Eric A. Schwartz

Voltage‐activated and calcium‐activated currents studied in solitary rod inner segments from the salamander retina

1. Solitary rod inner segments were obtained by enzymatic dissociation of the tiger salamander (Ambystoma tigrinum) retina. Their membrane currents were studied with the single‐pipette voltage‐clamp technique. Individual currents were isolated with the aid of pharmacological agents.

Hemi-gap-junction channels in solitary horizontal cells of the catfish retina

1. Solitary horizontal cells were isolated from catfish retinas and their membrane current was recorded with a whole‐cell voltage clamp. Reducing the extracellular Ca2+ concentration produced a current that could be suppressed by dopamine. This Ca(2+)‐ and dopamine‐sensitive current is hereafter termed I gamma. The voltage dependence, cytoplasmic regulation, and permeability of the I gamma channel suggest that it is half of a gap‐junction channel. 2. I gamma was voltage and time dependent. In the steady state, the current‐voltage relation displayed outward rectification at voltages more depolarized than 0 mV and a negative resistance region at voltages more hyperpolarized than ‐15 mV. The reversal potential was 3.3 +/‐ 1.5 mV when NaCl was the predominant extracellular salt and potassium‐D‐aspartate was the predominant intracellular salt. 3. The size of I gamma depended on the extracellular Ca2+ concentration. I gamma was maximal at external Ca2+ concentrations below 10 microM, half‐maximal at 220 microM‐Ca2+, and reduced to less than 4% of its maximum amplitude at external Ca2+ concentrations above 1 mM. Increasing the extracellular Ca2+ concentration reduced the amplitude of I gamma without changing the shape of the current‐voltage relation or the kinetics of inactivation. Thus, rectification does not result from a voltage‐dependent block by extracellular Ca2+. 4. Patches of cell membrane were voltage clamped in both the cell‐attached and excised‐patch configurations. In the cell‐attached configuration, the addition of dopamine to the solution outside the patch pipette blocked the opening of channels within the membrane patch. Thus, dopamine closes I gamma channels by initiating an intracellular messenger cascade. In the excised‐patch configuration, a maximum conductance of 145 pS was measured while Cs+ and tetraethylammonium+ (TEA+) were the only monovalent cations on both sides of the membrane. 5. The ability of dopamine to suppress I gamma was blocked by introducing an inhibitor of the cyclic AMP‐dependent protein kinase, PKI5‐24, into the cytoplasm. Thus, the action of dopamine is mediated by a pathway that includes the activation of a cyclic AMP‐dependent kinase. 6. I gamma was suppressed by nitroprusside, an agent which activates guanylate cyclase and increases the intracellular cyclic GMP concentration. The effect of nitroprusside was not altered by the intracellular application of PKI5‐24. Thus, nitroprusside suppresses I gamma through a pathway that does not include the activation of a cyclic AMP‐dependent kinase.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of an electrical synapse between solitary pairs of catfish horizontal cells by dopamine and second messengers.

1. Retinas from channel catfish were dissociated and the cells maintained in culture. Horizontal cells that normally receive input from cone photoreceptors were identified. The conductance of the electrical junction formed between a pair of ‘cone’ horizontal cells was measured by controlling the membrane voltage of each cell with a voltage clamp maintained through either a micropipette or a patch pipette. The two techniques yielded similar results. 2. Transjunctional current was measured while transjunctional voltage was stepped to values between +/‐ 60 mV. The current (measured 5 ms after a step) was proportional to voltage over the range tested. For steps to voltages greater than +/‐ 45 mV, the current exhibited a slight time‐dependent decline. 3. Dopamine decreased junctional conductance in a dose‐dependent fashion. A 50% reduction was obtained with 10 nM‐dopamine. The D1 agonist fenoldopam (100 nM) also decreased junctional conductance. The uncoupling produced by either agent was rapid and reversible. 4. The introduction of 100 microM‐cyclic AMP into one cell of a pair decreased junctional conductance by, on average, 40%. Forskolin (1‐10 microM), an activator of adenylate cyclase, decreased junctional conductance 50‐90%. 5. The introduction of 80 microM‐cyclic GMP into one cell of a pair decreased junctional conductance by, on average, 40%. Nitroprusside (1‐10 microM), an activator of guanylate cyclase, reduced junctional conductance 40‐65%. 6. The introduction of a peptide inhibitor specific for the cyclic AMP‐dependent protein kinase reversed a decrease in junctional conductance produced by superfusion with either dopamine (1 microM), fenoldopam (100 nM) or forskolin (5‐10 microM). 7. Intracellular Ca2+ concentration was measured with the fluorescent indicator Fura‐2. The intracellular Ca2+ concentration was increased by activation of a Ca2+ current. Junctional conductance remained constant as the internal Ca2+ concentration changed from 100 to 700 nM. 8. Intracellular pH was measured with the fluorescent indicator bis‐carboxyethylcarboxyfluorescein. The application of acetate (2.5 mM) reduced intracellular pH by 0.2‐0.3 units and decreased junctional conductance by approximately 50%. A subsequent application of fenoldopam did not alter intracellular pH, but decreased junctional conductance by more than 50%. 9. The sensitivity of the junctional conductance between isolated horizontal cells to dopamine is consistent with dopamine having a direct effect on coupling in intact retina. Dopamine regulates the activity of a cyclic AMP‐dependent protein kinase which in turn modulates junctional conductance. Changes in intracellular pH and Ca2+ concentration are not involved in mediating the effect of dopamine on coupling. Cyclic GMP and intracellular pH may participate in regulatory pathways independent of that used by cyclic AMP.

A cGMP-gated current can control exocytosis at cone synapses.

The voltage-gated Ca2+ current in cone photoreceptors operates over only a small part of the physiological voltage range produced by light and, consequently, appears insufficient for controlling transmitter release. We have used a whole-cell voltage clamp to measure membrane current and the capacitance change produced by exocytosis in solitary cone and rod photoreceptors isolated from the salamander retina. In both types of photoreceptor, Ca2+ influx through voltage-gated Ca2+ channels initiated exocytosis. In addition, Ca2+ influx through a cGMP-gated channel in the inner segment and synaptic processes of cones also initiated exocytosis. The cGMP-gated current sustained exocytosis over the entire physiological voltage range.

Kainate receptors mediate synaptic transmission between cones and ‘Off’ bipolar cells in a mammalian retina

Light produces a graded hyperpolarization in retinal photoreceptors, that decreases their release of synaptic neurotransmitter,. Cone photoreceptors use glutamate, as a neurotransmitter with which to communicate with two types of bipolar cell. Activation of metabotropic glutamate receptors in ‘On’ bipolar cells, initiates a second-messenger cascade that can amplify small synaptic inputs from cones. In contrast, it is not known how the ionotropic glutamate receptors that are activated in ‘Off’ bipolar cells, are optimized for transmitting small, graded signals. Here we show, by recording from a cone and a synaptically connected ‘Off’ bipolar cell in slices of retina from the ground squirrel, that transmission is mediated by glutamate receptors of the kainate-preferring subtype. In the dark, a cone releases sufficient neurotransmitter to desensitize most postsynaptic kainate receptors. The small postsynaptic current that persists (<5% of maximum) is quickly modulated by changes in presynaptic voltage. Since recovery from desensitization is slow (the decay time constant is roughly 500 milliseconds), little recovery can occur during the brief (roughly 100-millisecond) hyperpolarization that is produced in cones by a flash of light. By limiting the postsynaptic current, receptor desensitization prevents saturation of the ‘Off’ bipolar cells voltage response and allows the synapse to operate over the cones entire physiological voltage range.

Asynchronous transmitter release: control of exocytosis and endocytosis at the salamander rod synapse.

1. We have studied exocytosis and endocytosis in the synaptic terminal of salamander rods using a combination of Ca2+ imaging, capacitance measurement and the photolysis of Ca2+ buffers. 2. The average cytoplasmic Ca2+ concentration at the dark resting potential was 2‐4 microM. 3. An average cytoplasmic Ca2+ concentration of 2‐4 microM maintained a high rate of continuous exocytosis and endocytosis. 4. Changes in the rate of exocytosis were followed in less than 0.7 s by compensatory changes in the rate of endocytosis. 5. Vesicle cycling in the rod synapse is specialized for graded transmission and differs from that previously described for synapses that release synchronized bursts of transmitter.

A GABA Transporter Operates Asymmetrically and with Variable Stoichiometry

Membrane currents produced by the expression of a rat GABA transporter (GAT-1) stably transfected into HEK293 cells were characterized with a whole-cell voltage clamp. Three modes of function were identified: ex-gated currents produced by extracellular GABA, in-gated currents produced by intracellular GABA, and uncoupled currents produced in the absence of GABA. The ex-gated current was not the reversal of the in-gated current; moreover, the stoichiometry between GABA and co-ions was not always fixed. Each mode of function required a different set of ions on the two sides of the membrane. We made rapid solution changes and observed an allosteric effect of Na+ that only occurred at the extracellular surface. Thus, the GAT-1 transporter does not behave like a recirculating carrier but may be described as a pore with ion gates at either end that are controlled in part by allosteric sites.

Electrophysiology of glutamate and sodium co-transport in a glial cell of the salamander retina.

