79C. The fact that most of the amino acid substitutions that were made disrupted channel function in qualitatively similar ways suggests that serine in position 79 plays an important role in the pore. However, because none of these mutations alter the selectivity sequence for anions (Table I; unpublished data), channel permeability does not appear to be strongly dependent on the amino acid side chains at this position.
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). However, when we used the more rapid protocol, we were still unable to see evidence for a time-dependent change in conductance with wild-type mBest2. There was no difference in the rate of change of Erev and Gslope (Fig. 2 D). The apparent time constant for wild-type currents was 1.7 s, which is 10-fold faster than for S79T (Fig. 2 C).
S79T Has Reduced Sensitivity to SCN
The above experiments suggested that SCN may bind more slowly to the S79T channel than to wild-type channels. To determine whether this was reflected in a decreased steady-state affinity of SCN for the S79T channel, we measured the ability of SCN to block Cl conductance in S79T-expressing cells. Different amounts of Cl were replaced with equimolar amounts of SCN, and the currents were measured in both wild-type and S79T channels. As we have previously reported (Qu et al., 2004), in wild-type mBest2, SCN blocked both inward and outward currents with the same IC50 (9.8 ± 1.1 mM, n = 4; Fig. 3, C and D). In S79T channels, the IC50s were twofold larger: 18.5 ± 2.7 mM (n = 6) for outward current and 26.6 ± 6.0 mM for inward current (Fig. 3). This apparent lower affinity of the S79T channel is consistent with the slower reduction in channel conductance by SCN in the S79T mutant.
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) suggested that S79 was part of an anion binding site in the mBest2 channel. We then explored the involvement of adjacent residues. Fig. 4 shows an alignment of residues 75–95 of the vertebrate bestrophins. It has been suggested that this domain comprises the second TMD (Tsunenari et al., 2003). Nathans' lab has shown that mutation of the residues shown in bold italics in hBest1 (residues 75, 76, 77, 81, 82, 85, 88, 89, 91, 92, 93, 95, and 96) to cysteine produces no current (Tsunenari et al., 2003). We focused, therefore, on V78, F80, G83, F84, V86, and T87.
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45%. After washing out NEM, MTSET+ was added to the bath. This caused an increase in both inward and outward current, but the effect was significantly less than we found for cells that had not been exposed to NEM (Fig. 6 E). This suggests that a significant fraction of F80C residues had reacted with NEM so that these were now not available for reaction with MTSET+.
Mutation of F80 to Arg or Glu
As a further test of the electrostatic effects at position 80, we mutated F80 into Arg or Glu. When F80 was mutated into Glu, the currents were either inwardly rectifying or very small (Fig. 7 A). These results are consistent with the effect of MTSES– modification, which also made the current inwardly rectifying and reduced the conductance (Fig. 6). In contrast, when F80 was mutated into positively charged Arg, the currents outwardly rectified (Fig. 7 B). These results are consistent with the effects of MTSET+. Fig. 7 C plots the average rectification ratio (ratio of inward current at +50 mV to current at –50 mV) as a function of the charge located at position 80 in mBest2.
F80R Exhibits Voltage-dependent Block by DIDS
Currents in F80R-expressing cells were blocked by DIDS in a voltage-dependent manner (Fig. 8). With the F80R mutant, 100 µM DIDS blocked the current 90% at +100 mV, but only 45% at –100 mV. In contrast, with wild type and the F80E mutant the block was voltage independent (Fig. 8 A). The F80E mutant was somewhat less sensitive to DIDS than wild type (Fig. 8 A). To analyze the voltage dependence of block of F80R, we measured the effect of different DIDS concentrations at different potentials between –100 mV and +100 mV and analyzed the data according to Woodhull (1973). The currents were more sensitive to DIDS at more positive potentials (Fig. 8 C). The apparent Ki decreased from 38.6 µM at 10 mV to 16 µM at 100 mV (Fig. 8, D and E). According to the Woodhull equation, this predicts that the binding site for DIDS is 15% into the voltage field of the membrane. The voltage dependence of DIDS block provides additional evidence that F80 is in the conduction pathway of the channel.
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60%, whereas MTSES– stimulated the current approximately eightfold. The fact that F80C and V78C mutants respond oppositely to MTS reagents suggests that these residues have different proximities or relationships to the permeant anion. Because negative charge at position 80 inhibits current, this suggests that this position is closer to the narrowest region of the conduction pathway.
