Ion channels and ionotropic receptors in human embryonic stem cell derived neural progenitors.

Human neural progenitor cells differentiated from human embryonic stem cells offer a potential cell source for studying neurodegenerative diseases and for drug screening assays. Previously, we demonstrated that human neural progenitors could be maintained in a proliferative state with the addition of leukemia inhibitory factor and basic fibroblast growth factor. Here we demonstrate that 96 h after removal of basic fibroblast growth factor the neural progenitor cell culture was significantly altered and cell replication halted. Fourteen days after the removal of basic fibroblast growth factor, most cells expressed microtubule-associated protein 2 and TUJ1, markers characterizing a post-mitotic neuronal phenotype as well as neural developmental markers Cdh2 and Gbx2. Real-time PCR was performed to determine the ionotropic receptor subunit expression profile. Differentiated neural progenitors express subunits of glutamatergic, GABAergic, nicotinic, purinergic and transient receptor potential receptors. In addition, sodium and calcium channel subunits were also expressed. Functionally, virtually all the hNP cells tested under whole-cell voltage clamp exhibited delayed rectifier potassium channel currents and some differentiated cells exhibited tetrodotoxin-sensitive, voltage-dependent sodium channel current. Action potentials could also be elicited by currents injection under whole-cell current clamp in a minority of cells. These results indicate that removing basic fibroblast growth factor from the neural progenitor cell cultures leads to a post-mitotic state, and has the capability to produce excitable cells that can generate action potentials, a landmark characteristic of a neuronal phenotype. This is the first report of an efficient and simple means of generating human neuronal cells for ionotropic receptor assays and ultimately for electrically active human neural cell assays for drug discovery.

Cone properties of retinal margin cells in the monkey (Macaca mulatta).

PURPOSE: To characterize a cell population in the monkey retinal margin that was labeled with a cone-specific antibody and to determine the presence of additional markers. METHODS: Retinal whole-mount preparations from infant and adult rhesus monkeys (Macaca mulatta) were immunolabeled by incubation overnight with the primary antibodies 7G6, a cone-specific antibody; SV2, a synaptic-vesicle antibody; and opsin antibodies that recognize either the short or long/middle wavelength-sensitive opsins. RESULTS: The retinal margin cells labeled by 7G6 lay within 1 mm of the ora serrata and differed from 7G6-labeled cones in the central retina. The margin cells possessed a soma, a fiber process, and a terminal enlargement that lay in the plane of the retina; no outer segment was discernible. A total of 5400 and 7252 margin cones cells were found in each of two monkeys. The terminal enlargement and soma of the labeled margin cells also showed SV2 immunoreactivity. Surprisingly, opsin immunoreactivity extended throughout the margin cell, which is consistent with the absence of a discernible outer segment. CONCLUSIONS: Cells with immunoreactive cone properties were found in the margin of the monkey retina. The absence of an outer segment and the presence of somatic opsin and SV2 are reminiscent of features observed in the central cones of fetal monkey retinas. These results suggest that a subpopulation of cones in the retinal margin might fail to mature completely and thus retain juvenile characteristics into adulthood.

Mechanisms of glutamate metabolic signaling in retinal glial (Müller) cells.

Retinal Müller (glial) cells metabolize glucose to lactate, which is preferentially taken up by photoreceptor neurons as fuel for their oxidative metabolism. We explored whether lactate supply to neurons is a glial function controlled by neuronal signals. For this, we used subcellular fluorescence imaging and either amperometric or optical biosensors to monitor metabolic responses simultaneously from mitochondrial and cytosolic compartments of individual Müller cells from salamander retina. Our results demonstrate that lactate production and release is controlled by the combined action of glutamate and NH(4)(+), both at micromolar concentrations. Transport of glutamate by a high-affinity carrier can produce in Müller cells a rapid rise of glutamate concentration. In our isolated Müller cells, glutamine synthetase (GS) converted transported glutamate to glutamine that was released. This reaction, predominant when enough NH(4)(+) is available, was limited at micromolar concentrations of NH(4)(+), and more glutamate entered then as substrate into the mitochondrial tricarboxylic acid cycle (TCA). Increased production of glutamine by GS leads to increased utilization of ATP, some of which is generated glycolytically. Methionine sulfoximine, a specific inhibitor of GS, suppressed the stimulatory effect of glutamate and NH(4)(+) on glycolysis and induced massive entry of glutamate into the TCA cycle. The rate of glutamine production also determined the amount of pyruvate transaminated by glutamate to alanine. Lactate, alanine, and glutamine can be taken up and metabolized by photoreceptor neurons. We conclude that a major function of Müller glial cells is to nourish retinal neurons and to metabolize the neurotoxic ammonia and glutamate.

