Voltage-dependent Ca2+ channels, not ryanodine receptors, activate Ca2+-dependent BK potassium channels in human retinal pigment epithelial cells.

PURPOSE: In different tissues the activation of large conductance Ca2+-activated (BK) potassium channels has been shown to be coupled to voltage-gated Ca2+ channels as well as ryanodine receptors. As activation of BK channels leads to hyperpolarization of the cell, these channels provide a negative feedback mechanism for Ca2+-induced functions. Many cellular functions of the retinal pigment epithelium (RPE) are coupled to changes in [Ca2+]i. The aim of this study was to identify which Ca2+-entry pathway leads to the activation of BK channels in the RPE. METHODS: We used freshly isolated human RPE cells and the ARPE-19 cell line for the detection of transcripts of BK channel alpha subunits. Patch-Clamp measurements were used to characterize BK channels in ARPE-19 cells electrophysiologically. To monitor changes in [Ca2+]i ARPE-19 cells were loaded with Fura-2. RESULTS: Freshly isolated human RPE cells and ARPE-19 cells were shown to express BK channels. In ARPE-19 cells these channels were shown to be functionally active. Application of iberiotoxin led to a block of outward currents by 28.15%. At +50 mV ARPE-19 cells had a BK channel-mediated current density of 2.42 pA/pF. Activation of ryanodine receptors by caffeine led to a significant increase in [Ca2+]i by 34.16%. Nevertheless, caffeine-induced Ca2+ signals were not sufficient to activate BK channels. Instead, the activation of L-type Ca2+ channels by BayK 8644 caused a dramatic increase in BK channel activity and a shift of the reversal potential of the ARPE-19 cells by -22.6 mV. CONCLUSIONS: We have shown here for the first time that human RPE cells express BK channels. These channels are activated in RPE cells by increases in [Ca2+]i that are mediated by the opening of voltage gated L-type Ca2+ channels. As Ca2+ entering the RPE cells through these Ca2+ channels are known to be important for growth factor secretion and light-induced transepithelial transport, we speculate that BK channels coupled directly to these Ca2+ channels may provide a good tool for negative feedback control of the L-type Ca2+ channels.

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

BACKGROUND: The 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 Ca(2+) channels, the L-type channel. In order to provide a better base for the understanding of the underlying Ca(2+) signalling in these cells, we investigated the expression profile of voltage-dependent Ca(2+) channel subunits in RPE cells. METHODS: Using RT-PCR techniques with cDNA isolated from RPE cells, we investigated the expression pattern of Ca(2+) channel subunits. Furthermore, we analysed Ba(2+) currents through voltage-dependent Ca(2+) channels in RPE cells by the patch-clamp technique. RESULTS: We detected the expression of two L-type channel subtypes and the expression of two different T-type channel subtypes. As accessory subunits, they expressed beta2 and beta4 and all known alpha(2)delta 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 Ba(2+) 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. CONCLUSIONS: These data indicate the expression of a so far not detected subtype of voltage-dependent Ca(2+) 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 beta-islets of the pancreas.

Basal calcium entry in retinal pigment epithelial cells is mediated by TRPC channels.

PURPOSE: Ca(2+) is a major regulator of cell function. In the retinal pigment epithelium (RPE), intracellular free Ca(2+) concentration ([Ca(2+)](i)) is essential for the maintenance of normal retinal function. Therefore, accurate control of [Ca(2+)](i) is vital in these cells. Because Ca(2+) is permanently extruded from the cytosol, RPE cells need a basal Ca(2+) entry pathway that counteracts this Ca(2+) efflux. The purpose of this study was to identify the molecular basis of basal Ca(2+) entry into the RPE. METHODS: [Ca(2+)](i) was measured using Fura-2-loaded ARPE-19 cells. The expression pattern of TRPC channels was investigated by RT-PCR with RNA extracted from ARPE-19 cells and freshly isolated RPE cells from human donor eyes. RESULTS: In most cells, basal [Ca(2+)](i) is highly controlled by cell membranes that are only slightly permeable to Ca(2+) and by the activity of Ca(2+) pumps and transporters. The authors show here that RPE cells have a basal Ca(2+) conductance that is dose dependently blocked by La(3+). Basal [Ca(2+)](i) was also strongly reduced by the TRP channel blockers Gd(3+), Ni(2+), 2-APB, and SKF96365 and was insensitive to blockers of other Ca(2+) channels. In confirmation of this pharmacologic profile, RPE cells expressed TRPC1 and TRPC4 channels, as shown by RT-PCR experiments. CONCLUSIONS: Ca(2+) is needed for several permanently occurring regulatory processes in RPE cells. The Ca(2+) influx pathway identified in this study is essential to define a resting basal [Ca(2+)](i). This resting [Ca(2+)](i) may contribute, for example, to basal cytokine secretion essential for the maintenance of normal retinal function.

Functional studies of synthetic gramicidin hybrid ion channels in CHO cells.