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Br > SCN. None of the three mutations altered the permeability or conductance sequence significantly. The only possible exception to this generalization was in the F80R mutation, where GI > GBr was changed to GI = GBr. In all three mutations, PSCN/PCl was reduced, GSCN/GCl was enhanced, and GNO3/GCl was decreased. |
DISCUSSION |
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Mutations in Cl Channel Pores Generally Have Relatively Modest Effects on Selectivity
In identifying the amino acid residues that define the pore of the bestrophins, one would like to find a point mutation that dramatically alters the selectivity of the channel. The most dramatic example would be conversion of the channel from anion selective to cation selective. Such a change in selectivity has been reported for the A251E mutation in glycine receptors (Keramidas et al., 2002a). Such a dramatic switch in selectivity is possible to engineer in glycine receptors probably because their selectivity is largely due to electrostatic interactions of the permeant anion with basic residues in the channel pore. The converse substitutions convert the nAChR from cation selective to anion selective (Galzi et al., 1992). It has not been possible to convert voltage-gated and inwardly rectifying K channels into anion channels, but discrete mutations in any one of four residues in the K channel signature sequence have quantitatively dramatic effects on K selectivity. For example, mutation of either of the glycine residues in the TMTTVGYG sequence in the Shaker channel abolishes K selectivity (Heginbotham et al., 1994). K channels normally select K:Na by 100:1, but in the G to A mutant, Na and K have about equal permeabilities. Mutations in the signature sequence can be grouped into two groups, ones that have little effect on selectivity and ones that abolish selectivity among cations altogether. The mutations that abolish selectivity probably do so because they produce major structural changes in pore structure.
In contrast, mutational analysis of amino acids in Cl channel pores (with the exception of ligand-gated anion channels) does not produce such discrete outcomes, probably because these channels are not as highly selective as voltage-gated cation channels. In the ClC family, for example, four stretches of amino acids that are not contiguous in the primary sequence contribute to forming the selectivity filter (Jentsch et al., 2002). Mutation of almost every amino acid in these domains changes anion selectivity (Fahlke, 2001). In human ClC-1, for example, mutation of 17/19 amino acid residues in these regions alters channel selectivity (Fahlke et al., 1997). Moreover, the effects of these mutations are relatively modest. For example, one of the mutations (G233A) in the human ClC-1 channel that produces the largest changes in relative permeability increases PSCN/PCl only approximately eightfold and increases PNO3/PCl and PI/PCl only approximately threefold, compared with the 100-fold changes in Na/K selectivity produced by mutations in Shaker.
A similar situation exists with CFTR (Dawson et al., 1999). The pore of CFTR is comprised of residues in both TMD6 and TMD11. Indeed, although numerous mutations within the transmembrane regions of CFTR alter anion binding and single channel conductance, most mutations have rather little effect on anion selectivity. Even mutations that alter the selectivity sequence, such as F337A, do so by less than fourfold changes in relative permeabilities (Linsdell et al., 2000). These data have led Dawson and colleagues to propose that the detailed structure of the CFTR pore may not be a major factor determining anion selectivity (Dawson et al., 1999; Smith et al., 1999, 2001). Rather, they propose that permeation is determined largely by the ease with which an anion partitions into the channel, which is a function of how easily the anion exchanges its water of hydration with residues in the channel. As long as the pore provides an adequately hydrophilic environment for the permeating anion, small perturbations in channel structure may not alter the permeability.
Perturbation of Selectivity in mBest2 by Mutations in TMD2
Our results with mBest-2 are comparable to those reported for ClCs and CFTR with regard to the relatively modest changes that mutations confer on channel selectivity. We find that the largest effects of mutations in S79 and F80 on permeability were evident only with SCN as the permeant anion. PSCN/PCl was changed greater than fivefold, but NO3, I, and Br permeabilities were not significantly affected. This is similar to the effect of pore mutations in hClC-1 and CFTR that affected relative SCN permeability more than the relative permeability of smaller anions. Furthermore, in mBest2, there was not a single amino acid residue that exhibited priority in determining anion permeability: mutations in V78, S79, F80, G83, F84, V86, and T87 altered selectivity in similar manners. This suggests that, like CFTR and the ClCs, certain details of the structure of the pore may not be crucial in determining anion permeation. Permeation may simply depend on ions partitioning into a hydrophilic channel, and as long as the channel maintains this hydrophilic pore, permeation occurs relatively normally. It should be emphasized that cysteine mutations at 10 positions in TMD2 (75, 76, 77, 81, 82, 85, 88, 89, 91, and 92) result in nonfunctional channels. These mutations may result in disruption of the channel pore structure sufficiently so that the channel is not capable of acting as an aqueous pore for anions.
