Ion permeation through light-activated channels in rhabdomeric photoreceptors. Role of divalent cations

The receptor potential of rhabdomeric photoreceptors is mediated primarily by a Na influx, but other ions must also permeate through light-dependent channels to account for some properties of the photoresponse. We examined ion conduction in macroscopic and single- channel light-induced currents of Lima and Pecten photoreceptors. In the absence of Na, a fivefold change in extracellular K shifted the reversal voltage of the photocurrent (Vrev) by approximately 27 mV. Because the dependency of Vrev on [K]o was sub-Nernstian, and Vrev in each condition was more positive than Ek, some other ion(s) with a positive equilibrium potential must be implicated, in addition to K. We assessed the participation of calcium, an important candidate because of its involvement in light adaptation. Three strategies were adopted to minimize the impairments to cytosolic Ca homeostasis and loss of responsiveness that normally result from the required ionic manipulations: (a) Internal dialysis with Na-free solutions, to prevent reverse operation of the Na/Ca exchanger. (b) Rapid solution changes, temporally limiting exposure to potentially detrimental ionic conditions. (c) Single-channel recording, exposing only the cell- attached patch of membrane to the test solutions. An inward whole-cell photocurrent could be measured with Ca as the only extracellular charge carrier. Decreasing the [Ca]o to 0.5 mM reduced the response by 43% and displaced the reversal potential by -4.3 mV; the shift was larger (delta Vrev = -44 mV) when intracellular permeant cations were also removed. In all cases, however, the current carried by Ca was < 5% of that measured with normal [Na]o. Unitary light-activated currents were reduced in a similar way when the pipette contained only divalent cations, indicating a substantial selectivity for Na over Ca. The fall kinetics of the photoresponse was slower when external Ca was replaced by Ba, or when the membrane was depolarized; however, dialysis with 10 mM BAPTA failed to antagonize this effect, suggesting that mechanisms other than the Ca influx participate in the modulation of the time course of the photocurrent.     

Pressure injection of calcium both excites and adapts Limulus ventral photoreceptors

Single pressure injections of 1-2 mM calcium aspartate into the light-sensitive region of Limulus ventral photoreceptors resulted in a rapid, 20-40-mV depolarization lasting approximately 2 s. The depolarization closely followed the rise in intracellular free calcium caused by the injection, as indicated by aequorin luminescence. The depolarization was followed by reversible desensitization (adaptation) of responses to both light and inositol 1,4,5 trisphosphate. Similar single injections of calcium into the light-insensitive region of the receptor were essentially without effect, even though aequorin luminescence indicated a large, rapid rise in intracellular free calcium. The depolarization caused by injection of calcium arose from the activation of an inward current with rectification characteristics and a reversal potential between +10 and +20 mV that were similar to those of the light-activated conductance, which suggests that the same channels were activated by light and by calcium. The reversal potentials of the light- and calcium-activated currents shifted similarly when three-fourths of the extracellular sodium was replaced by sucrose, but were not affected by a similar replacement of sodium by lithium. The current activated by calcium was abolished by prior injection of a calcium buffer solution containing EGTA. The responses of the same cells to brief light flashes were slowed and diminished in amplitude, but were not abolished after the injection of calcium buffer. Light adaptation and prior injection of calcium diminished the calcium-activated current much less than they diminished the light-activated current.     

Electrophysiological properties of isolated photoreceptors from the eye of Lima scabra

Photoreceptor cells were enzymatically dissociated from the eye of the file clam, Lima scabra. Micrographs of solitary cells reveal a villous rhabdomeric lobe, a smooth soma, and a heavily pigmented intermediate region. Membrane voltage recordings using patch electrodes show resting potentials around -60 mV. Input resistance ranges from 300 M omega to greater than 1 G omega, while membrane capacitance is of the order of 50-70 pF. In darkness, quantum bumps occur spontaneously and their frequency can be increased by dim continuous illumination in a fashion graded with light intensity. Stimulation with flashes of light produces a depolarizing photoresponse which is usually followed by a transient hyperpolarization if the stimulus is sufficiently intense. Changing the membrane potential with current-clamp causes the early phase to invert around +10 mV, while the hyperpolarizing dip disappears around -80 mV. With bright light, the biphasic response is followed by an additional depolarizing wave, often accompanied by a burst of action potentials. Both Na and Ca ions are required in the extracellular solution for normal photoexcitation: the response to flashes of moderate intensity is greatly degraded either when Na is replaced with Tris, or when Ca is substituted with Mg. By contrast, quantum bumps elicited by dim, sustained light are not affected by Ca removal, but they are markedly suppressed in a reversible way in 0 Na sea water. It was concluded that the generation of the receptor potential is primarily dependent on Na ions, whereas Ca is probably involved in a voltage-dependent process that shapes the photoresponse. Light adaptation by repetitive flashes leads to a decrease of the depolarizing phase and a concomitant enhancement of the hyperpolarizing dip, eventually resulting in a purely hyperpolarizing photoresponse. Dark adaptation restores the original biphasic shape of the photoresponse.     

Cold-sensitive responses in the Paramecium membrane

A transient depolarization was recorded in response to the cooling of a deciliated Paramecium. The amplitude of the depolarization was almost proportional to the cooling rate. Therefore, the cells are sensitive to the rate of temperature change. The input resistance of the membrane transiently increased during the cooling. When constant current was applied to shift the resting membrane potential to a negative or positive level, the initial depolarization in response to cooling decreased, and the following hyperpolarization during cooling reversed to a gradual depolarization during a positive shift. The potential at which the reversal occurred was independent of K+ concentration and was slightly dependent on Ca2+ concentration (10 mV/log[Ca2+]o). The amplitude of the initial depolarization decreased with the increase in K+ and was not affected by Ca2+. These results are discussed in terms of changes in membrane conductances in response to cooling.     

Identification of a spontaneously active, Na+-permeable channel in guinea pig gallbladder smooth muscle

The action potential in gallbladder smooth muscle (GBSM) is caused by Ca2+ entry through voltage-dependent Ca2+ channels (VDCC), which contributes to the GBSM contractions. Action potential generation in GBSM is critically dependent on the resting membrane potential (about -50 mV), which is approximately 35 mV more positive of the K+ equilibrium potential. We hypothesized that a tonic, depolarizing conductance is present in GBSM and contributes to the regulation of the resting membrane potential and action potential frequency. GBSM cells were isolated from guinea pig gallbladders, and the whole cell patch-camp technique was used to record membrane currents. After eliminating the contribution of VDCC and K+ channels, we identified a novel spontaneously active cation conductance (I(cat)) in GBSM. This I(cat) was mediated predominantly by influx of Na+. Na+ substitution with N-methyl-D-glucamine (NMDG), a large relatively impermeant cation, caused a negative shift in the reversal potential of the ramp current and reduced the amplitude of the inward current at -50 mV by 65%. Membrane potential recordings with intracellular microelectrodes or in current-clamp mode of the patch-clamp technique indicated that the inhibition of I(cat) conductance by NMDG is associated with membrane hyperpolarization and inhibition of action potentials. Extracellular Ca2+, Mg2+, and Gd3+ attenuated the I(cat) in GBSM. Muscarinic stimulation did not activate the I(cat). Our results indicate that, in GBSM, an Na+-permeable channel contributes to the maintenance of the resting membrane potential and action potential generation and therefore plays a critical role in the regulation of GBSM excitability and contractility.     

Ionic mechanisms of two types of on-center bipolar cells in the carp retina. I. The responses to central illumination.

Properties of the depolarizing response of on-center bipolar cells to a light spot stimulus were studied in the carp retina. On-center bipolar cells were classified into two types, cone-dominant and rod-dominant, according to their major input from cones and rods. Cone-dominant bipolar cells responded to spectral light with the maximum amplitude near 625 nm, suggesting major input from red cones. The response was accompanied by a resistance increase and showed a reversal potential at -63 +/- 21 mV when the membrane was hyperpolarized by current. The results suggest that the photoresponse of cone-dominant cells is due to a decrease of gK and/or gCl, membrane conductances to potassium and chloride, respectively. Rod-dominant bipolar cells responded to spectral light with the maximum amplitude near 525 nm under scotopic conditions and near 625 nm under photopic conditions, providing evidence that they receive input from rods and red cones. In the scoptopic condition their response was accompanied by a resistance decrease and showed a reversal potential at 29 +/- 13 mV, whereas in the photopic condition the response in most of them was accompanied by a resistance increase, at least in their part and showed a reversal at -53 +/- 11 mV. The results suggest that the photoresponse activated by rod input is due to an increase in gNa. In the mesopic condition rod-dominant cells showed complex electrical membrane properties as the result of electric interaction between the above two differnt ionic mechanisms activated by rod and cone inputs.     

Chloride currents inChara—A patch-clamp study

Summary Ionic current steps were recorded with the patch-clamp technique from algal cells that had been prepared without enzyme treatment. Inward current steps with different conductance levels occurred, the lowest level being 7 pS. There were complex transitions between levels indicating either a lack of independence between single channels, or sublevels of a much larger conductance unit. The reversal potential was consistent with the permeant ion being Cl^–. Furthermore, when a different concentration of Cl^– was used in the patch electrode the reversal potential of the inward current shifted in a manner consistent with a Nernstian change in the Cl^– reversal potential. The frequency of the current steps was voltage dependent and suggestive of the hyperpolarization-activated Cl^– currents reported in voltage-clamp studies. Outward current steps, with conductances of 38 pS, were recorded when the membrane patch was depolarized by more than +120 mV. Their amplitude and frequency increased at more positive potentials. The current was probably carried by an efflux of cations through a different set of channels. The resting membrane potential, measured unambiguously without contamination from the tonoplast, was –190±5 mV.     

The light-sensitive conductance of melanopsin-expressing Joseph and Hesse cells in amphioxus

Two types of microvillar photoreceptors in the neural tube of amphioxus, an early chordate, sense light via melanopsin, the same photopigment as in "circadian" light detectors of higher vertebrates. Because in amphioxus melanopsin activates a G(q)/phospholipase C cascade, like phototransduction in arthropods and mollusks, possible commonalities in the photoconductance were investigated. Unlike other microvillar photoreceptors, reversal of the photocurrent can only be attained upon replacement of extracellular Na(+). In addition to Na(+), Ca(2+) is also permeant, as indicated by the fact that (a) in normal ionic conditions the photocurrent remains inward at V(m) > E(Na); (b) in Na-free solution a small residual inward photocurrent persists at V(m) near resting level, provided that Ca is present; and (c) V(rev) exhibits a modest shift with [Ca](o) manipulations. The unusual reversal is accounted for by an uncommonly low permeability of the light-dependent channels to K(+), as [K](o) only marginally affects the photocurrent amplitude and its reversal. Lanthanum and ruthenium red (RuR), two TRP channel antagonists, reversibly suppress the response to photostimulation of moderate intensity; therefore, the melanopsin-initiated cascade may recruit ion channels of the same family as those of rhabdomeric photoreceptors. With brighter lights, blockage declines, so that both La(3+) and RuR induce a right shift in the sensitivity curve without a reduction of its asymptote. Nonetheless, an effect on the transduction cascade, rather than the channels, was ruled out on the basis of the voltage dependency of the blockade and the lack of effects of intracellular application of the same substances. The mechanisms of action of these antagonists thus entail a state-dependent blockade, with a higher affinity for the channel in the closed conformation. Collectively, the results indicate a kinship of the light-sensitive channels of amphioxus with those of invertebrate rhabdomeric visual cells and support the representation of this lineage of photoreceptors among chordates.     

Properties of the membrane current of rod outer segments.

The membrane current of single rod outer segments in pieces of isolated toad retina was recorded with a glass suction electrode. Light evoked a slow net outward photocurrent consisting of a reduction in the steady inward dark current. In very dim light, the photocurrent broke up into discrete shot effects with a rounded shape and an amplitude of about 1 pA. These events were shown to result from photoisomerization of single rhodopsin molecules. The current in darkness showed fluctuations consisting of (a) discrete events apparently resulting from thermal isomerization of rhodopsin molecules, and (b) smaller amplitude shot effects shaped by two of the four rate processes of the light response.     

Properties of a tonically active, sodium-permeable current in mouse urinary bladder smooth muscle

Urinary bladder smooth muscle (UBSM) elicits depolarizing action potentials, which underlie contractile events of the urinary bladder. The resting membrane potential of UBSM is approximately -40 mV and is critical for action potential generation, with hyperpolarization reducing action potential frequency. We hypothesized that a tonic, depolarizing conductance was present in UBSM, functioning to maintain the membrane potential significantly positive to the equilibrium potential for K(+) (E(K); -85 mV) and thereby facilitate action potentials. Under conditions eliminating the contribution of K(+) and voltage-dependent Ca(2+) channels, and with a clear separation of cation- and Cl(-)-selective conductances, we identified a novel background conductance (I(cat)) in mouse UBSM cells. I(cat) was mediated predominantly by the influx of Na(+), although a small inward Ca(2+) current was detectable with Ca(2+) as the sole cation in the bathing solution. Extracellular Ca(2+), Mg(2+), and Gd(3+) blocked I(cat) in a voltage-dependent manner, with K(i) values at -40 mV of 115, 133, and 1.3 microM, respectively. Although UBSM I(cat) is extensively blocked by physiological extracellular Ca(2+) and Mg(2+), a tonic, depolarizing I(cat) was detected at -40 mV. In addition, inhibition of I(cat) demonstrated a hyperpolarization of the UBSM membrane potential and decreased the amplitude of phasic contractions of isolated UBSM strips. We suggest that I(cat) contributes tonically to the depolarization of the UBSM resting membrane potential, facilitating action potential generation and thereby a maintenance of urinary bladder tone.     

Na and Ca channels in a transformed line of anterior pituitary cells

The ionic conductances of GH3 cells, a transformed line from rat anterior pituitary, have been studied using the whole-cell variant of the patch-clamp technique (Hamill et al., 1981). Pipettes of very low resistance were used, which improved time resolution and made it possible to control the ion content of the cell interior, which equilibrated very rapidly with the pipette contents. Time resolution was further improved by using series resistance compensation and "ballistic charging" of the cell capacitance. We have identified and partially characterized at least three conductances, one carrying only outward current, and the other two normally inward. The outward current is absent when the pipette is filled with Cs+ instead of K+, and has the characteristics of a voltage-dependent potassium conductance. One of the two inward conductances (studied with Cs+ inside) has fast activation, inactivation and deactivation kinetics, is blocked by tetrodotoxin (TTX), and has a reversal potential at the sodium equilibrium potential. The other inward current activates more slowly and deactivates with a quick phase and a very slow phase after a short pulse. Either Ca++ or Ba++ serves as current carrier. During a prolonged pulse, current inactivates fairly completely if there is at least 5 mM Ca++ outside, and the amplitude of the current tails following the pulse diminishes with the time course of inactivation. When Ba++ entirely replaces Ca++ in the external medium, there is no inactivation, but deactivation kinetics of Ca channels vary as pulse duration increases: the slow phase disappears, the fast phase grows in amplitude. Inactivation (Ca++ outside) is unaltered by 50 mM EGTA in the pipette: inactivation cannot be the result of internal accumulation of Ca++.     

Prostaglandin E2 Activates Ca2+ Channels in Bovine Adrenal Chromaffin Cells

We have demonstrated that prostaglandin E2 (PGE2) treatment of bovine adrenal chromaffin cells results in a sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) in these cells. Because the continued elevation of [Ca2+]i was dependent on extracellular Ca2+ concentration, it can be assumed that the PGE2-induced [Ca2+]i increase is due, at least in part, to an opening of membrane Ca2+ channels. In this study, we used electrophysiological methods to examine the mechanism of the PGE2-induced [Ca2+]i increase directly. Puff application of PGE2 to the external medium resulted in a prolonged depolarization in about half of the chromaffin cells examined. In whole-cell voltage-clamp recordings, an increase in inward current was observed over a 6-7 min period following bath application of PGE2 (greater than or equal to 10 microM), even in the absence of external Na+. This inward current was abolished when the recordings were made with the cells in a Ca2(+)-free medium, but it was not inhibited by Mn2+, a blocker of voltage-dependent Ca2+ channels. In cell-attached patch-clamp configuration, PGE2 produced an increase in the opening frequency of inward currents. The reversal potential of the PGE2-induced currents was about +40 mV, which is close to the reversal potential of the Ca2+ channel. The opening frequency was not affected by membrane potential changes. In inside-out patch-clamp configuration, inositol 1,4,5-trisphosphate (2 microM) added to the cytoplasmic side activated the Ca2(+)-channel currents, but PGE2 was ineffective when applied to the cytoplasmic side. These results suggest that PGE2 activates voltage-independent Ca2+ channels in chromaffin cells through a diffusible second messenger, possibly inositol 1,4,5-trisphosphate.     

Chemosensitivity of single smooth muscle cells to acetylcholine, noradrenaline, and histamine in vitro

Electrical responses to acetylcholine, noradrenaline, and histamine were recorded from solitary smooth muscle cells. Iontophoresis of each transmitter elicited three fast responses: a hyperpolarization, a depolarization, or a biphasic hyperpolarization-depolarization. Each transmitter activated a specific receptor since responses were specifically blocked by antagonists, two transmitters elicited different responses in solitary cells, and desensitization of response to one transmitter did not cause desensitization of responses to other transmitters. Responses were due to increased ion conductances since input resistance decreased during responses and reversal potentials were measured for depolarizing responses (-5 mV) and hyperpolarizing responses (-60 mV). Regional differences in transmitter sensitivity were mapped on solitary cells. Biphasic responses were due to simultaneous activation of receptors mediating hyperpolarizing responses and receptors mediating depolarizing responses which were segregated in the cell membrane. Noradrenaline enhanced action potential amplitude by regulation of voltage-dependent ion conductances. Finally, noradrenaline and histamine elicited periodic hyperpolarizing potentials, which may be due to increased intracellular Ca++.     

Voltage-dependent ionic currents in taste receptor cells of the larval tiger salamander

Voltage-dependent membrane currents of cells dissociated from tongues of larval tiger salamanders (Ambystoma tigrinum) were studied using whole-cell and single-channel patch-clamp techniques. Nongustatory epithelial cells displayed only passive membrane properties. Cells dissociated from taste buds, presumed to be gustatory receptor cells, generated both inward and outward currents in response to depolarizing voltage steps from a holding potential of -60 or -80 mV. Almost all taste cells displayed a transient inward current that activated at -30 mV, reached a peak between 0 and +10 mV and rapidly inactivated. This inward current was blocked by tetrodotoxin (TTX) or by substitution of choline for Na+ in the bath solution, indicating that it was a Na+ current. Approximately 60% of the taste cells also displayed a sustained inward current which activated slowly at about -30 mV and reached a peak at 0 to +10 mV. The amplitude of the slow inward current was larger when Ca2+ was replaced by Ba2+ and it was blocked by bath applied CO2+, indicating it was a Ca2+ current. Delayed outward K+ currents were observed in all taste cells although in about 10% of the cells, they were small and activated only at voltages more depolarized than +10 mV. Normally, K+ currents activated at -40 mV and usually showed some inactivation during a 25-ms voltage step. The inactivating component of outward current was not observed at holding potentials more depolarized -40 mV. The outward currents were blocked by tetraethylammonium chloride (TEA) and BaCl2 in the bath or by substitution of Cs+ for K+ in the pipette solution. Both transient and noninactivating components of outward current were partially suppressed by CO2+, suggesting the presence of a Ca2(+)-activated K+ current component. Single-channel currents were recorded in cell-attached and outside-out patches of taste cell membranes. Two types of K+ channels were partially characterized, one having a mean unitary conductance of 21 pS, and the other, a conductance of 148 pS. These experiments demonstrate that tiger salamander taste cells have a variety of voltage- and ion-dependent currents including Na+ currents, Ca2+ currents and three types of K+ currents. One or more of these conductances may be modulated either directly by taste stimuli or indirectly by stimulus-regulated second messenger systems to give rise to stimulus-activated receptor potentials. Others may play a role in modulation of neurotransmitter release at synapses with taste nerve fibers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of Mechano-Electric Feedback on Scroll Wave Stability in Human Ventricular Fibrillation

Recruitment of stretch-activated channels, one of the mechanisms of mechano-electric feedback, has been shown to influence the stability of scroll waves, the waves that underlie reentrant arrhythmias. However, a comprehensive study to examine the effects of recruitment of stretch-activated channels with different reversal potentials and conductances on scroll wave stability has not been undertaken; the mechanisms by which stretch-activated channel opening alters scroll wave stability are also not well understood. The goals of this study were to test the hypothesis that recruitment of stretch-activated channels affects scroll wave stability differently depending on stretch-activated channel reversal potential and channel conductance, and to uncover the relevant mechanisms underlying the observed behaviors. We developed a strongly-coupled model of human ventricular electromechanics that incorporated human ventricular geometry and fiber and sheet orientation reconstructed from MR and diffusion tensor MR images. Since a wide variety of reversal potentials and channel conductances have been reported for stretch-activated channels, two reversal potentials, −60 mV and −10 mV, and a range of channel conductances (0 to 0.07 mS/µF) were implemented. Opening of stretch-activated channels with a reversal potential of −60 mV diminished scroll wave breakup for all values of conductances by flattening heterogeneously the action potential duration restitution curve. Opening of stretch-activated channels with a reversal potential of −10 mV inhibited partially scroll wave breakup at low conductance values (from 0.02 to 0.04 mS/µF) by flattening heterogeneously the conduction velocity restitution relation. For large conductance values (>0.05 mS/µF), recruitment of stretch-activated channels with a reversal potential of −10 mV did not reduce the likelihood of scroll wave breakup because Na channel inactivation in regions of large stretch led to conduction block, which counteracted the increased scroll wave stability due to an overall flatter conduction velocity restitution.     

Voltage-dependent large conductance channels in visceral smooth muscle.

The ionic conductances that underlie the resting membrane potential of visceral smooth muscle are not fully understood. Using the patch-clamp technique in the whole-cell configuration, single large conductance channels (LCCs) with unitary conductances of up to 400 pS were recorded in isolated smooth muscle cells of the opossum esophagus. These channels were active at physiological potentials (-100 to -40 mV) and opened with increasing frequency as the membrane potential was hyperpolarized. This voltage dependence gave rise to an inwardly rectifying macroscopic current which was half-maximally activated at -65 mV. The current through LCCs was carried by cations because reduction of external [NaCl] shifted the reversal potential of the LCC current towards the predicted Nernst potential for a nonselective cation current. These results suggest that LCCs may contribute to resting membrane potential in the circular muscle of the opossum esophagus.     

Inwardly rectifying K(+) channels in esophageal smooth muscle

The whole cell patch-clamp technique was used to investigate whether there were inwardly rectifying K(+) (K(ir)) channels in the longitudinal muscle of cat esophagus. Inward currents were observable on membrane hyperpolarization negative to the K(+) equilibrium potential (E(k)) in freshly isolated esophageal longitudinal muscle cells. The current-voltage relationship exhibited strong inward rectification with a reversal potential (E(rev)) of -76.5 mV. Elevation of external K(+) increased the inward current amplitude and positively shifted its E(rev) after the E(k), suggesting that potassium ions carry this current. External Ba(2+) and Cs(+) inhibited this inward current, with hyperpolarization remarkably increasing the inhibition. The IC(50) for Ba(2+) and Cs(+) at -60 mV was 2.9 and 1.6 mM, respectively. Furthermore, external Ba(2+) of 10 microM moderately depolarized the resting membrane potential of the longitudinal muscle cells by 6.3 mV while inhibiting the inward rectification. We conclude that K(ir) channels are present in the longitudinal muscle of cat esophagus, where they contribute to its resting membrane potential.     

The light-sensitive conductance of hyperpolarizing invertebrate photoreceptors: a patch-clamp study

Tight-seal recording was employed to investigate membrane currents in hyperpolarizing ciliary photoreceptors enzymatically isolated from the eyes of the file clam (Lima scabra) and the bay scallop (Pecten irradians). These two organisms are unusual in that their double retinas also possess a layer of depolarizing rhabdomeric cells. Ciliary photoreceptors from Lima have a rounded soma, 15-20 microns diam, and display a prominent bundle of fine processes up to 30 microns long. The cell body of scallop cells is similar in size, but the ciliary appendages are modified, forming small spherical structures that protrude from the cell. In both species light stimulation at a voltage near the resting potential gives rise to a graded outward current several hundred pA in amplitude, accompanied by an increase in membrane conductance. The reversal potential of the photocurrent is approximately -80 mV, and shifts in the positive direction by approximately 39 mV when the concentration of extracellular K is increased from 10 to 50 mM, consistent with the notion that light activates K-selective channels. The light-activated conductance increases with depolarization in the physiological range of membrane voltages (-30 to -70 mV). Such outward rectification is greatly reduced after removal of divalent cations from the superfusate. In Pecten, cell- attached recordings were also obtained; in some patches outwardly directed single-channel currents could be activated by light but not by voltage. The unitary conductance of these channels was approximately 26 pS. Solitary ciliary cells also gave evidence of the post stimulus rebound, which is presumably responsible for initiating the "off" discharge of action potentials at the termination of a light stimulus: in patches containing only voltage-dependent channels, light stimulation suppressed depolarization-induced activity, and was followed by a strong burst of openings, directly related to the intensity of the preceding photostimulation.     

三叉神经中脑核神经元膜的电学特性

用大鼠脑干脑片,给三叉神经中脑核79个神经元作了细胞内记录,测算了20个神经元膜的电学特性:静息电位-60.3±5.6mV;输入阻抗为10.5±5.4MΩ;时间常数1.3±0.5ms。电刺激可诱发动作电位,测算32个神经元的有关参数:阈电位-50—-55mV;波幅69.5±6.1mV;超射11.9±3.6mV;波宽0.8±0.2ms。TTX(0.3μmol/L)或无钠使之消失。通以长时程矩形波电流可引起200—250Hz的2—15个重复放电,但在通电停止前终止,TEA或4-AP可延长放电。膜电位-60—-55mV时在动作电位之后可看到阈下电位波动,它不受TTX的影响,无钙时消失,TEA或4-AP使波幅增大。静息电位去极化可使45个神经元中的40个发生外向整流作用,并被TEA,4-AP或无钙抑制,超极化则发生内向整流作用,Cs或无钠抑制之。灌流液中加入各种钾通道阻断药时神经元的稳态I-V曲线发生相应变化,提示I_(DR),l_A,I_(K(Ca))及I_Q可能都与静息时的膜电导有关。     

Light-activated ion channels in solitary photoreceptors of the scallop Pecten irradians.

Retinas from the scallop Pecten irradians were enzymatically dispersed, yielding a large number of isolated photoreceptors suitable for tight-seal recording. Whole-cell voltage clamp measurements demonstrated that the phototransducing machinery remained intact: quantum bumps could be elicited by dim illumination, while brighter flashes produced larger, smooth photocurrents. Single-channel currents specifically activated by light were recorded in cell-attached patches, and were almost exclusively confined to the rhabdomeric region. Their density is sufficiently high to account for the macroscopic photoresponse. Channel activation is graded with stimulus intensity in a range comparable to that of the whole-cell response, and can be recorded with illumination sufficiently dim to evoke only quantum bumps. Light-dependent channel openings are very brief, on average 1 ms or less at 20-22 degrees C, apparently not because of blockage by extracellular divalent cations. The mean open time does not change substantially with stimulus intensity. In particular, since dwell times are in the millisecond range even with the dimmest lights, the channel closing rate does not appear to be the rate-limiting step for the decay kinetics of discrete waves. The latency of the first opening after light onset is inversely related to light intensity, and the envelope of channel activity resembles the time course of the whole-cell photocurrent. Unitary currents are inward at resting potential, and have a reversal voltage similar to that of the macroscopic light response. Voltage modulates the activity of light-sensitive channels by increasing the opening rate and also by lengthening the mean open times as the patch is depolarized. The unitary conductance of the predominant class of events is approximately 48 pS, but at least one additional category of smaller-amplitude openings was observed. The relative incidence of large and small events does not appear to be related in a simple way to the state of adaptation of the cell.