Trafficking of molecules and metabolic signals in the retina.

Photoreceptors need the support of pigment epithelial (PE) and Müller glial cells in order to maintain visual sensitivity and neurotransmitter resynthesis. In rod outer segments (ROS), all-trans-retinal is transformed to all-trans-retinol by retinol dehydrogenase using NADPH. NADPH is restored in ROS by the pentose phosphate pathway utilizing high amounts of glucose supplied by choriocapillaries. The retinal formed is transported to PE cells where regeneration of 11-cis-retinal occurs. Müller cells take up and metabolize glucose predominantly to lactate which is massively released into the extracellular space (ES). Lactate is taken up by photoreceptors, where it is transformed to pyruvate which, in turn, enters the Krebs cycle in mitochondria of the inner segment. Stimulation of neurotransmitter release by darkness induces 130% rise in the amount of glutamate released into ES. Glutamate is transported into Müller cells where it is predominantly transformed to glutamine. Stimulation of photoreceptors induces an eightfold increase in glutamine formation. It appears, therefore, that there is a signaling function in the transfer of amino acids from Müller cells to photoreceptors. Work on the model-system of the honeybee retina demonstrated that photoreceptors release NH4+ and glutamate in a stimulus-dependent manner which, in turn, contribute to the biosynthesis of alanine in glia. Alanine released into the extracellular space is taken up and used by photoreceptors. Glial cells take glutamate by high-affinity transporters. This uptake induces a transient change in glial cell metabolism. The transformation of glutamate to glutamine is possibly also controlled by the uptake of NH4+ which directly affects cellular metabolism.

The intrinsic dynamics of retinal bipolar cells isolated from tiger salamander.

We studied how intrinsic membrane properties affect the gain and temporal pattern of response in bipolar cells dissociated from retinae of tiger salamanders. Currents specified by a pseudorandom binary sequence, an m-sequence, superimposed on various means, were injected into the cells. From the resultant membrane voltage response for each mean current, impulse responses were estimated. From each impulse response, transfer function, gain, and time constant were calculated. The bipolar cells acted as quasilinear adaptive filters whose gain and response speed are determined by the mean input current. Near resting potential, gain. and time constant were maximum. Dynamics were slow and low-pass, characterized by an approximately exponential impulse response. With depolarization, gains were reduced sharply, responses were much faster, and dynamics became band-pass, as indicated by an undershoot in the impulse response. For any given mean current, the shape of the impulse response did not depend on the amplitude of the m-sequence currents. Thus, bipolar cells behaved in a quasilinear fashion. The adaptive behavior was eliminated by blocking a potassium current, which implicates the role of a voltage-gated potassium conductance. Computer simulations on a model neuron including a delayed-rectifier reconstructed the observed behavior, and provided insight into other, less readily observable, parameters. Thus, bipolar cells, even when isolated, possess mechanisms which regulate, with unsuspected elaborateness, the sensitivities and dynamics of their responsiveness. Implications for adaptation and neuronal processing are discussed.

Immunohistochemical analysis of the neurotrophins BDNF and NT-3 and their receptors trk B, trk C, and p75 in the developing chick retina.

The neurotrophins are trophic and mitogenic factors critical for the development of specific classes of neurons in the central and peripheral nervous systems. In the retina, BDNF and NT-3 have been shown to promote the survival of differentiated ganglion cells (Rodriguez-Tebar et al., 1989; De La Rosa et al., 1994). NT-3 has also been demonstrated to support the survival of amacrine cells and facilitates the differentiation of retinal neurons in culture (De La Rosa et al., 1994). Here, we examine immunohistochemically the expression of BDNF and NT-3 proteins, their cognate receptors, trk B and trk C, respectively, and the p75 neurotrophin receptor in the developing chick retina. At E8, the earliest stage of retinal development examined, all of these proteins exhibit diffuse expression throughout the width of the retina, with the strongest reactivity in the innermost layers. A gradual restriction in expression to ganglion cells and amacrine cells, the staining of which is most prominent at E15, is followed by a downregulation of expression with the strongest immunoreactivity persisting in the ganglion cell layer. Overlapping patterns of expression throughout embryonic development indicate a colocalization of the neurotrophins and their receptors, although NT-3 and p75 alone are present in the inner plexiform layer and only p75 is observed in the outer plexiform layer. Although some of the immunoreactivity for BDNF, NT-3, and their receptors in retina may reflect trophic mechanisms operating in association with the optic tectum and isthmo-optic nucleus, the colocalization of ligands and receptors in retina strengthens the assertion that these neurotrophins function locally during development.

