Olaf Strauß

The presence of bestrophin-1 modulates the Ca2+ recruitment from Ca2+ stores in the ER

Bestrophin-1, mainly analyzed in overexpression experiments, functions as Ca2+-dependent Cl– channel. Analysis of endogenously expressed bestrophin-1 suggested an influence on intracellular Ca2+. The aim of the study is to analyze the influence of endogenously expressed bestrophin-1 on Ca2+ homeostasis. Primary cultures of retinal pigment epithelial (RPE) cells were established from wild-type and bestrophin-1-deficient mice. Intracellular free Ca2+ ([Ca2+]i) was recorded by Ca2+ imaging; through immunocytochemistry and differential centrifugation, subcellular localization of bestrophin-1 was analyzed. RPE cells of bestrophin-1-deficient mice showed higher levels of resting [Ca2+]i than cells from wild-type mice. In cells from knockout mice and wild-type mice, ATP led to increases in [Ca2+]i subsequent to phospholipase C activation. ATP-induced Ca2+ in bestrophin-1-deficient mice rose faster and decayed slower. In cells from wild-type mice, ATP led to [Ca2+]i increase via depletion of Ca2+ from thapsigargin-sensitive stores. In cells from bestrophin-1-deficient mice, ATP-dependent increase in [Ca2+]i resulted in 40% of cells from depletion of bafilomycin-sensitive and in 60% from thapsigargin-sensitive Ca2+ stores. After differential centrifugation, bestrophin-1 was found in fractions enriched of ClC-3 Cl channel and myosin-7A. Co-localization analysis of bestrophin-1, with β-catenin or pan-cadherin, in fresh sections of porcine retina, revealed bestrophin-1 in the basolateral membrane. A portion of endogenously expressed bestrophin-1,localized in the endoplasmic reticulum, influenced uptake of Ca2+ into Ca2+ stores. Therefore, bestrophin-1 possibly conducts Cl– as counter ion for Ca2+ uptake into cytosolic Ca2+ stores.

Ca2+-Channels in the RPE

The retinal pigment epithelium closely interacts with photoreceptors and helps to maintain the activity of photoreceptors. Investigations using patch-clamp techniques on cultured or freshly isolated retinal pigment epithelial cells from various species demonstrated the expression of voltage-dependent Ca2+ channels with characteristics of L-type channels. Since retinal pigment epithelial cells rarely display changes of the membrane potential which lead to the activation of these Ca2+ channels, their function seemed to be unclear. Recent findings shed light onto the possible role of these Ca2+ channels. First of all, the subtype of these ion channels could be identified as neuroendocrine subtype of L-type channels. Recent studies demonstrated that the neuroendocrine subtype of L-type channels is regulated by serine/ threonine kinases and protein tyrosine kinases. These phosphorylation-dependent regulatory mechanisms lead to Ca2+ fluxes into the cell which are independent of changes in the membrane potential and induced by a shift in the voltage-dependence ofthese ion channels. The regulation modality implied that L-type Ca2+ channels play an important role in signal transduction pathways which are important for a communication between retinal pigment epithelium and photoreceptors. L-type Ca2+ channels in the retinal pigment epithelium seem to be involved in the regulation of secretion of various factors, in growth factor-dependent intracellular signalling and in the regulation of the phagocytosis of photoreceptor outer membranes. Thus, voltage-dependent Ca2+ channels in the retinal pigment epithelium are of importance for the function of photoreceptors.

Effect of bestrophin-1 on L-type Ca2+ channel activity depends on the Ca2+ channel beta-subunit.

Bests vitelliforme macular degeneration is an inherited retinal degeneration associated with a reduction of the light-peak in the patients electro-oculogram. Bestrophin-1, the product of the disease-promoting/forming gene can function as regulator of voltage-dependent L-type Ca(2+) channels in the retinal pigment epithelium (RPE). Since mice deficient for either β4-subunits or Ca(V)1.3 subunits show reduced light-peaks, the regulatory function of bestrophin-1 on heterologously expressed Ca(2+) channels composed of the pore-forming Ca(V)1.3 and the auxiliary β4-subunit was analyzed. Precipitation of β4-subunits led to co-precipitation with bestrophin-1 and subsequent analysis of subcellular localization showed co-localization of bestrophin-1, Ca(V)1.3 and β4-subunit in the cell membrane. Ca(V)1.3 currents in the presence of β4-subunits and bestrophin-1 showed accelerated time-dependent activation and decreased current density compared to currents measured in the absence of bestrophin-1. In the presence of the β3-subunit, which is not expressed in the RPE bestrophin-1 did not modulate Ca(V)1.3 activity. Deletion of a cluster of proline-rich motifs in the C-terminus of bestrophin-1 reduced its co-immuno precipitation with the β4-subunit and strongly reduced the Ca(V)1.3 activity. Cells co-expressing bestrophin-1 lacking the proline-rich motifs and Ca(V)1.3 subunits showed less efficient trafficking of bestrophin-1 into the cell membrane. In summary, we conclude that bestrophin-1 modulates L-type channels of the RPE via proline-rich motif-dependent interaction with β4-subunits. A disturbed interaction reduces the currents of the Ca(V)1.3 subunits. This mechanism could open new ways to understand changes in the patients electro-oculogram and functional alterations of the RPE leading to retinal degeneration.

Interaction of bestrophin-1 and Ca2+ channel β-subunits: identification of new binding domains on the bestrophin-1 C-terminus.

Bestrophin-1 modulates currents through voltage-dependent L-type Ca2+ channels by physically interacting with the β-subunits of Ca2+ channels. The main function of β-subunits is to regulate the number of pore-forming CaV-subunits in the cell membrane and modulate Ca2+ channel currents. To understand the influence of full-length bestrophin-1 on β-subunit function, we studied binding and localization of bestrophin-1 and Ca2+ channel subunits, together with modulation of CaV1.3 Ca2+ channels currents. In heterologeous expression, bestrophin-1 showed co-immunoprecipitation with either, β3-, or β4-subunits. We identified a new highly conserved cluster of proline-rich motifs on the bestrophin-1 C-terminus between amino acid position 468 and 486, which enables possible binding to SH3-domains of β-subunits. A bestrophin-1 that lacks these proline-rich motifs (ΔCT-PxxP bestrophin-1) showed reduced efficiency to co-immunoprecipitate with β3 and β4-subunits. In the presence of ΔCT-PxxP bestrophin-1, β4-subunits and CaV1.3 subunits partly lost membrane localization. Currents from CaV1.3 subunits were modified in the presence of β4-subunit and wild-type bestrophin-1: accelerated time-dependent activation and reduced current density. With ΔCTPxxP bestrophin-1, currents showed the same time-dependent activation as with wild-type bestrophin-1, but the current density was further reduced due to decreased number of Ca2+ channels proteins in the cell membrane. In summary, we described new proline-rich motifs on bestrophin-1 C-terminus, which help to maintain the ability of β-subunits to regulate surface expression of pore-forming CaV Ca2+-channel subunits.

Physiological features of primary cultures and subcultures of human retinal pigment epithelial cells before and after cryopreservation for cell transplantation

Abstract · Background: One striking disadvantage of in vitro culturing of human retinal pigment epithelial (RPE) cells is the loss of epithelial differentiation and specific cell function during culture. This may be one of the main reasons for the failure of RPE cell transplantation. The aim of this study was to evaluate cell culture conditions ensuring the maintenance of differentiation and function of RPE cells after subcultivation and storage in liquid nitrogen. · Methods: Enzymatically isolated cells were seeded onto coated culture dishes, cultured with a specially formulated improved growth medium until confluence and then cryopreserved in liquid nitrogen for 16–66 months. HLA class I and II typing was performed before cryopreservation and after thawing. Expression of Ca2+ channels in primary, first-passage and cryopreserved RPE cells was studied using the patch-clamp technique. · Results: After cryopreservation no loss of any HLA antigen was detectable in 12 of 14 cell strains studied. Patch-clamp experiments demonstrated that high-threshold L-type Ca2+ channels, which are typical for freshly isolated cells, could be detected in first-passage and cryopreserved RPE cells only when improved culture conditions were employed, not in conventionally cultured cells. The characteristics of these channels showed little change in subcultured cells compared to primary cultures. · Conclusion: This is the first study showing the maintenance of adult human RPE-specific cell differentiation and characteristics in vitro after primary culture and after cryopreservation using improved cell culture methods. The optimization and quality control of cell culture is an important prerequisite for successful cell transplantation.

