Olaf Strauss

TMEM16B, A Novel Protein with Calcium-Dependent Chloride Channel Activity, Associates with a Presynaptic Protein Complex in Photoreceptor Terminals

Photoreceptor ribbon synapses release glutamate in response to graded changes in membrane potential evoked by vast, logarithmically scalable light intensities. Neurotransmitter release is modulated by intracellular calcium levels. Large Ca2+-dependent chloride currents are important regulators of synaptic transmission from photoreceptors to second-order neurons; the molecular basis underlying these currents is unclear. We cloned human and mouse TMEM16B, a member of the TMEM16 family of transmembrane proteins, and show that it is abundantly present in the photoreceptor synaptic terminals in mouse retina. TMEM16B colocalizes with adaptor proteins PSD95, VELI3, and MPP4 at the ribbon synapses and contains a consensus PDZ class I binding motif capable of interacting with PDZ domains of PSD95. Furthermore, TMEM16B is lost from photoreceptor membranes of MPP4-deficient mice. This suggests that TMEM16B is a novel component of a presynaptic protein complex recruited to specialized plasma membrane domains of photoreceptors. TMEM16B confers Ca2+-dependent chloride currents when overexpressed in mammalian cells as measured by halide sensitive fluorescent protein assays and whole-cell patch-clamp recordings. The compartmentalized localization and the electrophysiological properties suggest TMEM16B to be a strong candidate for the long sought-after Ca2+-dependent chloride channel in the photoreceptor synapse.

Ion channels in the RPE.

In close interaction with photoreceptors, the retinal pigment epithelium (RPE) plays an essential role for visual function. The analysis of RPE functions, specifically ion channel functions, provides a basis to understand many degenerative diseases of the retina. The invention of the patch-clamp technique significantly improved the knowledge of ion channel structure and function, which enabled a new understanding of cell physiology and patho-physiology of many diseases. In this review, ion channels identified in the RPE will be described in terms of their specific functional role in RPE physiology. The RPE expresses voltage- and ligand-gated K(+), Cl(-), and Ca(2+)-conducting channels. K(+) and Cl(-) channels are involved in transepithelial ion transport and volume regulation. Voltage-dependent Ca(2+) channels act as regulators of secretory activity, and ligand-gated cation channels contribute to RPE function by providing driving forces for ion transport or by influencing intracellular Ca(2+) homoeostasis. Collectively, activity of these ion channels determines the physiology of the RPE and its interaction with photoreceptors. Furthermore, changes in ion channel function, such as mutations in ion channel genes or a changed regulation of ion channel activity, have been shown to lead to degenerative diseases of the retina. Increasing knowledge about the properties of RPE ion channels has not only provided a new understanding of RPE function but has also provided greater understanding of RPE function in health and disease.

The Ternary Rab27a–Myrip–Myosin VIIa Complex Regulates Melanosome Motility in the Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) contains melanosomes similar to those found in the skin melanocytes, which undergo dramatic light‐dependent movements in fish and amphibians. In mammals, those movements are more subtle and appear to be regulated by the Rab27a GTPase and the unconventional myosin, Myosin VIIa (MyoVIIa). Here we address the hypothesis that a recently identified Rab27a‐ and MyoVIIa‐interacting protein, Myrip, promotes the formation of a functional tripartite complex. In heterologous cultured cells, all three proteins co‐immunoprecipitated following overexpression. Rab27a and Myrip localize to the peripheral membrane of RPE melanosomes as observed by immunofluorescence and immunoelectron microscopy. Melanosome dynamics were studied using live‐cell imaging of mouse RPE primary cultures. Wild‐type RPE melanosomes exhibited either stationary or slow movement interrupted by bursts of fast movement, with a peripheral directionality trend. Nocodazole treatment led to melanosome paralysis, suggesting that movement requires microtubule motors. Significant and similar alterations in melanosome dynamics were observed when any one of the three components of the complex was missing, as studied in ashen‐ (Rab27a defective) and shaker‐1 (MyoVIIa mutant)‐derived RPE cells, and in wild‐type RPE cells transduced with adenovirus carrying specific sequences to knockdown Myrip expression. We observed a significant increase in the number of motile melanosomes, exhibiting more frequent and prolonged bursts of fast movement, and inversion of directionality. Similar alterations were observed upon cytochalasin D treatment, suggesting that the Rab27a–Myrip–MyoVIIa complex regulates tethering of melanosomes onto actin filaments, a process that ensures melanosome movement towards the cell periphery.