Early emergence of photoreceptor mosaicism in the primate retina revealed by a novel cone-specific monoclonal antibody.

We have examined the emergence of the photoreceptor mosaic in fetal macaque monkeys by using a novel monoclonal antibody, 7G6, that recognizes all cones in the adult primate retina. In the fetal retina, however, between embryonic (E) day 80 and E130, some opsin-positive cones were not labeled by 7G6. Double-labeling experiments revealed that though long and middle wavelength-sensitive fetal cones are 7G6-positive, a subset of short wavelength-sensitive cones are delayed in their acquisition of 7G6 immunoreactivity. Heterogeneity in cone labeling with 7G6 was evident at all fetal ages. The onset of 7G6 immunoreactivity, surprisingly, precedes both the expression of the cone opsins and the formation of synaptic contacts in the outer plexiform layer. Specifically, a population of cones was labeled in the periphery of the E65 retina, concomitant with ongoing cone genesis. Moreover, early-differentiating 7G6-positive cones are organized into a regular array in immature, peripheral regions of the fetal retina indicating that the spatial arrangement of cones is initiated either during active cone proliferation or the initial differentiation of these cells. These results suggest that the periodic spacing of cones in the primate retina emerges autonomously within the photoreceptor layer, prior to the formation of synaptic connections within the retina or with the brain.

Ionic conductances of monkey solitary cone inner segments.

1. The membrane properties of cone inner segments dissociated enzymatically from monkey retina were studied under voltage-clamp conditions using patch pipettes in the whole-cell clamp configuration. 2. A noninactivating, voltage-gated calcium current was evoked at potentials positive to -60 mV and peaked between -30 and -20 mV when barium was substituted for calcium. Cadmium (50 microM) but not nickel (50 microM) blocked the current. 3. A large calcium-activated anion current (IAn) was observed when the membrane potential was set to a level between -60 and 30 mV. The reversal potential of IAn was 0 mV with chloride as the sole anion and about -30 and -40 mV when methanesulfonate and D-aspartate, respectively, replaced intracellular chloride to set the equilibrium potential for chloride at -50 mV. IAn inactivated and oscillated when the membrane potential was maintained at depolarized levels, contrary to calcium-activated anionic currents seen in photoreceptors of other species. 4. A sustained-type potassium current was activated by depolarizations positive to -50 mV. The time course of activation and deactivation were voltage dependent. This potassium current was partially blocked by 20 mM tetraethylammonium ions. 5. A transient potassium current was activated by depolarizations positive to -20 mV. This current was blocked by 4-aminopyridine (2 mM) and inactivated with a time constant of approximately 500 ms. The amplitude in response to voltage steps to 45 mV was decreased by prepulses to voltages more positive than -30 mV. 6. Hyperpolarization negative to -65 mV activated an inward current that was completely blocked by external cesium (10 mM). The reversal potential suggested a conductance mechanism permeable to both sodium and potassium ions. 7. A calcium-activated potassium current, which was found in salamander photoreceptors, was not detected. 8. The presence of these conductances is expected to influence the membrane potential and the time course of the light response in monkey cones.

Process outgrowth and synaptic varicosity formation by adult photoreceptors in vitro.

