H. Criss Hartzell

Molecular Physiology of Bestrophins: Multifunctional Membrane Proteins Linked to Best Disease and Other Retinopathies

This article reviews the current state of knowledge about the bestrophins, a newly identified family of proteins that can function both as Cl(-) channels and as regulators of voltage-gated Ca(2+) channels. The founding member, human bestrophin-1 (hBest1), was identified as the gene responsible for a dominantly inherited, juvenile-onset form of macular degeneration called Best vitelliform macular dystrophy. Mutations in hBest1 have also been associated with a small fraction of adult-onset macular dystrophies. It is proposed that dysfunction of bestrophin results in abnormal fluid and ion transport by the retinal pigment epithelium, resulting in a weakened interface between the retinal pigment epithelium and photoreceptors. There is compelling evidence that bestrophins are Cl(-) channels, but bestrophins remain enigmatic because it is not clear that the Cl(-) channel function can explain Best disease. In addition to functioning as a Cl(-) channel, hBest1 also is able to regulate voltage-gated Ca(2+) channels. Some bestrophins are activated by increases in intracellular Ca(2+) concentration, but whether bestrophins are the molecular counterpart of Ca(2+)-activated Cl(-) channels remains in doubt. Bestrophins are also regulated by cell volume and may be a member of the volume-regulated anion channel family.

Chloride Channels: Often Enigmatic, Rarely Predictable

Until recently, anion (Cl(-)) channels have received considerably less attention than cation channels. One reason for this may be that many Cl(-) channels perform functions that might be considered cell-biological, like fluid secretion and cell volume regulation, whereas cation channels have historically been associated with cellular excitability, which typically happens more rapidly. In this review, we discuss the recent explosion of interest in Cl(-) channels, with special emphasis on new and often surprising developments over the past five years. This is exemplified by the findings that more than half of the ClC family members are antiporters, and not channels, as was previously thought, and that bestrophins, previously prime candidates for Ca(2+)-activated Cl(-) channels, have been supplanted by the newly discovered anoctamins and now hold a tenuous position in the Cl(-) channel world.

ADF/cofilin-mediated actin dynamics regulate AMPA receptor trafficking during synaptic plasticity

Dendritic spines undergo actin-based growth and shrinkage during synaptic plasticity, in which the actin depolymerizing factor (ADF)/cofilin family of actin-associated proteins are important. Elevated ADF/cofilin activities often lead to reduced spine size and immature spine morphology but can also enhance synaptic potentiation in some cases. Thus, ADF/cofilin may have distinct effects on postsynaptic structure and function. We found that ADF/cofilin-mediated actin dynamics regulated AMPA receptor (AMPAR) trafficking during synaptic potentiation, which was distinct from actins structural role in spine morphology. Specifically, elevated ADF/cofilin activity markedly enhanced surface addition of AMPARs after chemically induced long-term potentiation (LTP), whereas inhibition of ADF/cofilin abolished AMPAR addition. We found that chemically induced LTP elicited a temporal sequence of ADF/cofilin dephosphorylation and phosphorylation that underlies AMPAR trafficking and spine enlargement. These findings suggest that temporally regulated ADF/cofilin activities function in postsynaptic modifications of receptor number and spine size during synaptic plasticity.

Synaptic excitation and inhibition resulting from direct action of acetylcholine on two types of chemoreceptors on individual amphibian parasympathetic neurones

1. Synaptic transmission was studied in visually identified parasympathetic ganglion cells that modulate the heart beat of the mudpuppy Necturus maculosus).

Anoctamin/TMEM16 family members are Ca2+‐activated Cl− channels

Ca2+‐activated Cl− channels (CaCCs) perform many important functions in cell physiology including secretion of fluids from acinar cells of secretory glands, amplification of olfactory transduction, regulation of cardiac and neuronal excitability, mediation of the fast block to polyspermy in amphibian oocytes, and regulation of vascular tone. Although a number of proteins have been proposed to be responsible for CaCC currents, the anoctamin family (ANO, also known as TMEM16) exhibits characteristics most similar to those expected for the classical CaCC. Interestingly, this family of proteins has previously attracted the interest of both developmental and cancer biologists. Some members of this family are up‐regulated in a number of tumours and functional deficiency in others is linked to developmental defects.

Voltage- and calcium-dependent gating of TMEM16A/Ano1 chloride channels are physically coupled by the first intracellular loop

Ca2+-activated Cl− channels (CaCCs) are exceptionally well adapted to subserve diverse physiological roles, from epithelial fluid transport to sensory transduction, because their gating is cooperatively controlled by the interplay between ionotropic and metabotropic signals. A molecular understanding of the dual regulation of CaCCs by voltage and Ca2+ has recently become possible with the discovery that Ano1 (TMEM16a) is an essential subunit of CaCCs. Ano1 can be gated by Ca2+ or by voltage in the absence of Ca2+, but Ca2+- and voltage-dependent gating are very closely coupled. Here we identify a region in the first intracellular loop that is crucial for both Ca2+ and voltage sensing. Deleting 448EAVK in the first intracellular loop dramatically decreases apparent Ca2+ affinity. In contrast, mutating the adjacent amino acids 444EEEE abolishes intrinsic voltage dependence without altering the apparent Ca2+affinity. Voltage-dependent gating of Ano1 measured in the presence of intracellular Ca2+ was facilitated by anions with high permeability or by an increase in [Cl−]e. Our data show that the transition between closed and open states is governed by Ca2+ in a voltage-dependent manner and suggest that anions allosterically modulate Ca2+-binding affinity. This mechanism provides a unified explanation of CaCC channel gating by voltage and ligand that has long been enigmatic.