Regulation of L-type calcium channels by protein tyrosine kinase and protein kinase C in cultured rat and human retinal pigment epithelial cells.

The effect of protein tyrosine kinases (PTKs) on L‐type calcium channel currents was studied in cultured rat and human retinal pigment epithelial cells. Barium currents through L‐type channels were measured in the perforated patch‐clamp technique and identified by using the L‐type calcium channel opener Bay K8644 (10−6 M). Application of the PTK blockers genistein (5 × 10−6 M) or lavendustin A (5×10−6 M) led to a decrease of L‐type currents. The inactive genistein analog daidzein (10−5 M) showed no effect on calcium channels. Intracellular application of pp60c‐src (30 U/ml) via the patch‐pipette during the conventional whole‐cell configuration led to an increase of L‐type currents. The protein kinase A and protein kinase G blocker H9 (10−6 M) showed no effect on L‐type currents; genistein reduced the current in the presence of H9. The protein kinase C (PKC) blocker chelerythrine (10−5 M) reduced the L‐type current; additional inhibition of PTK by lavendustin showed an additional reduction of currents. Intracellular application of myristoylated PKC substrate (5×10−5 M) for PKC inhibition led to a fast rundown of L‐type current amplitudes. Intracellularly applied myristoylated PKC substrate (10−4 M) together with pp60c‐src showed no effect on L‐type current. Up‐regulation of PKC by 10−6 M phorbol‐12‐myristate‐13‐acetate (PMA) had no effect on the L‐type current amplitude. However, genistein in cells pretreated with PMA led to an increase of the L‐type currents. Intracellular application of pp60c‐src in PMA‐treated cells led to a reduction of L‐type currents. We conclude that in the resting cell, PTK and PKC regulate L‐type calcium channels in an additive manner. L‐type channels appeared as a site of integration of PTK activation and of PKC‐dependent pathways. The activity of PKC determines whether PTK decreases or increases L‐type channel activity.—Strauss, O., Mergler, S., Wiederholt, M. Regulation of L‐type calcium channels by protein tyrosine kinase and protein kinase C in cultured rat and human retinal pigment epithelial cells. FASEB J. 11, 859–867 (1997)

The Royal College of Surgeons Rat: An Animal Model for Inherited Retinal Degeneration with a Still Unknown Genetic Defect

The Royal College of Surgeons (RCS) rat is the first known animal with inherited retinal degeneration. Despite the fact that the genetic defect is not known, the RCS rat is widely used for research in hereditary retinal dystrophies. This review tries to summarize observations which have been made in the RCS rat and to make an attempt to formulate candidate genes which may the cause for the retinal degeneration in this rat strain. The genetic defect in RCS rats causes the inability of the retinal pigment epithelium (RPE) to phagocytose shed photoreceptor outer segments. In normal rats or humans, this circadian process is regulated by both the cyclic adenosine monophosphate (cAMP) and the calcium/ inositol phosphate systems. The calcium/inositol phosphate system seems to be linked to the phagocytosis receptors which recognize photoreceptor outer membranes to initialize phagocytosis. The cAMP system appeared as modulator of the regulation of phagocytosis. An increase in the intracellular cAMP concentration is an ‘off’ signal for phagocytosis. In RPE cells from RCS rats many observations have been made which indicate a changed second messenger metabolism concerning both the cAMP and the calcium/inositol phosphate systems. The genetic defect seems to concern a protein which is involved in the initialization of a second messenger pathway. We conclude that the genes coding for the phagocytosis receptor or for proteins which are linked to receptors (for example G proteins) are good candidates for defective genes in RCS rats.

Heat-sensitive TRPV channels in retinal pigment epithelial cells: regulation of VEGF-A secretion.