To assess the regenerative capability of the photoreceptor synapse, we have isolated and cultured photoreceptors from the mature salamander retina. Both rod and cone photoreceptors were able to regenerate processes within 3 d of plating. Cells extended numerous actin-containing filopodia as well as a few neuritic processes. The neurites contained microtubules and formed synaptic vesicle-filled varicosities, as shown by immunostaining for tubulin and synaptic vesicle proteins and by electron microscopy. Furthermore, regenerated varicosities were capable of depolarization-induced vesicle labeling, suggesting that they can recycle synaptic vesicles and release neurotransmitter by synaptic vesicle exocytosis. Differences were observed between rod and cone cell synaptic regeneration in vitro, which resembled structural differences between their synaptic terminals in situ: rod cells formed multiple synaptic vesicle-filled varicosities along neurites at a distance from the soma, whereas cone cells tended to accumulate synaptic vesicles within the soma. The regeneration of neurites and synaptic vesicle-filled varicosities was abolished by microtubule depolymerizing agents, suggesting a role for microtubule-based vesicle transport in the formation of varicosities. Finally, process outgrowth and varicosity formation were independent of cell-cell contact and, indeed, proceeded in the complete absence of other cells. These findings suggest not only that differentiated photoreceptors are capable of synaptic renewal but that the regeneration of presynaptic-like terminals is an intrinsic ability of rod and cone cells.

Monoclonal antibody substrates facilitate long-term regenerative growth of adult primate retinal neurons.

A culture system for adult monkey and human retinal cells was developed utilizing monoclonal antibodies as substrates. Monoclonal antibodies were made using monkey retinal membranes as immunogen, and screening for the ability to bind and facilitate growth of dissociated retinal neurons when used as a culture substrate. Two antibodies, MR1 and 9B5, which recognize immunohistochemically distinct cell surface antigens, were found to work effectively as a culture substrate for dissociated monkey retinal cells. A variety of dissociated monkey retinal neurons and glial cells grew extensive processes on MR1 and 9B5, and continued to express cell-specific antigens. In addition, many multipolar neurons remained electrically excitable after several days in culture. Dissociated human retinal cells did not cross-react with MR1 but grew well on 9B5. These results extend previous work showing that antibody substrates can support the growth of neurons derived from other vertebrate species (e.g., neonatal rat, adult salamander). The long-term survival and growth of primate cells in culture supports the current view that neurons in the adult mammalian central nervous system (CNS) may regenerate under permissive circumstances. Further, these data provide evidence that direct support by non-neuronal cells is not required and that regeneration in vitro may be mediated by suitably adhesive non-cellular substrates.

Induction of class I major histocompatibility complex antigens on adult primate retinal neurons.

The expression of class I major histocompatibility complex (MHC) antigens was examined on adult monkey and human retinal cells following injury caused by dissociation. In a panning assay, neurons and glia demonstrated a rapid induction of class I MHC antigens following enzymatic dissociation, and expression was sustained on regenerating neurons as well as on glia in culture. Class I MHC antigen expression was also enhanced following optic nerve crush of monkey eyes in vivo. These results suggest that the functions of class I MHC antigens may be relevant to primate CNS neurons and glia following injury.

Glutamate and 2-amino-4-phosphonobutyrate evoke an increase in potassium conductance in retinal bipolar cells.

Although there is general agreement that L-glutamate can produce a depolarizing inward current to account for the hyperpolarizing (OFF) bipolar cell response, the conductance mechanism underlying the depolarizing (ON) response has been difficult to establish satisfactorily. To investigate the ionic bases of the center responses, we studied the whole-cell currents controlled by L-glutamate and its analogues in solitary bipolar cells from salamander retina. We report here two groups of isolated bipolar cells: one group responded to L-glutamate with the previously described inward current [Attwell, D., Mobbs, P., Tessier-Lavigne, M. & Wilson, M. (1987) J. Physiol. (London) 387, 125-161] and a second group showed an outward current that reversed at about -70 mV. Both were associated with an increase in membrane conductance. In addition, DL-2-amino-4-phosphonobutyrate, a compound diagnostic for ON-bipolar cell activity [Slaughter, M. M. & Miller, R. F. (1981) Science 211, 182-185], elicited outward currents that closely resembled those seen in response to L-glutamate and, furthermore, that were shown to arise from an increase in conductance to potassium ions. Thus the presence of two distinct conductances controlled by L-glutamate in solitary cells would provide one mechanism for generating the ON and OFF light responses at the bipolar cell level in the intact retina.

Fibroblast behavior at aqueous interfaces with perfluorocarbon, silicone, and fluorosilicone liquids.

