Showing papers by "Erwin Neher published in 1992"

Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells

Abstract: In synapses, a rise in presynaptic intracellular calcium leads to secretory vesicle fusion in less than a millisecond, as indicated by the short delay from excitation to postsynaptic signal. In nonsynaptic secretory cells, studies at high time resolution have been limited by the lack of a detector as fast and sensitive as the postsynaptic membrane. Electrochemical methods may be sensitive enough to detect catecholamines released from single vesicles. Here, we show that under voltage-clamp conditions, stochastically occurring signals can be recorded from adrenal chromaffin cells using a carbon-fibre electrode as an electrochemical detector. These signals obey statistics characteristic for quantal release; however, in contrast to neuronal transmitter release, secretion occurs with a significant delay after short step depolarizations. Furthermore, we identify a pedestal or 'foot' at the onset of unitary events which may represent the slow leak of catecholamine molecules out of a narrow 'fusion pore' before the pore dilates for complete exocytosis.

Calcium gradients and buffers in bovine chromaffin cells

Abstract: 1. Digital imaging and photometry were used in conjunction with the fluorescent Ca2+ indicator, Fura-2, to examine intracellular Ca2+ signals produced by depolarization of single adrenal chromaffin cells. 2. Depolarization with a patch pipette produced radial gradients of Ca2+ within the cell, with Ca2+ concentration highest in the vicinity of the plasma membrane. These gradients dissipated within a few hundred milliseconds when the voltage-gated Ca2+ channels were closed. 3. Dialysis of Fura-2 into the chromaffin cell caused concentration-dependent changes in the depolarization-induced Ca2+ signal, decreasing its magnitude and slowing its recovery time course. These changes were used to estimate the properties of the endogenous cytoplasmic Ca2+ buffer with which Fura-2 competes for Ca2+. 4. The spatially averaged Fura-2 signal was well described by a model assuming fast competition between Fura-2 and an endogenous buffer on a millisecond time scale. Retrieval of calcium by pumps and slow buffers occurs on a seconds-long time scale. No temporal changes indicative of buffers with intermediate kinetics could be detected. 5. Two independent estimates of the capacity of the fast endogenous Ca2+ buffer suggest that 98-99% of the Ca2+ entering the cell normally is taken up by this buffer. This buffer appears to be immobile, because it does not wash out of the cell during dialysis. It has a low affinity for Ca2+ ions, because it does not saturate with 1 microM-Ca2+ inside the cell. 6. The low capacity, affinity and mobility of the endogenous Ca2+ buffer makes it possible for relatively small amounts of exogenous Ca2+ buffers, such as Fura-2, to exert a significant influence on the characteristics of the Ca2+ concentration signal as measured by fluorescence ratios. On the other hand, even at moderate Fura-2 concentrations (0.4 mM) Fura-2 will dominate over the endogenous buffers. Under these conditions radiometric Ca2+ concentration signals are largely attenuated, but absolute fluorescence changes (at 390 nm) accurately reflect calcium fluxes.

Calcium requirements for secretion in bovine chromaffin cells.

Abstract: 1. Measurements of membrane capacitance and intracellular Ca2+ concentration, [Ca2+]i, were used to examine the Ca2+ dependence of secretion in single adrenal chromaffin cells. 2. Intracellular dialysis of Ca2+, through a patch pipette, promoted secretion; the rate of secretion increased monotonically as [Ca2+]i was elevated, while the total amount of secretion reached a maximum at 1.5 microM-Ca2+ and declined at high [Ca2+]i. 3. Release of Ca2+ from internal stores, using bradykinin or ionomycin, transiently elevated [Ca2+]i and the rate of secretion. 4. Considering responses to both Ca2+ dialysis and release from internal stores, it appears that the rate of secretion increases over a range of [Ca2+]i levels above 0.2 microM and saturates at concentrations greater than 10 microM, if at all. Secretion appears to have a Hill coefficient for Ca2+ of about 2. At [Ca2+]i greater than 1-2 microM, prolonged elevation of [Ca2+]i, via dialysis, produced lower rates of secretion than transient elevation of [Ca2+]i caused by release from internal stores. This may have been caused by a depletion of readily releasable chromaffin granules during prolonged elevation of [Ca2+]i. 5. Brief depolarizing pulses produced transient rises in both [Ca2+]i and the rate of secretion. The ability of these pulses to evoke secretion 'washed out' during prolonged intracellular dialysis, due to both reduced Ca2+ influx and a diminished ability of the cell to secrete in response to a given Ca2+ load. 6. The kinetics of the secretory response depended upon the size of the depolarization-induced Ca2+ load; small rises in [Ca2+]i increased membrane capacitance only during the depolarization, while larger rises in [Ca2+]i produced increases both during and following the depolarization. The secretory responses that outlasted the depolarization appeared to be due to persistent elevation of [Ca2+]i. Secretory responses were sometimes followed by a slower decline in membrane capacitance, probably due to endocytosis of membrane. 7. Comparison of the rates of secretion measured during depolarization to those produced by Ca2+ dialysis or release from internal stores suggests that [Ca2+]i at secretory sites can exceed 10 microM during depolarization. The spatially averaged measurements of [Ca2+]i indicate much smaller levels of [Ca2+]i; thus, there must be pronounced spatial gradients of [Ca2+]i during depolarization.

Dihydropyridine-sensitive and omega-conotoxin-sensitive calcium channels in a mammalian neuroblastoma-glioma cell line.

Abstract: 1. Pharmacological and kinetic properties of high-voltage-activated (HVA) Ca2+ channel currents were studied using the whole-cell and perforated patch-clamp methods in a mouse neuroblastoma and rat glioma hybrid cell line, NG108-15, differentiated by dibutyryl cyclic AMP or by prostaglandin E1 and theophylline. 2. The HVA currents were separated into two components by use of two organic Ca2+ channel antagonists, omega-conotoxin GVIA (omega CgTX) and a dihydropyridine (DHP) compound, nifedipine. One current component, IDHP, was blocked by nifedipine (Kd = 8.2 nM) and was resistant to omega CgTX. Conversely, the other component, I omega CgTX, was irreversibly blocked by omega CgTX and was resistant to DHPs. Thus, IDHP could be studied in isolation by a short application of omega CgTX, while I omega CgTX could be studied in the presence of nifedipine. 3. The voltage for half-activation of IDHP was smaller than that of I omega CgTX by 13 mV. IDHP was activated at potentials that were subthreshold for voltage-dependent K+ currents of the cell, whereas I omega CgTX was not. 4. Time courses of activation and deactivation of IDHP were faster than those of I omega CgTX. 5. Voltage-dependent inactivation was small for both IDHP and I omega CgTX at any potential. 6. Ca(2+)-dependent inactivation of IDHP was faster and more prominent than that of I omega CgTX. The time course of the Ca(2+)-dependent inactivation of IDHP, but not I omega CgTX, was slowed as the membrane potential was made more positive between -20 and 30 mV, although amplitude of the current was increased. 7. Alkaline earth metal ions carried the two components of IHVA in the same order: Ba2+ greater than Sr2+ greater than Ca2+. 8. Metal ions blocked the two components of IHVA in the same order of potency: Gd3+ greater than La3+ greater than Cd2+ greater than Cu2+ greater than Mn2+ greater than Ni2+. 9. An alkylating agent, N-ethylmaleimide (NEM, 0.1 mM), selectively augmented IDHP by 30%. 10. During the course of cellular differentiation induced by dibutyryl cyclic AMP, IDHP appeared earlier than I omega CgTX. 11. These results indicate that two classes of Ca2+ channels contribute to the HVA currents of this cell line. The DHP-sensitive channel is more apt to generate Ca2+ spikes and Ca2+ plateau potentials than the omega CgTX-sensitive channel.

Volume-sensitive chloride conductance in bovine chromaffin cell membrane.

Abstract: 1. Bovine chromaffin cells were inflated by pressure applied through a pipette or swollen during intracellular perfusion with hypertonic solutions. Effects of such procedures on electrical properties of the membrane were studied by a combination of the tight-seal whole-cell patch-clamp technique and Fura-2 fluorescence measurements of free intracellular calcium concentration ([Ca2+]i). 2. Application of air pressure (about +5 cmH2O or 490 Pa) through the patch pipette caused an increase in the cell volume and concomitant development of an inwardly directed transient current at the holding potential of -60 mV. The current gradually increased to a peak value and subsequently decayed almost to its initial level within 5-10 min. A short pulse of pressure (5-10 s) was sufficient to elicit the whole sequence of events. 3. Intracellular free Ca2+ ion concentration, [Ca2+]i, steeply increased at the beginning of the pressure pulse to about 0.2 microM and either stayed at this level or decayed back to the more usual value of approximately 0.1 microM. 4. Similar changes in the transmembrane current and [Ca2+]i were observed during intracellular perfusion of cells with hypertonic solutions (30-50 mosM difference relative to the bath solution) or during extracellular application of hypotonic solution. 5. Swelling of non-perfused cells by extracellular application of hyposmotic solution caused the appearance of inward currents in cell-attached membrane patches held at a fixed potential -30 mV relative to the cell's resting potential. The kinetics of the current resembled those of the whole-cell current. 6. Intracellular introduction of guanosine triphosphate (GTP, 300 microM) significantly prolonged the duration (from 62 +/- 10 s, n = 5, to 98 +/- 8 s, n = 4, when measured at the level of half-amplitude), while introduction of the non-hydrolysable analogue of guanosine diphosphate (GDP), guanosine 5'-O-(2-thiodiphosphate) (GDP beta S, 300 microM), decreased the maximal rate of increase (from 11.4 +/- 2.6 pA/s, n = 6, to 3.2 +/- 2.1 pA/s, n = 10) of the current activated by pressure. 7. Lowering of the intracellular free Ca2+ ion concentration by introduction of 10 mM-EGTA did not significantly affect the current amplitude or time course. However, a rapid increase in the [Ca2+]i to micromolar levels (by activation of the voltage-operated calcium channels during membrane depolarization) could terminate development of the current activated by pressure and cause its fast decay to zero-current level.(ABSTRACT TRUNCATED AT 400 WORDS)

Patch clamp techniques: an overview.

Abstract: Publisher Summary This chapter provides an overview of the patch clamp techniques. The patch clamp technique provides the experimental means for merging the tools of modern molecular and cellular biology with those of electrophysiology. Using various recording configurations, it is possible to dissect the mechanisms of channel modulation. In cell-attached recording, modulation of channel activity in response to bath-applied agonist generally indicates a second-messenger mechanism. Candidate messengers can be tested directly on excised patches or in whole-cell recording. Current research on signaling pathways seeks to establish the functionally meaningful mechanisms through selective activation or inhibition of a portion of the pathway. A variety of patch clamp configurations, combined with single-channel resolution, provides a powerful experimental approach from the molecular level, in which channel genes are altered and expressed, to the cellular level, in which posttranslational signaling mechanisms are elucidated, to the systems level, in which cellular interactions in intact or slice preparations are revealed.

Ca(2+)-activated K+ channels modulate muscarinic secretion in cat chromaffin cells.

Abstract: 1. This study was aimed at testing the hypothesis that Ca(2+)-dependent K+ channels regulate the release of catecholamines mediated by muscarinic stimulation of cat adrenal chromaffin cells. Two parameters were measured: the secretory response to brief pulses of methacholine (100 microM for 10 s) in intact cat adrenal glands perfused at a high rate with oxygenated Krebs solution; and the changes in cytosolic Ca2+ concentrations, [Ca2+]i, produced by puff applications of methacholine pulses (also 100 microM for 10 s) in isolated single cat adrenal chromaffin cells loaded with Fura-2. 2. A pulse of methacholine released 805 +/- 164 ng of catecholamines (mean of thirty-two pulses). d-Tubocurarine (DTC) increased the secretory response in a concentration-dependent manner. The maximum increase (around 1000 ng catecholamines over control values) was reached at 100 microM-DTC and the EC50 was around 10 microM. 3. The secretory responses to methacholine alone, or to the combination of methacholine plus DTC, were strongly dependent on the extracellular Ca2+ concentration, [Ca2+]o. Thus Ca2+o removal from the perfusing solution for 5-10 min abolished catecholamine release. 4. At 0.1 microM, isradipine (an L-type Ca2+ channel blocker) inhibited by 71% the secretory response to DTC plus methacholine. At 1 microM, Bay K 8644 (an L-type Ca2+ channel activator) increased 2-fold the secretory response to DTC plus methacholine (2746 ng of catecholamines). 5. Apamin (1 microM) increased 3.5-fold the secretory response to methacholine pulses (from 500 to 1800 ng of catecholamines). 6. Methacholine pulses enhanced [Ca2+]i from the resting level of 100 nM to a peak of 1000 nM which quickly declined to basal level. DTC (100 microM) enhanced by 20% the [Ca2+]i peak and substantially prolonged its duration. 7. Apamin (1 microM) increased by 60% the [Ca2+]i peak evoked by methacholine, and delayed the initiation of decline of the [Ca2+]i peak. 8. These results are compatible with the idea that muscarinic stimulation depolarizes the cat adrenal chromaffin cell through an unidentified mechanism. Depolarization is probably counteracted by activation of Ca2+i-dependent K+ channels. Therefore, inhibition of these channels enhances depolarization and firing of action potentials which activate voltage-dependent L-type Ca2+ channels to increase further the Ca2+i signal and the secretory response. Thus Ca2+i-dependent K+ channels, probably of the small-conductance type (SK), seem to be involved in the modulation of muscarinic-evoked catecholamine release responses in cat adrenal chromaffin cells.