PURPOSE Choroidal neovascularization in age-related macular degeneration is caused, to a large extent, by increased secretion of vascular endothelial growth factor (VEGF)-A by the retinal pigment epithelium (RPE). The purpose of the study was to identify pathways that lead to increased VEGF secretion by the RPE. METHODS Ca(2+) signaling was studied in ARPE-19 and human RPE cells in primary culture by means of Ca(2+) imaging. Membrane conductance was measured in the whole-cell configuration of the patch-clamp technique. VEGF-A secretion was measured by using ELISA. RESULTS Freshly isolated RPE cells or ARPE-19 cells were shown to express TRPV1, -2, -3, and -4 channels. Increasing the temperature or stimulation by IGF-1 increased the VEGF-A secretion rate in both cell types. These effects were both sensitive to the TRPV channel blocker ruthenium red (20 μM). The heat-inducible Ca(2+) signals were blocked by the TRPV channel blockers La(3+) and ruthenium red by 68% and 52%, respectively. In contrast, high concentrations of 2-APB (3 mM) increased [Ca(2+)](i), whereas the TRPV1 channel opener capsaicin and the TRPV3 channel opener camphor had no effect. Reduction of TRPV2 expression by siRNA attenuated the heat-evoked Ca(2+) response. In addition, a heat-activated inwardly rectifying current was measured that was completely blocked by ruthenium red. IGF-1 also increased whole-cell current with a corresponding increase in [Ca(2+)](i), which was blocked by the PI3-kinase blocker LY294002. CONCLUSIONS The data strongly suggest that TRPV2 channels expressed by the RPE are involved in the Ca(2+) signaling that mediates both heat-dependent and IGF-1 (via PI3-kinase activation)-induced VEGF secretion.

Altered regulation of L-type channels by protein kinase C and protein tyrosine kinases as a pathophysiologic effect in retinal degeneration

The effect of protein tyrosine kinases (PTK) on L‐type calcium channels in cultured retinal pigmented epithelium (RPE) from rats with retinal dystrophy was investigated. Barium currents through Bay K 8644 (10–6M) sensitive L‐type channels were measured using the patch‐clamp technique. The current density of L‐type currents is twice as high and the inactivation time constants are much slower than in cells from nondystrophic control rats. Application of the PTK blockers genistein, lavendustin A, and herbimycin A (all 5× 10–6 M) led to an increase of L‐type currents. Intracellular application of pp60c‐src (30 U/ml) via the patch pipette led to a transient decrease of L‐type currents. The protein kinase A (PKA) and PKG blocker H9 (10–6 M) showed no effect on L‐type currents. However, the protein kinase C blocker chelerythrine (10–5 M) reduced these currents. Up‐regulation of PKC by 10–6 M 4β‐phorbol‐12 myristate‐13 acetate (PMA) led to a decrease of L‐type currents. Additional application of genistein led to a further decrease of these currents. However, intracellular application of pp60c‐src in PMA‐treated cells led to a transient increase of L‐type currents. Investigating the calcium response to bFGF application showed that RPE cells from RCS rats used different pathways than control RPE cells to increase cytosolic free calcium. This different pathway does not involve the activation of L‐type channels. The present study with RPE cells from rats with retinal dystrophy shows a changed integration of PTK and PKC in channel regulation. Considering the altered response to bFGF in RCS‐RPE cells, this disturbed regulation of L‐type channels by tyrosine kinases is involved in the etiology of retinal degeneration in RCS rats.—Mergler, S., Steinhausen, K., Wiederholt, M., Strauss, O. Altered regulation of L‐type channels by protein kinase C and protein tyrosine kinases as a pathophysiologic effect in retinal degeneration. FASEB J. 12, 1125–1134 (1998)

Disease-associated missense mutations in bestrophin-1 affect cellular trafficking and anion conductance