Tmem16A Encodes the Ca2+-activated Cl− Channel in Mouse Submandibular Salivary Gland Acinar Cells

Activation of an apical Ca2+-dependent Cl− channel (CaCC) is the rate-limiting step for fluid secretion in many exocrine tissues. Here, we compared the properties of native CaCC in mouse submandibular salivary gland acinar cells to the Ca2+-gated Cl− currents generated by Tmem16A and Best2, members from two distinct families of Ca2+-activated Cl− channels found in salivary glands. Heterologous expression of Tmem16A and Best2 transcripts in HEK293 cells produced Ca2+-activated Cl− currents with time and voltage dependence and inhibitor sensitivity that resembled the Ca2+-activated Cl− current found in native salivary acinar cells. Best2−/− and Tmem16A−/− mice were used to further characterize the role of these channels in the exocrine salivary gland. The amplitude and the biophysical footprint of the Ca2+-activated Cl− current in submandibular gland acinar cells from Best2-deficient mice were the same as in wild type cells. Consistent with this observation, the fluid secretion rate in Best2 null mice was comparable with that in wild type mice. In contrast, submandibular gland acinar cells from Tmem16A−/− mice lacked a Ca2+-activated Cl− current and a Ca2+-mobilizing agonist failed to stimulate Cl− efflux, requirements for fluid secretion. Furthermore, saliva secretion was abolished by the CaCC inhibitor niflumic acid in wild type and Best2−/− mice. Our results demonstrate that both Tmem16A and Best2 generate Ca2+-activated Cl− current in vitro with similar properties to those expressed in native cells, yet only Tmem16A appears to be a critical component of the acinar Ca2+-activated Cl− channel complex that is essential for saliva production by the submandibular gland.

Explaining Calcium-Dependent Gating of Anoctamin-1 Chloride Channels Requires a Revised Topology

Rationale: Ca2+-activated Cl channels play pivotal roles in the cardiovascular system. They regulate vascular smooth muscle tone and participate in cardiac action potential repolarization in some species. Ca2+-activated Cl channels were recently discovered to be encoded by members of the anoctamin (Ano, also called Tmem16) superfamily, but the mechanisms of Ano1 gating by Ca2+ remain enigmatic. Objective: The objective was to identify regions of Ano1 involved in channel gating by Ca2+. Methods and Results: The Ca2+ sensitivity of Ano1 was estimated from rates of current activation, and deactivation in excised patches rapidly switched between zero and high Ca2+ on the cytoplasmic side. Mutation of glutamates E702 and E705 dramatically altered Ca2+ sensitivity. E702 and E705 are predicted to be in an extracellular loop, but antigenic epitopes introduced into this loop are not accessible to extracellular antibodies, suggesting this loop is intracellular. Cytoplasmically applied membrane-impermeant sulfhydryl reagents alter the Ca2+ sensitivity of Ano1 E702C and E705C as expected if E702 and E705 are intracellular. Substituted cysteine accessibility mutagenesis of the putative re-entrant loop suggests that E702 and E705 are located adjacent to the Cl conduction pathway. Conclusions: We propose an alternative model of Ano1 topology based on mutagenesis, epitope accessibility, and cysteine-scanning accessibility. These data contradict the popular re-entrant loop model by showing that the putative fourth extracellular loop (ECL 4) is intracellular and may contain a Ca2+ binding site. These studies provide new perspectives on regulation of Ano1 by Ca2+.

Volume sensitivity of the bestrophin family of chloride channels

Bestrophins are a newly identified family of Cl− channels. Mutations in the founding member of the family, human bestrophin‐1 (hBest1), are responsible for a form of early onset macular degeneration called Best vitelliform macular dystrophy. The link between dysfunction of  hBest1 and macular degeneration remains unknown. Because retinal pigmented epithelium (RPE) cells may be subjected to varying osmotic pressure due to light‐dependent changes in the ionic composition of the subretinal space and because RPE cells may undergo large volume changes during phagocytosis of shed photoreceptor discs, we investigated whether bestrophin currents were affected by cell volume. When hBest1 and mBest2 were overexpressed in HEK 293, HeLa, and ARPE‐19 cells, a new Ca2+‐activated Cl− current appeared. This current was very sensitive to cell volume. A 20% increase in extracellular osmolarity caused cell shrinkage and a ∼70–80% reduction in bestrophin current. Decreases in extracellular osmolarity increased the bestrophin currents slightly, but this was difficult to quantify due to simultaneous activation of endogenous volume‐regulated anion channel (VRAC) current. To determine whether a similar current was present in mouse RPE cells, the effect of hyperosmotic solutions on isolated mouse RPE cells was examined. Mouse RPE cells exhibited an endogenous Cl− current that resembled the expressed hBest1 in that it was decreased by hypertonic solution. We conclude that bestrophins are volume sensitive and that they could play a novel role in cell volume regulation of RPE cells.

Characterization of the oligomeric structure of the Ca(2+)-activated Cl- channel Ano1/TMEM16A.

Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca2+-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl− secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Förster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca2+, suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis.