Voltage- and calcium-dependent inactivation of calcium channels in Lymnaea neurons.

Ca(2+) channel inactivation in the neurons of the freshwater snail, Lymnaea stagnalis, was studied using patch-clamp techniques. In the presence of a high concentration of intracellular Ca(2+) buffer (5 mM EGTA), the inactivation of these Ca(2+) channels is entirely voltage dependent; it is not influenced by the identity of the permeant divalent ions or the amount of extracellular Ca(2+) influx, or reduced by higher levels of intracellular Ca(2+) buffering. Inactivation measured under these conditions, despite being independent of Ca(2+) influx, has a bell-shaped voltage dependence, which has often been considered a hallmark of Ca(2+)-dependent inactivation. Ca(2+)-dependent inactivation does occur in Lymnaea neurons, when the concentration of the intracellular Ca(2+) buffer is lowered to 0.1 mM EGTA. However, the magnitude of Ca(2+)-dependent inactivation does not increase linearly with Ca(2+) influx, but saturates for relatively small amounts of Ca(2+) influx. Recovery from inactivation at negative potentials is biexponential and has the same time constants in the presence of different intracellular concentrations of EGTA. However, the amplitude of the slow component is selectively enhanced by a decrease in intracellular EGTA, thus slowing the overall rate of recovery. The ability of 5 mM EGTA to completely suppress Ca(2+)-dependent inactivation suggests that the Ca(2+) binding site is at some distance from the channel protein itself. No evidence was found of a role for serine/threonine phosphorylation in Ca(2+) channel inactivation. Cytochalasin B, a microfilament disrupter, was found to greatly enhance the amount of Ca(2+) channel inactivation, but the involvement of actin filaments in this effect of cytochalasin B on Ca(2+) channel inactivation could not be verified using other pharmacological compounds. Thus, the mechanism of Ca(2+)-dependent inactivation in these neurons remains unknown, but appears to differ from those proposed for mammalian L-type Ca(2+) channels.

Measurement of calcium channel inactivation is dependent upon the test pulse potential.

We have developed two methods to measure Ca2+ channel inactivation in Lymnaea neurons-one method, based upon the conventional double-pulse protocol, uses currents during a moderately large depolarizing pulse, and the other uses tail currents after a very strong activating pulse. Both methods avoid contamination by proton currents and are unaffected by rundown of Ca2+ current. The magnitude of inactivation measured differs for the two methods; this difference arises because the measurement of inactivation is inherently dependent upon the test pulse voltage used to monitor the Ca2+ channel conductance. We discuss two models that can generate such test pulse dependence of inactivation measurements-a two-channel model and a two-open-state model. The first model accounts for this by assuming the existence of two types of Ca2+ channels, different proportions of which are activated by the different test pulses. The second model assumes only one Ca2+ channel type, with two closed and open states; in this model, the test pulse dependence is due to the differential activation of channels in the two closed states by the test pulses. Test pulse dependence of inactivation measurements of Ca2+ channels may be a general phenomenon that has been overlooked in previous studies.

Modulation of membrane currents by cyclic AMP in cleavage-arrested Drosophila neurons.

A number of Drosophila learning mutants have defective intracellular second-messenger systems. In an effort to develop techniques that will allow direct measurement of the effects of these mutations on whole-cell neuronal membrane currents, the perforated-patch whole-cell (PPWC) technique has been applied to cleavage-arrested cultured embryonic Drosophila neurons. This technique permits the measurement of membrane currents without disturbing the intracellular environment. As a result of the maintenance of the intracellular environment, Drosophila neuron currents are found to be much more stable than when measured using the conventional whole-cell (CWC) patch-clamp technique. Ca2+ channel currents, which typically 'wash out' within a few minutes of the beginning of CWC recording, are stable for the duration of the seal (tens of minutes) when measured using the PPWC technique. Since the learning mutations dunce and rutabaga disrupt cyclic AMP signalling, the action of externally applied dibutyryl cyclic AMP (db-cAMP) and theophylline on Ca2+ and K+ channel currents were studied. db-cAMP and theophylline enhanced the Ba2+ current, carried by Ca2+ channels, but had no effect on the K+ current in the cleavage-arrested neurons. However, the large variability and reduction in density of Ba2+ and K+ currents raise questions about the suitability of using these cleavage-arrested cells as models for Drosophila neurons.

Ca2+ channel Ca(2+)-dependent inactivation in a mammalian central neuron involves the cytoskeleton.

Ca2+ channel inactivation was investigated in acutely isolated hippocampal pyramidal neurons from adult rats and found to have a component dependent on intracellular Ca2+. Ca(2+)-dependent inactivation was indentified as the additional inactivation of channel current observed when Ca2+ replaced Ba2+ as the current carrying ion, and was found to be an independent process from that of Ba2+ current inactivation based on three lines of evidence: (1) no correlation between Ca(2+)-dependent inactivation and Ba2+ current inactivation was found, (2) only Ca(2+)-dependent inactivation was reduced by intracellular application of Ca2+ chelators, and (3) only Ca(2+)-dependent inactivation was sensitive to compounds which alter the cytoskeleton. Drugs which stabilize (taxol and phalloidin) and destabilize (colchicine and cytochalasin B) the cytoskeleton altered the development and recovery from Ca(2+)-dependent inactivation, indicating that the neuronal cytoskeleton may mediate Ca2+ channel sensitivity to intracellular Ca2+. Ca(2+)-dependent inactivation was not associated with a particular subset of Ca2+ channels, suggesting that all Ca2+ channels in these neurons are inactivated by intracellular Ca2+.

A cytoskeletal mechanism for Ca2+ channel metabolic dependence and inactivation by intracellular Ca2+.

Many different types of voltage-dependent Ca2+ channels inactivate when intracellular ATP declines or intracellular Ca2+ rises. An inside-out, patch-clamp technique was applied to the Ca2+ channels of Lymnaea neurons to determine the mechanism(s) underlying these two phenomena. Although no evidence was found for a phosphorylation mechanism, agents that act on the cytoskeleton were found to alter Ca2+ channel activity. The cytoskeletal disrupters colchicine and cytochalasin B were found to speed Ca2+ channel decline in ATP, whereas the cytoskeletal stabilizers taxol and phalloidin were found to prolong Ca2+ channel activity without ATP. In addition, cytoskeletal stabilizers reduced Ca(2+)-dependent channel inactivation, suggesting that both channel metabolic dependence and Ca(2+)-dependent inactivation result from a cytoskeletal interaction.

Photo-released intracellular Ca2+ rapidly blocks Ba2+ current in Lymnaea neurons.

1. The effect of intracellular Ca2+ on Ba2+ current flowing through voltage-dependent Ca2+ channels was studied using the whole-cell patch-clamp technique on isolated neurons from the snail Lymnaea stagnalis. Intracellular Ca2+ was increased by flash photolysis of the caged Ca2+ compound DM-nitrophen and measured with the optical indicator fluo-3. 2. After the highest intensity flashes, peak Ba2+ current was blocked by 42% with a time constant of 5 ms. The onset of the block followed a similar time course whether channels were activated or closed. The Ba2+ current surviving after the flash had the same voltage dependence of activation and rate of inactivation as did the total Ba2+ current before the flash. 3. Recovery of the Ba2+ current from block was nearly complete and occurred with a time constant of 16 s. Multiple episodes of photolysis-induced block could be studied in the same cell when 7-10 min were allowed between flashes. In some cells, recovery from block was accompanied by a transient enhancement of the current above the pre-block magnitude. 4. Neurons greatly reduced the ability of photolysis to increase Ca2+, both by unloading the DM-nitrophen before flashes were applied and by rapidly buffering the photolytically released Ca2+. Maximal flashes on extracellular droplets of the DM-Ca2+ solution created a Ca2+ jump from 110 nM to 40 microM. In contrast, the same flashes on DM-Ca(2+)-loaded neurons resulted in a Ca2+ transient starting from a baseline of 36 nM to a peak of 130 nM. This intracellular Ca2+ transient decayed with three time constants (120 ms, 2 s and 13 s). 5. Endogenous buffer(s) binds Ca2+ rapidly. When intracellular Ca2+ was monitored within 2 ms of the flash, no rapid Ca2+ spike due to binding of photo-released Ca2+ could be detected. Addition of dibromo-BAPTA to the intracellular solution reduced the block by one third, which is consistent with the measured reduction of intracellular Ca2+. This indicates that the endogenous buffer can bind Ca2+ as rapidly as dibromo-BAPTA and as fast as Ca2+ is released by photolysis. 6. The Ca2+ dependence of the block, obtained by varying flash intensity, indicates some saturation by 130 nM. A simple two-state model of the block consistent with both the time course of block and recovery and the concentration dependence gave a dissociation constant of approximately 50 nM and forward rate constant of 7 x 10(8) M-1 s-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of single calcium channels in Drosophila embryonic nerve and muscle cells.

Voltage-activated Ca channels play a central role in synaptic transmission, control of cell excitability, and many other cellular processes. It is now clear that there are multiple types of Ca channels with various modes of modulation. Drosophila offers exceptional advantages for studying the molecular basis of the diversity and modulation of Ca channels. As a step in this study, we have characterized the single-channel Ba currents recorded from cell-attached patches on cultured embryonic Drosophila nerve and muscle cells. The voltage dependence and selectivity of the channels carrying these Ba currents identify them as Ca channels. All Ca channels found in Drosophila neurons appear to have the same voltage dependence of activation and similar single-channel conductance, 12-17 pS (100 mM Ba2+). However, the kinetic properties of individual Ca channels vary greatly. The mean open time of individual channels ranges from 2 msec to less than 0.2 msec. Some channels completely inactivate during the first half of a 90 msec depolarization, while others are more active in the second half. Many channels open during almost every depolarization, while others open in less than 20% of the depolarizations. Channels with longer open times tend to inactivate and open during a small fraction of depolarizations. When these kinetic properties were quantified, a continuum of values was found, instead of the clustering of values that might be expected for discrete types of channels. Muscle Ca channels form a more homogeneous class than do the neuronal Ca channels. The muscle Ca channel conductance is 18 pS. These channels do not inactivate during 90 msec depolarizations and open during a majority of depolarizations applied. Muscle Ca channels are similar to a subset of neuronal Ca channels. When a purified toxin from the spider Hololena curta is applied to neurons, the number of active Ca channels is reduced, and those channels still active open in a small fraction of depolarization. Since channels that open in a small fraction of depolarizations tend to inactivate, these data support the hypothesis that this toxin selectively blocks noninactivating neuronal Ca channels. This differential toxin sensitivity and the much larger variability observed in kinetic properties of neuronal, compared to muscle, Ca channels suggest that there are at least two types of neuronal Ca channels in Drosophila.

Spider toxins selectively block calcium currents in Drosophila.

Toxins from spider venom, originally purified for their ability to block synaptic transmission in Drosophila, are potent and specific blockers of Ca2+ currents measured in cultured embryonic Drosophila neurons using the whole-cell, patch-clamp technique. Differential actions of toxins from two species of spiders indicate that different types of Drosophila neuronal Ca2+ currents can be pharmacologically distinguished. Hololena toxin preferentially blocks a non-inactivating component of the current, whereas Plectreurys toxin blocks both inactivating and non-inactivating components. These results suggest that block of a non-inactivating Ca2+ current is sufficient to block neurotransmitter release at Drosophila neuromuscular junction.

Characterization of proton currents in neurones of the snail, Lymnaea stagnalis.

1. Internal perfusion voltage-clamp and inside-out patch-clamp techniques were used to study the voltage-dependent H+ currents in snail neurone cell bodies. 2. In whole cells the voltage-activated outward H+ current was measured 60 ms after stepping to +40 mV with an internal pH (pHi) of 5.9 and no internal K+([K+]i = 0), and the delayed K+ current was measured 60 ms after stepping to +40 mV with pHi = 7.3 and [K+]i = 74 mM. The mean H+ and K+ current densities were 14.6 +/- 7.8 and 38.2 +/- 14.0 nA/nF, respectively, giving a mean ratio of the H+ to K+ current of 0.4 +/- 0.2. There is not a strong correlation between the densities of the two kinds of outward currents found in different cells. 3. Inside-out patch studies reveal that the H+ and K+ currents are distributed quite differently in the membrane. While 85% of all patches had K+ current, only five out of thirty-eight patches studied had H+ currents. In those five patches the H+ currents measured at +30 mV ranged from 10.7 to 21.0 pA, and the ratio of the H+ and K+ currents at +30 mV was 0.83 +/- 0.38. The mean H+ and K+ currents for all thirty-eight patches were 1.9 +/- 4.9 and 10.5 +/- 7.9 pA, respectively. 4. The current distribution patterns demonstrate that the H+ current does not flow through the delayed K+ current channels even though the two currents have similar voltage dependence and time course. 5. The relative ability of various extracellular divalent cations to block the H+ current was found to be Cu2+ approximately equal to Zn2+ greater than Ni2+ greater than Cd2+ greater than Co2+ greater than Mn2+ greater than Mg2+ = Ca2+ = Ba2+. Since 100 microM-Zn2+ blocks the H+ current more than it blocks the Ca2+ current, it can be used to reduce the contamination of Ca2+ current measurements by the H+ current. 6. The magnitude of the H+ current has a stronger temperature sensitivity than does the magnitude of the delayed K+ current. The Q10 of the H+ current magnitude is 2.1 +/- 0.4, while the Q10 of the K+ current magnitude is 1.4 +/- 0.04. This suggests a higher activation energy may be involved in the conduction of the H+ current than for K+ current. 7. The smooth time course of the H+ current measured in patches indicates that the size of the unitary H+ current is very small.(ABSTRACT TRUNCATED AT 400 WORDS)

Voltage-independent barium-permeable channel activated in Lymnaea neurons by internal perfusion or patch excision.

Isolated nerve cells from Lymnaea stagnalis were studied using the internal-perfusion and patch-clamp techniques. Patch excision frequently activated a voltage-independent Ba2+-permeable channel with a slope conductance of 27 pS at negative potentials (50 mM Ba2+). This channel is not seen in patches on healthy cells and, unlike the voltage-dependent Ca channel, is not labile in isolated patches. The activity of the channel in inside-out patches is unaffected by intracellular ATP, Ca2+ below 1 mM or the catalytic subunit of cAMP-dependent protein kinase but is reversibly blocked by millimolar intracellular Ca2+ or Ba2+. The channel can be activated in on-cell patches by either internal perfusion with high Ca2+ or the long-term internal perfusion of low Ca2+ solutions not containing ATP. These channels may carry the inward Ca2+ current which causes a regenerative increase in intracellular Ca+ when snail neurons are perfused with high Ca2+ solutions. High internal Ca2+, or long periods of internal perfusion with ATP-free solutions, induces an increase in a resting (-50 mV) whole-cell Ba2+ conductance. This conductance can be turned off by returning the intracellular perfusate to a low Ca2+ solution containing ATP and Mg2+. The activity of this channel appears to have an opposite dependence on intracellular conditions to that of the voltage-dependent Ca channel.

Ionic currents of Drosophila neurons in embryonic cultures.

Drosophila offers a unique opportunity to determine how the genome codes for ionic channels in an organized nervous system. Considerable progress has already been made in studying the molecular biology of Drosophila K channels. In order for similar progress to be made on neuronal voltage-dependent Ca channels, a physiological preparation is needed in which the function of these channels can be directly studied. The patch-clamp studies reported here show that cultures of embryonic Drosophila cells (Seecof and Unanue, 1968) meet this need. These cultures provide the first opportunity to study with voltage-clamp techniques the Ca and Na currents of Drosophila neurons. The focus of these studies is on the Ca current; however, descriptions of the K and Na currents are also given since they help to characterize the cells studied and the quality of the voltage clamp. The voltage-dependent K, Na, and Ca currents of Drosophila neurons are very similar to those of molluscan neurons and other better studied neurons. The K currents are the largest currents in these neurons, averaging over 300 pA at +20 mV. There are 2 classes of Ca-independent K currents, inactivating currents that are 4-AP sensitive, and noninactivating currents that are insensitive to 4-AP. A large fraction of the K currents are located in the somal membrane. The Na currents are TTX sensitive and probably located in the processes. The peak amplitudes of the Ca currents vary from 0 to over 100 pA in these neurons, averaging 40 pA. With 5 mM external Ca2+ or Ba2+, the Ba currents are about twice as large as the Ca currents. Although 100 microM Cd2+ completely blocks the Ca current, organic blockers have very little effect. Variable inactivation characteristics and sensitivity to washout suggest the possibility of multiple types of Ca channels. A search for single-channel Ba currents in the somal membrane was unsuccessful.

Effects of intracellular pH and calcium activity on ion currents in internally perfused neurons of the snail Lymnaea stagnalis.

The suction pipet method of intracellular dialysis and voltage clamp of cells has proven extremely useful in analysing the electrical properties of cells too small for the application of conventional microelectrode techniques and in larger cells for studying the effects of alterations in the internal ionic composition. Using neurons of the snail Lymnaea stagnalis, we have analysed several problems involved in the latter application of this technique and present several solutions to them. One major problem centers around the degree of control over the ionic composition of the cytoplasm achieved by altering the pipet solution. Using ion-sensitive microelectrodes during internal dialysis, we found that the efficiency of exchange between pipet and cytoplasm was much poorer for highly buffered ions such as H+ and Ca2+, than for K+, for example. Special precautions are described that can help this situation. The second problem involves the study of the effects of low internal pH on ion-channel properties. We summarize evidence for a specific voltage-dependent hydrogen ion channel, current through which becomes prominent at low internal pH. We analyse how the presence of this heretofore unrecognized current can seriously confuse the results of experiments designed to study the effects of low internal pH on other voltage-dependent currents.

Membrane currents of internally perfused neurones of the snail, Lymnaea stagnalis, at low intracellular pH.

The effects of low intracellular pH (pHi) on the membrane currents of snail neurone somata were studied using the internal perfusion and ion-sensitive micro-electrode techniques. Recordings with pH-sensitive micro-electrodes made while the pH of the perfusion solution was changed between 7.3 and 6.3 indicated that only with high buffer concentrations (100 mM) could pHi be changed effectively. H+ was slower to exchange into the cytoplasm than an unbuffered ion such as K+. When pHi was decreased to 5.9, large outward H+ currents could be recorded at voltages positive to -30 mV. The time course and amplitude of these currents were such that they did not affect the measurement of the peak amplitude of the fast transient K+ current (A-current), but severely contaminated both Ca2+ and delayed K+ current measurements. Low pHi blocked the A-current. The titration curve was consistent with the binding of two H ions to a site with a pK of 6.05 to block the channel. Low pHi appeared to block the slow inactivation of the delayed outward current without greatly changing its peak amplitude. However, when correction was made for the increase of H+ current at low pHi, the effect of internal H+ was found to be a block of the delayed K+ current with no consistent effect on inactivation. The Ca2+ current was also decreased at low pHi, but we were unable to determine whether this was a direct effect of pHi or secondary to a rise in internal free [Ca2+]. If no correction was made for H+ currents, the block of the Ca2+ current appeared greater and more reversible than it actually was. We conclude that under certain conditions, such as low pHi, the H+ current is a significant fraction of the total outward current in snail neurones, and may also be in a variety of other cells. The H+ currents must be accounted for under such conditions in order to study accurately the properties of K+ and Ca2+ currents.

Intracellular factors for the maintenance of calcium currents in perfused neurones from the snail, Lymnaea stagnalis.

Isolated nerve cell bodies from Lymnaea stagnalis were internally perfused and voltage-clamped. The magnitude of the Ca2+ current was monitored while perfusing with various intracellular solutions. When the intracellular perfusate was unenriched (containing only inorganic ions, 100 mM-HEPES and 5 mM-EGTA), the Ca2+ current was found to 'wash out', falling to half of its maximum value approximately 30-40 min from the beginning of perfusion. Stopping the flow of the perfusing solution increased this half-time to more than 50 min. The current-voltage relationship changed only slightly during wash-out. The addition of 2 mM-ATP and 1 mM-Mg2+ to the internal perfusate prevented, and even reversed, wash-out of the Ca2+ current. Both ATP and Mg2+ were necessary for maximal effect. Such current loss as occurred in the presence of ATP and Mg2+ was associated with a decrease in the capacitance of the cell and probably resulted from membrane being pulled into the pipette. The rate of inactivation of the Ca2+ current increased during perfusion with an unenriched internal solution, but decreased to initial values when ATP and Mg2+ were added to the internal perfusate. Although intracellular Mg2+ was necessary for the prevention of wash-out, levels higher than 1 mM had a blocking effect on the Ca2+ current. Certain factors that promote cyclic AMP-dependent protein phosphorylation (internal: cyclic AMP, theophylline and catalytic subunit of cyclic AMP-dependent protein kinase; external: dibutyryl cyclic AMP, 8-bromo cyclic AMP and forskolin) had no effect on the magnitude of the Ca2+ current in cells perfused with ATP and Mg2+. Externally applied theophylline blocked the Ca2+ current. The mechanism through which ATP and Mg2+ act to prevent wash-out of the Ca2+ current may be to enhance the ability of the cell to lower the Ca2+ concentration near the inner surface of the plasma membrane. This would prevent both the reversible block of Ca2+ current by intracellular Ca2+ and an irreversible loss of current due to high levels of intracellular Ca2+.

Permeation and interaction of divalent cations in calcium channels of snail neurons.

We have studied the current-carrying ability and blocking action of various divalent cations in the Ca channel of Lymnaea stagnalis neurons. Changing the concentration or species of the permeant divalent cation shifts the voltage dependence of activation of the Ca channel current in a manner that is consistent with the action of the divalent cation on an external surface potential. Increasing the concentration of the permeant cation from 1 to 30 mM produces a twofold increase in the maximum Ca current and a fourfold increase in the maximum Ba current; the maximum Ba current is twice the size of the maximum Ca current for 10 mM bulk concentration. Correcting for the changing surface potential seen by the gating mechanism, the current-concentration relation is almost linear for Ba2+, and shows only moderate saturation for Ca2+; also, Ca2+, Ba2+, and Sr2+ are found to pass through the channel almost equally well. These conclusions are obtained for either of two assumptions: that the mouth of the channel sees (a) all or (b) none of the surface potential seen by the gating mechanism. Cd2+ blocks Lymnaea and Helix Ca channels at concentrations 200 times smaller than those required for Co2+ or Ni2+. Ca2+ competes with Cd2+ for the blocking site; Ba2+ binds less strongly than Ca2+ to this site. Mixtures of Ca2+ and Ba2+ produce an anomalous mole fraction effect on the Ca channel current. After correction for the changing surface potential (using either assumption), the anomalous mole fraction effect is even more prominent, which suggests that Ba2+ blocks Ca current more than Ca2+ blocks Ba current.

Metabolic abnormalities in the cancer patient.

Many malnourished patients with cancer fail to gain weight with what appears to be adequate nutritional support. Metabolic abnormalities resulting from remote effects of the tumor on host metabolism have been postulated to increase energy requirements in such cancer patients. In the current study, 44 patients with lung cancer who had significant weight loss (16 +/- 2% of usual body weight) were studied under metabolic ward conditions. Whole body glucose production rates were significantly elevated in cancer patients compared to age-matched healthy controls. Blood glucose levels 2 hours after a standard oral glucose challenge were also significantly increased, but insulin levels were not different at this time. Fasting glucose and insulin levels were not different. Fasting plasma alanine levels were significantly decreased in these patients, while branched-chain amino acids were not different. Increased alanine flux for gluconeogenesis is likely to reflect a basic metabolic abnormality in patients with cancer and could be explained on the basis of a resistance to insulin action in such patients.

Intracellular calcium ions and calcium currents in perfused neurones of the snail, Lymnaea stagnalis.

Neuronal somata of Lymnaea stagnalis were internally perfused and voltage clamped using the suction pipette method. The cells were exposed to internal solutions buffered to various concentrations of Ca2+ while the cytoplasmic Ca2+ activity [( Ca2+]i) was monitored with a Ca2+ -sensitive micro-electrode. [Ca2+]i was usually about 10(-7) M when the cell was perfused with a solution buffered to any level of Ca2+ from 9 X 10(-7) to below 10(-8) M. With internal solutions buffered to 10(-6) M-Ca2+ or greater, [Ca2+]i increased rapidly and overshot the perfusate Ca2+ activity by up to two orders of magnitude. It was thus virtually impossible to hold [Ca2+]i steady at any levels other than about 10(-7) M or 10(-4) M using internal perfusion of simple ionic internal solutions. The excess Ca2+ which caused the overshoot of [Ca2+]i entered the cell from the external solution through Cd2+ -sensitive channels. Cd2+ in the external solution prevented or reversed the overshoot of [Ca2+]i and brought [Ca2+]i to near the perfusate level. ATP added to the internal solution also prevented [Ca2+]i from overshooting the perfusate level during perfusion with high-Ca2+ buffers. By monitoring [Ca2+]i with a Ca2+ -sensitive micro-electrode, we were able to estimate the relationship between [Ca2+]i and the Ca2+ current (ICa) measured under voltage clamp. ICa was completely blocked as [Ca2+]i was raised to 10(-6) M. We believe that the discrepancy between our data and other estimates of the ICa vs. [Ca2+]i relationship using internal perfusion of molluscan nerve cells results from the incorrect assumption that [Ca2+]i is controlled adequately during internal perfusion.

Rapidly activating hydrogen ion currents in perfused neurones of the snail, Lymnaea stagnalis.

Cells from the circumoesophageal nerve ring of the pond snail Lymnaea stagnalis were internally perfused with solutions containing Cs aspartate, EGTA and pH buffers. Time-dependent, voltage-dependent 'residual' outward currents were observed at positive potentials. They were found to be carried largely by H+. The outward H+ currents were reduced by high internal pH, low external pH, external Cd2+ and 4-aminopyridine. External tetraethylammonium ions reduced the H+ currents but had a more effective blocking action on the K+ currents in these cells. All five agents reduced the maximum H+ conductance. In addition Cd2+, low external pH and high internal pH were found to shift the voltage dependence of the H+ current to more positive potentials. There was no significant difference between H+ currents recorded with the internal pCa2+ about 7 and those recorded with the internal pCa2+ near 5. It is likely that the H+ channel described here provides the basis for the increase in H+ permeability described by Thomas & Meech (1982) in depolarized Helix neurones. As judged by their sensitivity to different antagonists, H+ channels are unlike any other previously described channel. They are highly selective for protons and we suggest that their role in molluscan neurones is to compensate for the rapid intracellular acidification which is generated by trains of action potentials (Ahmed & Connor, 1980).

Calcium current activation kinetics in neurones of the snail Lymnaea stagnalis.

Both the activation kinetics and the magnitude of the Ca current in Lymnaea are strongly dependent on temperature. The Q10 for the reciprocal of the activation time constant is 4.9 +/- 0.2 and the Q10 for the maximum current is 2.3 +/- 0.1. By lowering the temperature to 7-10 degrees C, we have been able to resolve the Ca tail currents. The block of Ca current by Cd2+ is voltage dependent, being more effective at more positive potentials. As determined from the magnitude of the tail currents, the Ca permeability is not maximally activated until the membrane potential is greater than +70 mV. The Ca permeability is half activated in the range 30-35 mV. The open-channel current-voltage relation for the Ca current is in rough agreement with the prediction of the constant-field equation. There is no indication of current saturation at negative potentials for potentials down to -60 mV. The Ca tail current decays with at least two time constants, one 200-400 microseconds and the other 2-4 ms. Although these time constants are not strongly voltage dependent, the ratio of the amplitude of the fast component of the tail current to that of the slow component is much larger at -60 mV than at 0 mV. The time course of the Ba tail current is very similar to that of the Ca tail current. The time course of the activation of the Ca current follows m2 kinetics and does not show evidence for a Cole-Moore-type shift for holding potentials between -50 and -110 mV. During a second positive pulse applied 1 ms after the first, the Ca current activates more rapidly, without the delay characteristic of the Ca current of a single positive pulse. The activation of the Ca current can be represented by a linear sequential model. The simplest model that describes both the turn-on and the turn-off of the Ca current must have at least three closed states, followed by a single open state.