Ion Channels for Communication Between and Within Cells (Nobel Lecture)

Abstract: Erwin Neher Max-Planck-lnstitut fur biophysikalische Chemie D-3400 Gottingen Germany Around 1970 the fundamental signal mechanisms for communication between cells of the nervous system were known. Hodgkin and Huxley(1952) had provided the basis for understanding the nerve action poten- tial. The concept of chemical transmission at synapses had received its experimental verification by detailed studies on excitatory and inhibitory postsynaptic po- tentials (see Katz, 1966, for a concise description of the electrical signals in nerve and muscle). The ques- tion of the molecular mechanisms underlying these signals was still open, however. Hodgkin and Huxley (1952) used the concept of voltage-operated gates for a formal description of conductance changes, and by 1970 the terms Nat channel and K’channel were used frequently (see review by Hille, 1970), although no direct evidence for the existence of channels was availablefrom biological preparations.Thiswasdiffer- ent for the case of artificial membranes. Miller and Rudin (1963) introduced “black-lipid membranes” as experimental model systems, which in many respects resemble the bimolecular lipid membrane of living cells. These membranes are rather good insulators. However, when they are doped with certain antibiot- ics or proteins they become electrically conductive. R. C. Bean et al. (1969) and Hladky and Haydon (1970) showed that some of these dopants induce discrete, step-like changes in conductance when they are added in trace amounts. All the evidence suggested that the conductance changes observed represent the insertion of single pore-like structures into the mem- branes. In biological membranes similar measurements were not possible at the time, since the methods avail- able for recording currents in living cells typically had background noise levels higher by about a factor of a hundred than the “single-channel currents” ob- served in bilayers (see Figure 1). Indirect methods, however, provided strong evidence that channels similar in conductance to those in artificial mem- branes should be operative in nerve and muscle cells. Early attempts to count the number of Na’ channels by tetrodotoxin binding indicated that the contribu- tion of a single channel to Na+ conductance might be as much as 500 pS. Later, the technique of noise analysis (Katz and Miledi, 1972; Neher and Stevens, 1977) provided more accurate numbers. Anderson and Stevens (1973) estimated the conductance contri- bution of single acetylcholine-activated channels

Nobel lecture. Ion channels for communication between and within cells.

Abstract: Around 1970 the fundamental signal mechanisms for communication between cells of the nervous system were known. Hodgkin and Huxley (1952) had provided the basis for understanding the nerve action potential. The concept of chemical transmission at synapses had received its experimental verification by detailed studies on excitatory and inhibitory postsynaptic potentials (see Katz, 1966, for a concise description of the electrical signals in nerve and muscle). The question of the molecular mechanisms underlying these signals was still open, however. Hodgkin and Huxley (1952) used the concept of voltage-operated gates for a formal description of conductance changes, and by 1970 the terms Na-channel and K-channel were used frequently (see review by Hille, 1970), although no direct evidence for the existence of channels was available from biological preparations. This was different for the case of artificial membranes. Miiller and Rudin (1963) introduced \"black-lipid membranes\" as experimental model systems, which in many respects resemble the bimolecular lipid membrane of living cells. These membranes are rather good insulators. However, when they are doped with certain antibiotics or proteins they become electrically conductive. Bean et al. (1969) and Hladky and Haydon (1970) showed that some of these dopants induce discrete, steplike changes in conductance when they are added in trace amounts. All the evidence suggested that the conductance changes observed represent the insertion of single pore-like structures into the membranes.