The suggestion that the specific structure of the channel pore is relatively unimportant in selectivity is supported by our observation that qualitatively similar effects on channel permeability are produced by substitutions at positions 79 and 80 with amino acids having a diversity of side chains. For example, PSCN/PCl and GSCN/GCl were similar in the S79A, S79E, and S79R mutants. The ability of mBest2 to continue to function as a Cl channel, albeit with different selectivity than the wild type, in the light of presumed major changes in the putative pore domain suggest that either TMD2 is not actually the pore domain or that the mechanisms of selectivity do not rely on the amino acid side chains, at least when they are altered one at a time.
The role of electrostatic interactions in the mBest2 pore remains unclear. Within the putative TMD2, the only positively charged residue is R92. Nathans and coworkers have reported that the R92C mutation in hBest1 produced no current (Tsunenari et al., 2003). The fact that introduction of charge at position F80 produces changes in rectification suggest that F80 is in close proximity to the permeant anion, but the absence of a charged residue in the native protein in TMD2 suggests that either the electrostatic environment of the pore may not be important in permeation or that charged residues are contributed from other regions of the mBest2 protein that we have not yet investigated in detail.
Channel Gating
Because we have measured macroscopic currents, we do not know whether the changes in relative conductance that occur as a consequence of mutations are caused by changes in single channel conductance or channel gating. However, the observation that the time course of change in conductance in the S79T mutant lags significantly behind the change in Erev provides support to the idea that the decreased conductance produced by SCN is due at least partially to changes in channel gating. One would predict that, if the small GSCN/GCl in wild-type mBest2 was a consequence of a smaller flux of SCN relative to Cl though the channel, the effect of SCN would occur instantaneously. That is, if GSCN/GCl is due entirely to the binding of SCN to a site in the selectivity filter of the channel, this should occur as soon as SCN begins to permeate the channel, as evidenced by the shift in the Erev. However, the fact that this change takes about a minute to reach completion in the S79T mutant suggests that it takes some time for SCN to reach its binding site. If this is the case, the binding site may not be in the permeation pathway and that GSCN/GCl may be determined by binding of SCN to an allosteric site in the channel, possibly on the cytoplasmic side. The observation that block of current by SCN is not detectably voltage dependent supports the suggestion that the SCN binding site is not in the selectivity filter. However, the observation that DIDS blocks the current induced by the F80R mutant in a voltage-dependent manner argues that F80 is located in the voltage field of the membrane.
Topology of mBest2
Tsunenari et al. (2003) have proposed a topology model for hBest1 based on insertion of N-linked glycosylation sites and TEVP protease cleavage sites as well as the effects of membrane-impermeant sulfhydryl reagents on hBest1 currents. From these data, they propose that hBest1 has four TMDs and that TMD2 crosses the membrane from outside to inside in the NH2- to COOH-terminal direction. However, some of their data is inconsistent with this interpretation. Their data show clearly that residues C69 and N99 are accessible to the extracellular space, whereas their model places these residues at the opposite ends of TMD2. Their evidence is based on modification of hBest1 currents by the membrane-impermeant sulfhydryl reagent MTSET+. Modification of the native C69 residue with extracellular MTSET+ inhibits hBest1 current, so C69 is extracellular. The C69A mutant is unaffected by MTSET+, confirming that the effect of MTSET+ is on C69. N99 is also thought to be extracellular because extracellular MTSET+ inhibits the current produced by the C69A/N99C mutant. Intracellular cysteine does not quench the effect of MTSET+ on the C69A/N99C mutant, showing that MTSET+ does not permeate the channel. It seems impossible that both C69 and N99 are extracellular if this sequence is truly transmembrane.