Bestrophin-1, an integral membrane protein encoded by the BEST1 gene, is localized predominantly to the basolateral membrane of the retinal pigment epithelium. Mutations in the BEST1 gene have been associated with Best vitelliforme macular dystrophy (BMD), a central retinopathy with autosomal dominant inheritance and variable penetrance. Over 120 disease-causing mutations are known, the majority of which result in amino acid substitutions within four mutational hot-spot regions in the highly conserved N-terminal half of the protein. Although initially thought to impair Cl− channel function, the molecular pathology of BEST1 mutations is still controversial. We have analyzed the subcellular localization of 13 disease-associated BEST1 mutant proteins in polarized MDCK II cells, an established model of apical to basolateral protein sorting. Immunostaining demonstrated that nine of the 13 mutant proteins failed to integrate into the cell membrane. The defective proteins were predominantly retained in the cytoplasm, whereas wild-type bestrophin-1 revealed cell membrane localization. Functional analysis of I− fluxes in HEK-293 cells showed that all mutants exhibited a significant reduction in anion conductance. Our data indicate that defective intracellular trafficking could be a common cause of BMD accompanied by impaired anion conductance, representing a loss of anion channel function that is probably due to mistargeting of mutant protein.

Endogenous Gas6 and Ca2+-channel activation modulate phagocytosis by retinal pigment epithelium

Mutation or loss of MerTK as well as deficiency of alphavbeta5-integrins, gives rise to retinal-degeneration due to inefficient phagocytosis of photoreceptor outer-segment fragments by the retinal pigment epithelium (RPE). This study shows that Gas6 expressed endogenously by human RPE promotes phagocytosis. The RPE expresses Gas6 more highly in vivo and in serum-reduced conditions in vitro than in high-serum conditions, suggesting a negative-feedback control. An antibody-blockage approach revealed that Gas6-expressing RPE phagocytizes photoreceptor outer-segment fragments due to stimulation of MerTK by endogenous Gas6 in vitro. MerTK- and Gas6-antibodies reduced phagocytosis. Blocking L-type Ca(2+)-channels with nifedipine inhibited MerTK dependent phagocytosis in vitro. Application of integrin inhibitory, soluble, RGD-containing peptides or soluble vitronectin reduced L-type Ca(2+)-channel currents in RPE. Herbimycin A, which reduces phosphorylation of integrin receptor-associated proteins and decreases L-type Ca(2+)-channel currents in RPE, eliminates the inhibiting vitronectin effect and abolishes phagocytosis. Thus, Gas6-promoted phagocytosis was inhibited by L-type Ca(2+)-channel blockage, which in turn may be activated by integrin receptor stimulation. These results suggest that L-type Ca(2+)-channels could be regulated downstream of both MerTK and alphavbeta5-integrin, indicating that the binding and uptake mechanisms of phagocytosis are part of a converging pathway.

Involvement of Protein Tyrosine Kinase in the InsP3-Induced Activation of Ca2+-Dependent Cl− Currents in Cultured Cells of the Rat Retinal Pigment Epithelium

Abstract. This combined study of patch-clamp and intracellular Ca2+ ([Ca2+]i) measurement was undertaken in order to identify signaling pathways that lead to activation of Ca2+-dependent Cl− channels in cultured rat retinal pigment epithelial (RPE) cells. Intracellular application of InsP3 (10 μm) led to an increase in [Ca2+]i and activation of Cl− currents. In contrast, intracellular application of Ca2+ (10 μm) only induced transient activation of Cl− currents. After full activation by InsP3, currents were insensitive to removal of extracellular Ca2+ and to the blocker of ICRAC, La3+ (10 μm), despite the fact that both maneuvers led to a decline in [Ca2+]i. The InsP3-induced rise in Cl− conductance could be prevented either by thapsigargin-induced (1 μm) depletion of intracellular Ca2+ stores or by removal of Ca2+ prior to the experiment. The effect of InsP3 could be mimicked by intracellular application of the Ca2+-chelator BAPTA (10 mm). Block of PKC (chelerythrine, 1 μm) had no effect. Inhibition of Ca2+/calmodulin kinase (KN-63, KN-92; 5 μm) reduced Cl−-conductance in 50% of the cells investigated without affecting [Ca2+]i. Inhibition of protein tyrosine kinase (50 μm tyrphostin 51, 5 μm genistein, 5 μm lavendustin) reduced an increase in [Ca2+]i and Cl− conductance. In summary, elevation of [Ca]i by InsP3 leads to activation of Cl− channels involving cytosolic Ca2+ stores and Ca2+ influx from extracellular space. Tyrosine kinases are essential for the Ca2+-independent maintenance of this conductance.