1. Müller cells were isolated from salamander retinas and their membrane voltage was controlled with a whole‐cell voltage clamp. External D‐aspartate, L‐aspartate and L‐glutamate each induced a membrane current. D‐Glutamate, kainate, quisqualate and N‐methyl‐D‐aspartate were more than 100x less effective than L‐aspartate. Kynurenic acid had no effect on the current produced by L‐glutamate, L‐aspartate or D‐aspartate. 2. The current induced by an acidic amino acid (AAA) was completely dependent on the presence of external Na+. Neither Li+, Cs+, choline nor TEA+ were able to substitute for Na+. The relationship between external Na+ concentration and current amplitude can be explained if the binding of three Na+ ions enabled transport. The apparent affinity constant for Na+ binding was 41 mM. Altering K+, H+ and Cl‐ concentrations demonstrated that these ions are not required for transport. 3. The shape of the current‐voltage relation did not depend on the external amino acid concentration. The relationship between D‐aspartate concentration and current amplitude can be described by the binding of D‐aspartate to a single site with an apparent affinity constant of 20 microM. 4. Influx and efflux of AAA were not symmetric. Although influx was electrogenic, efflux did not produce a current. Moreover, influx stimulated efflux; but efflux inhibited influx. 5. Removing external Na+ demonstrated that Na+ carried a current in the absence of an AAA. Li+ was a very poor substitute for Na+. This current may be due to the uncoupled movement of Na+ through the transporter. The relationship between the external Na+ concentration and the amplitude of the uncoupled current can be explained if the binding of two or three Na+ ions enabled the translocation of Na+ in the absence of an AAA. The apparent affinity constant for Na+ binding was approximately 90 mM. 6. The temperature dependence of the AAA‐induced current had a Q10 between 8 and 18 degrees C of 1.95. The Q10 is consistent with a rate constant for influx of 10(4) s‐1 (at ‐70 mV and 20 degrees C). The maximum rate of influx was measured following a concentration jump produced by the photolysis of ‘caged’ L‐glutamate. The onset of the observed current was limited by the 1.3 ms resolution of the recording system. Hence, the rate constant for influx must be faster than 10(3) s‐1.(ABSTRACT TRUNCATED AT 400 WORDS)

Ions required for the electrogenic transport of GABA by horizontal cells of the catfish retina.

1. Solitary horizontal cells were isolated from catfish retinas. Membrane currents activated by extracellular and intracellular GABA were characterized during a whole‐cell voltage clamp. 2. Extracellular GABA activated two currents: a GABAA current, and an ‘influx’ current mediated by a GABA transporter. The influx current was studied after the GABAA current was blocked with 0.5 mM picrotoxin. The influx current required extracellular Na+ and Cl‐. Extracellular Na+ could not be replaced by another alkali metal cation. 3. The influx current also depended upon the identity of ions in the intracellular solution. Either an intracellular alkali metal cation or Cl‐ was required to produce an influx current. 4. The influx current was inward at ‐75 mV and decreased as the membrane was depolarized towards +20 mV. When the membrane was depolarized beyond +25 mV, the polarity of the current depended upon the ion composition of the intracellular solution and could be inward, zero or outward. 5. The introduction of GABA into a cell during the course of an experiment produced an outward current. This ‘efflux’ current was small at ‐75 mV and increased with depolarization. The efflux current required intracellular Na+ and Cl‐. Intracellular Na+ could not be replaced by another alkali metal cation. 6. The efflux current also depended upon the identity of ions in the extracellular solution. An extracellular alkali metal cation was required to produce an efflux current. Removing extracellular Cl‐ did not affect the efflux current. 7. The outward movement of GABA produced a local accumulation in extracellular GABA concentration that could be detected by the activation of the GABAA current. GABA efflux only occurred during conditions that produced an efflux current. Electroneutral efflux did not occur. 8. In the absence of GABA, extracellular alkali metal cations produced a ‘leakage’ current. The leakage current was inward at ‐75 mV and decreased as the membrane was depolarized towards +20 mV. When the membrane was depolarized beyond +25 mV, the polarity of the leakage current depended, like the GABA influx current, upon the ion composition of the intracellular solution and could be inward, zero or outward. The addition of GABA to the intracellular solution produced an efflux current and suppressed the leakage current. 9. We conclude that the transporter mediates electrogenic influx, efflux and leakage. Each mode of operation depends upon ions on both sides of the membrane. Influx and efflux are not symmetrical.

l-Glutamate conditionally modulates the K+ current of miller glial cells

L-Glutamate inhibits the K+ conductance that dominates the electrical behavior of a Müller glial cell. The effect of glutamate is enhanced by simultaneous exposure to dopamine. L-Glutamate acts at a metabotropic receptor that controls the K+ conductance through two pathways. A rapid pathway produces a partial inhibition in less than 2 s. Thereafter, a slow pathway progressively inhibits the conductance with a half-time of minutes. Pathways initiated by L-glutamate and dopamine appear to converge on and stimulate adenylyl cyclase. A subsequent step is the activation of a cAMP-dependent protein kinase, PKA. The local overflow of L-glutamate from active synapses may functionally remove K+ channels from nearby glial membranes. A uniform rise in extracellular L-glutamate concentration, as might occur during pathological conditions, should suppress a glial cells K+ conductance and allow other voltage-dependent processes to be influenced by depolarization.