Perfluorocarbon, silicone, and fluorosilicone liquids with potential for use as vitreous substitutes in the management of complex retinal detachment were evaluated for surface reactivity by assessing the behavior of anchorage-dependent fibroblasts plated at the phase boundary between these compounds and culture medium. Low-viscosity perfluorcarbons were alumina-treated to remove polar impurities. On perfluorodecalin, perfluorodimethylcyclohexane, perfluorotrimethylcyclohexane, perfluoroethylcyclohexane, perfluorooctane, perfluoroperhydrophenanthrene, perfluoromethyladamantane, perfluorodimethyladamantane, the highly viscous perfluoropolyether liquids Krytox TLF7067 and 6354, and dimethylsiloxane liquids of a variety of viscosities, most cells did not attach; the few that did attach exhibited minimal spreading behavior and did not achieve the flattened spindle-shape morphology which is a prerequisite to normal proliferative activity. Conversely, on perfluoromethyldecaline, perfluorofluorene, perfluorotributylamine, the perfluoropolyether K-6 hexamer, trifluoropropylmethylsiloxane (fluorosilicone), and diphenyldimethylsiloxane, some cells became fusiform-shaped and exhibited proliferation, the extent of which varied with the compound. The association of alumina treatment of perfluorocarbon liquids with a reduction in cell growth was indicative of a relationship between the presence of residual hydrogen-containing impurities and the capacity for cellular attachment and growth. This correlation was demonstrated also in experiments in which cell attachment and growth was facilitated by the addition of hydrogen-rich monohydroperfluorooctane to alumina-treated perfluorooctane. In conclusion, evidence for the presence of surface active impurities in liquid vitreous substitute materials can be obtained by observing the behavior of attachment-dependent cells plated at the boundary between these compounds and culture medium.

Immunolabelling by a newt retinal pigment epithelium antibody during retinal development and regeneration.

The binding of RPE-1, a mouse monoclonal antibody selective for newt retinal pigment epithelium, was followed in eyes undergoing embryonic development and retinal regeneration. Using the indirect immunofluorescence technique on frozen sections, we observed bright and continuous labelling exclusively in the retinal pigment epithelium (RPE) of normal adult newts, but labelling became diminished near the ora serrata region and stopped abruptly at the ciliary margin. During development, labelling was not detected in the retinal pigment epithelium (RPE) until the formation of photoreceptor outer segments and was not observed in any other ocular tissue. There was no correlation between the appearance of pigment in retinal pigment epithelial cells and their labelling with the RPE-1 antibody. Furthermore, albino salamander embryos showed the same pattern of labelling with RPE-1 as that seen in age-matched pigmented animals. During retinal regeneration, RPE cells were labelled less intensely, but heavy labelling was observed in the newly formed retinal cells. With time, labelling in regenerated retina receded, so that by the end of regeneration, labelling by RPE-1 was once more restricted to the RPE cells. The identification of RPE-1 as a marker for postmitotic retinal neurons about to undergo differentiation provides a promising approach for further studies of regeneration with the help of molecular tools.

Growth and synapse formation among major classes of adult salamander retinal neurons in vitro.

Adult neurons, isolated from the salamander retina, were maintained in low-density cell culture and examined for synapse formation by electrophysiological and electron microscopic techniques. Morphologically identifiable rod, cone, horizontal, bipolar, and amacrine/ganglion cells survived for many months, grew processes, and formed numerous cell contacts. Intracellular recordings showed the presence of a variety of voltage- and time-dependent conductances and both electrical and chemical transmission among these cells. At the ultrastructural level, gap junctions, monad ribbon synapses, and conventional synapses, like those present in the intact retina, were observed in sibling cultures. Thus, all major classes of adult retinal neurons, in addition to ganglion cells, are able to regenerate processes and reform synapses. The regenerated synaptic contacts are functional and structurally diverse.

Divalent cations directly affect the conductance of excised patches of rod photoreceptor membrane.

Phototransduction in rod cells is likely to involve an intracellular messenger system that links the absorption of light by rhodopsin to a change in membrane conductance. The direct effect of guanosine 3',5'-monophosphate (cGMP) on excised patches of rod outer segment membrane strongly supports a role for cGMP as an intracellular messenger in phototransduction. It is reported here that magnesium and calcium directly affect the conductance of excised patches of rod membrane in the absence of cGMP and that magnesium, applied to intact rod cells, blocks a component of the cellular light response. The divalent cation-suppressed conductance in excised patches showed outward rectification and cation-selective permeability resembling those of the light-suppressed conductance measured from the intact rod cell. The divalent cation-suppressed conductance was partly blocked by a concentration of the pharmacological agent L-cis-diltiazem that blocked all of the cGMP-activated conductance. Divalent cations may act, together with cGMP, as an intracellular messenger system that mediates the light response of the rod photoreceptor cell.