There are differences between hBest1 and mBest2 that raise additional questions. First, although position 69 is Cys in both mBest2 and hBest1, MTSET+ inhibits the hBest1 current via C69 (Tsunenari et al., 2003) but has no effect on wild-type mBest2 current (Qu et al., 2004). Second, although mutation of N99 to Cys in hBest1 results in a current that is sensitive to MTSET+, the wild-type residue in mBest2 at position 99 is Cys, but wild-type mBest2 is not sensitive to MTSET. Is it possible that mBest2 and hBest1 have different topologies or do the mutations cause major structural rearrangements that are confounding the interpretations?
Our data with DIDS block of the F80R mutated channel suggest that this residue is 15% of the way into the voltage field from the outside of the cell. Furthermore, we find that residues from 78 to 87 are accessible to MTS reagents applied from the outside of the cell. These data suggest that this region of mBest2 is in an outer vestibule of the channel.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health grants GM60448 and EY014852 to H.C. Hartzell and American Heart Association Scientist Development Grant 0430204N to Z. Qu.
David C. Gadsby served as editor.
Submitted: 21 May 2004
Accepted: 18 August 2004
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V. M. Milenkovic, A. Rivera, F. Horling, and B. H. F. Weber Insertion and Topology of Normal and Mutant Bestrophin-1 in the Endoplasmic Reticulum Membrane J. Biol. Chem., January 12, 2007; 282(2): 1313 - 1321. [Abstract] [Full Text] [PDF]
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 L. G. Palmer and G. Frindt Cl- channels of the distal nephron Am J Physiol Renal Physiol, December 1, 2006; 291(6): F1157 - F1168. [Abstract] [Full Text] [PDF]
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K. Yu, Y. Cui, and H. C. Hartzell The Bestrophin Mutation A243V, Linked to Adult-Onset Vitelliform Macular Dystrophy, Impairs Its Chloride Channel Function Invest. Ophthalmol. Vis. Sci., November 1, 2006; 47(11): 4956 - 4961. [Abstract] [Full Text] [PDF]
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L.-T. Chien, Z.-R. Zhang, and H. C. Hartzell Single Cl- Channels Activated by Ca2+ in Drosophila S2 Cells Are Mediated By Bestrophins J. Gen. Physiol., August 28, 2006; 128(3): 247 - 259. [Abstract] [Full Text] [PDF]
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 S. Pifferi, G. Pascarella, A. Boccaccio, A. Mazzatenta, S. Gustincich, A. Menini, and S. Zucchelli Bestrophin-2 is a candidate calcium-activated chloride channel involved in olfactory transduction PNAS, August 22, 2006; 103(34): 12929 - 12934. [Abstract] [Full Text] [PDF]
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T. Tsunenari, J. Nathans, and K.-W. Yau Ca2+-activated Cl- Current from Human Bestrophin-4 in Excised Membrane Patches J. Gen. Physiol., May 30, 2006; 127(6): 749 - 754. [Abstract] [Full Text] [PDF]
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Z. Qu, L.-T. Chien, Y. Cui, and H. C. Hartzell The anion-selective pore of the bestrophins, a family of chloride channels associated with retinal degeneration. J. Neurosci., May 17, 2006; 26(20): 5411 - 5419. [Abstract] [Full Text] [PDF]
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L. Y. Marmorstein, J. Wu, P. McLaughlin, J. Yocom, M. O. Karl, R. Neussert, S. Wimmers, J. B. Stanton, R. G. Gregg, O. Strauss, et al. The Light Peak of the Electroretinogram Is Dependent on Voltage-gated Calcium Channels and Antagonized by Bestrophin (Best-1) J. Gen. Physiol., April 24, 2006; 127(5): 577 - 589. [Abstract] [Full Text] [PDF]
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 C. Hartzell, Z. Qu, I. Putzier, L. Artinian, L.-T. Chien, and Y. Cui Looking Chloride Channels Straight in the Eye: Bestrophins, Lipofuscinosis, and Retinal Degeneration Physiology, October 1, 2005; 20(5): 292 - 302. [Abstract] [Full Text] [PDF]
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O. Strauss The Retinal Pigment Epithelium in Visual Function Physiol Rev, July 1, 2005; 85(3): 845 - 881. [Abstract] [Full Text] [PDF]
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