The function of a gramicidin hybrid ion channel in living Chinese hamster ovary (CHO) cells was investigated by the patch clamp method. The synthetic ion channel 1 consists of two cyclohexyl ether amino acids that link two mini-gramicidin strands. With 1 at a concentration of 1.0 microM, an increase in the whole-cell membrane conductance was observed after 1.37 min. The conductance showed larger currents when Cs(+) was used as charge carrier than when Na(+) and K(+) were used. In single-channel recordings with Cs(+) as charge carrier, the substance showed comparable single-channel amplitudes in the membrane of living cells and artificial black lipid bilayers. In addition to functioning as a cation channel, compound 1 appeared to be a water channel. Exposure of the CHO cells to an extracellular hypoosmotic solution did not substantially change the cell volume. Extracellular hypoosmotic conditions in the presence of 1 increased the cell size to 146.5 % that of the control. Thus, the synthetic hybrid channel 1 can function as a cation channel with some Cs(+) specificity, and as a water channel in CHO cells.

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.

Expression of bestrophin-1, the product of the VMD2 gene, modulates voltage-dependent Ca2+ channels in retinal pigment epithelial cells.

Mutations in the VMD2 gene cause Best's disease, an inherited form of macular degeneration. The reduction in the light-peak amplitude in the patient's electro-oculogram suggests that bestrophin-1 influences the membrane conductance of the retinal pigment epithelium (RPE). Systemic application of the L-type Ca2+ channel blocker nimodipine reduced the light-peak amplitude in the rat electroretinogram but not a- and b-waves. Expression of bestrophin-1 in a RPE cell line (RPE-J) led to changes in L-type channel properties. Wild-type bestrophin-1 induced an acceleration of activation kinetics of Ba2+ currents through L-type Ca2+ channels and a shift of the voltage-dependent activation to more negative values, closer to the resting potential of RPE cells. Expression of bestrophin-1 with Best disease-causing mutations led to comparable shifts in voltage-dependent activation but different effects on activation and inactivation kinetics. Bestrophin W93C exhibited slowed activation and inactivation, and bestrophin R218C accelerated the activation and inactivation. Thus, transfection of RPE cells with bestrophin-1 distinctively changed L-type Ca2+ channel kinetics and voltage-dependence. On the basis of these data, we propose that presence of bestrophin-1 influences kinetics and voltage-dependence of voltage-dependent Ca2+ channels and that these effects might open new ways to understand the mechanisms leading to retinal degeneration in Best's disease.

Extracellular potassium effects are conserved within the rat erg K+ channel family.

The biophysical properties of native cardiac erg1 and recombinant HERG1 channels have been shown to be influenced by the extracellular K(+) concentration ([K(+)](o)). The erg1 conductance, for example, increases dramatically with a rise in [K(+)](o). In the brain, where local [K(+)](o) can change considerably with the extent of physiological and pathophysiological neuronal activity, all three erg channel subunits are expressed. We have now investigated and compared the effects of an increase in [K(+)](o) from 2 to 10 mm on the three rat erg channels heterologously expressed in CHO cells. Upon increasing [K(+)](o), the voltage dependence of activation was shifted to more negative potentials for erg1 (DeltaV(0.5) = -4.0 +/- 1.1 mV, n = 28) and erg3 (DeltaV(0.5) = -8.4 +/- 1.2 mV, n = 25), and was almost unchanged for erg2 (DeltaV(0.5) = -2.0 +/- 1.3 mV, n = 6). For all three erg channels, activation kinetics were independent of [K(+)](o), but the slowing of inactivation by increased [K(+)](o) was even more pronounced for erg2 and erg3 than for erg1. In addition, with increased [K(+)](o), all three erg channels exhibited significantly slower time courses of recovery from inactivation and of deactivation. Whole-cell erg-mediated conductance was determined at the end of 4 s depolarizing pulses as well as with 1 s voltage ramps starting from the fully activated state. The rise in [K(+)](o) resulted in increased conductance values for all three erg channels which were more pronounced for erg2 (factor 3-4) than for erg1 (factor 2.5-3) and erg3 (factor 2-2.5). The data demonstrate that most [K(+)](o)-dependent changes in the biophysical properties are well conserved within the erg K(+) channel family, despite gradual differences in the magnitude of the effects.

Biophysical properties of heteromultimeric erg K+ channels.

The three ether-à-go-go-related gene (erg) K(+) channel subunits are able to form heteromultimers within their subfamily. The functional importance of this finding is indicated by in situ hybridization experiments showing that the different erg subunits have overlapping expression patterns in several regions of the brain. To investigate the biophysical properties of heteromultimeric erg channels, concatemers of two erg subunits were constructed and expressed heterologously in Chinese hamster ovary (CHO) cells. The resulting currents were measured using the patch-clamp technique. The heteromultimers exhibited an intermediate potential dependence of activation compared with the corresponding wild-type (WT) erg channels. In contrast, the time course of activation was clearly dominated by the faster activating subunit. The kinetics of recovery from inactivation and the deactivation kinetics of all heteromultimers were similar to those of WT erg1 channels, the rat homologue of the human erg1 K(+) channel (HERG), even if erg1 was not part of the concatemer. Taken together, the biophysical properties of heteromultimeric erg channels result in larger current amplitudes upon both depolarization and repolarization. Thus, through heteromeric assembly erg channels may contribute significantly to different physiological functions such as setting and stabilizing the resting membrane potential and modulation of action potential frequency.