Thyronamine induces TRPM8 channel activation in human conjunctival epithelial cells

3-Iodothyronamine (T1AM), an endogenous thyroid hormone (TH) metabolite, induces numerous responses including a spontaneously reversible body temperature decline. As such an effect is associated in the eye with increases in basal tear flow and thermosensitive transient receptor potential melastatin 8 (TRPM8) channel activation, we determined in human conjunctival epithelial cells (IOBA-NHC) if T1AM also acts as a cooling agent to directly affect TRPM8 activation at a constant temperature. RT-PCR and quantitative real-time PCR (qPCR) along with immunocytochemistry probed for TRPM8 gene and protein expression whereas functional activity was evaluated by comparing the effects of T1AM with those of TRPM8 mediators on intracellular Ca(2+) ([Ca(2+)]i) and whole-cell currents. TRPM8 gene and protein expression was evident and icilin (20μM), a TRPM8 agonist, increased Ca(2+) influx as well as whole-cell currents whereas BCTC (10μM), a TRPM8 antagonist, suppressed these effects. Similarly, either temperature lowering below 23°C or T1AM (1μM) induced Ca(2+) transients that were blocked by this antagonist. TRPM8 activation by both 1µM T1AM and 20μM icilin prevented capsaicin (CAP) (20μM) from inducing increases in Ca(2+) influx through TRP vanilloid 1 (TRPV1) activation, whereas BCTC did not block this response. CAP (20μM) induced a 2.5-fold increase in IL-6 release whereas during exposure to 20μM capsazepine this rise was completely blocked. Similarly, T1AM (1μM) prevented this response. Taken together, T1AM like icilin is a cooling agent since they both directly elicit TRPM8 activation at a constant temperature. Moreover, there is an inverse association between changes in TRPM8 and TRPV1 activity since these cooling agents blocked both CAP-induced TRPV1 activation and downstream rises in IL-6 release.

Angiotensin-2-Mediated Ca2+ Signaling in the Retinal Pigment Epithelium: Role of Angiotensin-Receptor- Associated-Protein and TRPV2 Channel

Angiotensin II (AngII) receptor (ATR) is involved in pathologic local events such as neovascularisation and inflammation including in the brain and retina. The retinal pigment epithelium (RPE) expresses ATR in its AT1R form, angiotensin-receptor-associated protein (Atrap), and transient-receptor-potential channel-V2 (TRPV2). AT1R and Atrap co-localize to the basolateral membrane of the RPE, as shown by immunostaining. Stimulation of porcine RPE (pRPE) cells by AngII results in biphasic increases in intracellular free Ca2+inhibited by losartan. Xestospongin C (xest C) and U-73122, blockers of IP3R and PLC respectively, reduced AngII-evoked Ca2+response. RPE cells from Atrap−/− mice showed smaller AngII-evoked Ca2+peak (by 22%) and loss of sustained Ca2+elevation compared to wild-type. The TRPV channel activator cannabidiol (CBD) at 15 µM stimulates intracellular Ca2+-rise suggesting that porcine RPE cells express TRPV2 channels. Further evidence supporting the functional expression of TRPV2 channels comes from experiments in which 100 µM SKF96365 (a TRPV channel inhibitor) reduced the cannabidiol-induced Ca2+-rise. Application of SKF96365 or reduction of TRPV2 expression by siRNA reduced the sustained phase of AngII-mediated Ca2+transients by 53%. Thus systemic AngII, an effector of the local renin-angiotensin system stimulates biphasic Ca2+transients in the RPE by releasing Ca2+from cytosolic IP3-dependent stores and activating ATR/Atrap and TRPV2 channels to generate a sustained Ca2+elevation.

The Role of Bestrophin-1 in Intracellular Ca 2+ Signaling

Mutations in the BEST1 gene lead to a variety of retinal degenerations, among them Bests vitelliforme macular degeneration. To clarify the mechanism of the disease, the understanding of the function of BEST1 gene product, bestrophin-1, is mandatory. In overexpression studies bestrophin-1 appeared to function as a Ca(2+)-dependent Cl channel. On the other hand, bestrophin-1 is able to participate in intracellular Ca(2+) signaling. Endogenously expressed bestrophin-1 largely localized to the cytosolic compartment close to the basolateral membrane of the retinal pigment epithelium (RPE) as it can be shown using differential centrifugation, immunohistochemistry, and transmission electron microscopy. To elucidate a cytosolic function of bestrophin-1, we explored the store-operated Ca(2+) entry in short-time cultured porcine RPE cells. Depletion of cytosolic Ca(2+)stores by SERCA inhibition led to activation of Orai-1 Ca(2+) channels. This resulted in an influx of extracellular Ca(2+) into the cell which was reduced when bestrophin-1 expression was knocked down using siRNA techniques. Quantification of Ca(2+) which can be released from cytosolic Ca(2+) stores revealed that after reduction of bestrophin-1 expression less Ca(2+) is stored in ER Ca(2+) stores. Thus, bestrophin-1 functions as an intracellular Cl channel which helps to accumulate and to release Ca(2+) from stores by conducting the counterion for Ca(2+).

Expression profile of voltage-dependent Ca2+ channel subunits in the human retinal pigment epithelium

BackgroundThe secretion of a variety of factors by the retinal pigment epithelium (RPE) is essential for the structural integrity of the neuronal retina and choroid, but also plays a pivotal role in the etiology of diseases such as choroidal neovascularisation. A recent study showed that the secretory activity of the RPE is regulated by the activity of a certain type of voltage-dependent Ca2+ channels, the L-type channel. In order to provide a better base for the understanding of the underlying Ca2+ signalling in these cells, we investigated the expression profile of voltage-dependent Ca2+ channel subunits in RPE cells.MethodsUsing RT-PCR techniques with cDNA isolated from RPE cells, we investigated the expression pattern of Ca2+ channel subunits. Furthermore, we analysed Ba2+ currents through voltage-dependent Ca2+ channels in RPE cells by the patch-clamp technique.ResultsWe detected the expression of two L-type channel subtypes and the expression of two different T-type channel subtypes. As accessory subunits, they expressed β2 and β4 and all known α2δ subunits. In general, we were able to confirm these data with cDNA from the ARPE-19 cell line. They only showed some differences in their expression pattern of accessory subunits. Since the expression of T-type channels was so far unknown in RPE cells, we confirmed their expression in the RPE using cDNA isolated from freshly isolated human RPE cells. Furthermore, the patch-clamp analysis of Ba2+ currents showed a heterogeneous pattern of voltage-dependent inward currents in RPE cells. In some cells, typical slowly inactivating L-type currents were detected, whereas in other cells fast inactivating T-type currents could be detected.ConclusionsThese data indicate the expression of a so far not detected subtype of voltage-dependent Ca2+ channels, the T-type channels. Together with the expression of L-type channels, RPE cells show a comparable expression pattern to that of other secretory cells, such as β-islets of the pancreas.

Flufenamic acid enhances current through maxi-K channels in the trabecular meshwork of the eye.

Purpose. Flufenamic acid relaxes trabecular meshwork, a smooth muscle-like tissue involved in the regulation of ocular outflow in the eye. In this study, we attempted to determine if ionic channels are involved in this response. Methods. Cultured human (HTM) and bovine (BTM) trabecular meshwork cells were investigated using the patch-clamp technique. Results. In trabecular meshwork, flufenamic acid (10 -5 M) reversibly stimulated outward current to 406 ± 71% of initial outward current level in BTM (n = 10) and 294 ± 75% of initial current level in HTM (n = 12) in all cells investigated; no significant differences emerged. The response was dosage-dependent. Replacement of potassium in all solutions eliminated the response to flufenamic acid (n = 4, BTM). Blocking K ATP channels with glibenclamide (10 -5 M, n = 6) and small-conductance calcium-activated potassium channels with apamin (10 -6 M, n = 5) had no effect. A direct effect on calcium channels could also not be detected. Blockage of the large-conductance calcium-activated potassium channel (maxi-K) by iberiotoxin (10 -7 M) suppressed 87 ± 9% (n = 6; HTM) and 91 ± 10% (n = 6; BTM) of the response. Depleting the cells of calcium did not significantly alter the response to flufenamic acid. Conclusions. Flufenamic acid stimulates maxi-K channels in trabecular meshwork of both human and bovine origin. This should lead to hyperpolarization, closure of L-type channels and lowered cytosolic calcium levels, possibly explaining the relaxation observed in response to this substance.