Control of the light-regulated current in rod photoreceptors by cyclic GMP, calcium, and l-cis-diltiazem.

The effect of calcium ions on the cGMP-activated current of outer segment membrane was examined by the excised-patch technique. Changes in the extracellular calcium concentration had marked effects on the cGMP-activated current, while changes in intracellular calcium concentration were ineffective. Changes in calcium concentration in the absence of cGMP had little, if any, effect on membrane conductance. These results suggest that both intracellular cGMP and extracellular calcium can directly affect the conductance underlying the light response in rod cells. The pharmacological agent l-cis-diltiazem reversibly inhibited the cGMP-activated current when applied to the intracellular side of an excised patch. When superfused over intact rod cells, l-cis-diltiazem reversibly blocked much of the normal light response. The isomer, d-cis-diltiazem, did not significantly affect either patches or intact rod cells. Thus, the light-regulated conductance has binding sites for both calcium and cGMP that may interact during the normal light response in rod cells and a site specific for l-cis-diltiazem that can be used to identify and further study the conductance mechanism.

Rod cells dissociated from mature salamander retina: ultrastructure and uptake of horseradish peroxidase.

To test the effects of isolation on adult neurons, we investigated the fine structure and synaptic activity of rod cells dissociated from the mature salamander retina and maintained in vitro. First, freshly isolated rod cells appeared remarkably similar to their counterparts in the intact retina: the outer segment retained its stack of membranous disks and the inner segment contained its normal complements of organelles. Some reorganization of the cell surface, however, was observed: (a) radial fins, present at the level of the cell body, were lost; and (b) the apical and distal surfaces of the inner and outer segments, respectively became broadly fused. Second, the synaptic endings or pedicles retained their presynaptic active zones: reconstruction of serially sectioned pedicles by using three-dimensional computer graphics revealed that 73% of the synaptic ribbons remained attached to the plasmalemma either at the cell surface or along its invaginations. Finally, tracer experiments that used horseradish peroxidase demonstrated that dissociated rod cells recycled synaptic vesicle membrane in the dark and thus probably released transmitter by exocytosis. Under optimal conditions, a maximum of 40% of the synaptic vesicles within the pedicle were labeled. As in the intact retina, uptake of horseradish peroxidase was suppressed by light. Thus, freshly dissociated receptor neurons retained many of their adult morphological and physiological characteristics. In long-term culture, the photoreceptors tended to round up; however, active zones were present even 2 wk after removal of the postsynaptic processes.

Control of the generator current in solitary rods of the Ambystoma tigrinum retina.

THe current suppressed by light, the generator current, was studied in solitary salamander (Ambystoma tigrinum) rod photoreceptors with the single-micropipette voltage-clamp technique. The effects of Ca, cyclic GMP, and voltage were measured while voltage- and Ca-activated currents of the inner segment were blocked with Co, Cs, and TEA (tetraethylammonium). The generator current was increased more than 5-fold by lowering the external Ca concentration from 1.5 mM to 10 microM. The generator current could be decreased approximately 1/2 by injecting Ca into an outer segment. Injection of EGTA quickly increased the generator current approximately 2-fold. After injection ceased, the increase was quickly reversed. The generator current could be increased more than 5-fold by injecting cyclic GMP or 8Br-cyclic GMP. Injection of protons, the pH buffer bicine (N,N-bis[2-hydroxyethyl]glycine), or GMP did not produce a change in the generator current. The current-voltage curve for the generator current was influenced by external Co: in 3 mM-Co the current-voltage curve had a negative resistance between -45 and -90 mV; in 0.1 mM-Co the current-voltage curve paralleled the voltage axis between -45 and -90 mV. The difference is attributed to a voltage-dependent block by Co. Susceptibility to the blocking action of Co was reduced by lowering internal or external Ca concentration, or by injecting cyclic GMP. When rods were bathed in a medium containing 7-100 microM-Ca, a step depolarization produced a time-dependent decline in current. Because the reversal potential remained constant, the decline is attributed to an inactivation. The extent of inactivation was reduced by increasing the concentration of external Ca or injecting cyclic GMP.