Chloride channels activated by hyperpolarization in Aplysia neurones

The outside-out configuration of the patch-clamp technique was used in order to characterize the elementary events underlying the previously described hyperpolarization-activated Cl current of Aplysia neurones (1,2). A few membrane patches bathed with K-free internal and external solutions showed channels characterized by long duration (up to several seconds) bursts of activity which were clearly activated by hyperpolarization. I–V curves of elementary current obtained for three different values of the Cl equilibrium potential show that these channels are selective for Cl ions.During the bursts, the current fluctuates between three levels: the resting (closed) level, a level of maximum conductance (10–15 pS) and a half-conductance level. This behaviour is similar to that of the Cl channels extracted from Torpedo electroplaques (3).     

Three acetylcholine receptors in Aplysia neurones

1. In the pleural ganglion of Aplysia californica, groups of cells were identified that respond differently to an iontophoretic injection of ACh. The anterior neurones are excited by ACh, whereas the medial cells are inhibited. The inhibitory response is biphasic, consisting of a short-latency, rapid component and a longer-latency, slow component. Homologous responses (e.p.s.p.s in the anterior cells and a two-component inhibitory response in the medial cells) are evoked in these same cell groups by stimulation of an identifiable presynaptic neurone.2. The e.p.s.p. and the corresponding ACh potential are completely eliminated by hexamethonium which has no effect on either of the inhibitory potentials. Both the e.p.s.p. and the rapid i.p.s.p. (and the corresponding ACh potentials) are blocked by tubocurarine, dihydro-beta-erythroidine, strychnine and brucine. These drugs have no effect on the slow inhibitory potential, whether elicited synaptically or by ACh injection. The slow response can be selectively blocked by methylxylocholine, tetraethylammonium (TEA), and phenyltrimethylammonium (PTMA). Since the three types of potentials were found to be differentially affected by ACh antagonists, it was concluded that the various responses are due to activation of three different ACh receptors.3. Of the cholinomimetics tested, only carbamylcholine imitates all three actions of ACh. Nicotinic agents, which were shown to activate the two curare-sensitive receptors, have no stimulating effect on the curare-insensitive receptor. This latter receptor can be selectively stimulated by arecoline. The cholinomimetics were shown to have a secondary blocking effect on the receptor(s) they stimulate.4. Muscarine, even at high doses, is ineffective as either a stimulating or a blocking agent on any of the three receptor types. The muscarinelike drugs oxotremorine, methacholine, and pilocarpine have only weak and non-specific cholinomimetic action on these receptors. Their blocking effects are likewise negligible.5. The two curare-sensitive receptors, which are presumably the same as those described by Tauc & Gerschenfeld (1962), respond like vertebrate nicotinic receptors to both cholinomimetics and cholinolytics. The third receptor type, on the other hand, has a unique pharmacological profile. It is unaffected by both nicotine and muscarine, and is blocked neither by curare nor by atropine. Knowing that it can be stimulated by arecoline and blocked by methylxylocholine, TEA and PTMA does not facilitate its incorporation into the classical scheme of cholinergic receptors.     

Selective blocking action of tetraethylammonium ion on three types of acetylcholine receptors in Aplysia ganglion cells

The ganglion cells of Aplysia have three types of acetylcholine (ACh)-receptors in their membranes. The activation of one type produces Na+-dependent depolarization (DNa-type). The other receptors are HK- and HC1-types, the activation of which produces either K+- and Cl- dependent hyperpolarization, respectively. ACh-induced membrane current (IACh) was measured under the voltage clamp at different holding potential levels (V) of the membrane. The effects of tetraethylammonium (TEA) were analyzed quantitatively on each type of receptor activity by observing the changes in dose-inhibition curves and IACh-V relations obtained under voltage clamp. TEA exhibited a dose dependent block on both DNa- and HK-type of receptor activities but no effect on HCl-type. The mode of blocking action was found to be competitive in HK-type but noncompetitive in DNa-type. In the DNa-type receptor, 3 mM TEA shifted the reversal potential of ACh-induced current response from +5 to +40 mV, and caused a decrease in the slope of the IACh-V curve. In HK-type, TEA simply decreased the slope of IACh-V curve without changing the reversal potential of the ACh-induced response. It was concluded that TEA competed that with ACh for common binding sites at the receptor of the HK-type, but not of the DNa-type, and that in DNa-type, TEA made a binding with an allosteric site of the ACh-receptor and depressed the increase in membrane permeability toward Na+ as well as K+.     

Two types of acid-sensing ion channel currents in rat hippocampal neurons

Two types of acid-sensing ion channel (ASIC)-like currents in cultured rat hippocampal neurons were recorded and their characteristics were studied by using a whole-cell recording technique. The results revealed that the ASIC-like currents, induced by a quick drop of the extracellular pH, decayed with different time constants (τ) of 229 ± 16 (Type I) and 1209 ± 56 ms (Type II). The ASIC-like currents displayed different sensitivities to extracellular proton (pH_0.5 was 6.17 ± 0.04 for Type I and 5.70 ± 0.07 for Type II) and amiloride, a specific ASIC channel blocker (IC₅₀ was 1.19 ± 0.37 μM for Type I and 0.14 ± 0.02 μM for Type II). Among all the 360 recorded neurons, ASIC-like currents were induced in 314 neurons (87.2%). In the neurons expressing ASICs, Type I currents were evoked from 269 neurons (85.7%) and Type II currents were induced only from 45 neurons (14.3%). As these ASIC-like currents presented various electrophysiological and pharmacological properties, further experiments should be conducted to decipher the complex subunit composition of ASICs in the hippocampus.     

The effect of the tetraethylammonium ion on the delayed currents of frog skeletal muscle

1. A method which permitted control of the membrane potential near the end of a muscle fibre and measurement of an approximation of the membrane current was used to investigate the effects of the tetraethylammonium (TEA) ion on the delayed outward potassium current obtained on depolarizing.2. Assuming R(i) to be 250 Omega cm and a fibre diameter of 80 mu, the mean value for the maximum potassium conductance (g(K)) was 23.2 +/- 3.2 mmho.cm(-2).3. 58 mM-TEA, in two series of experiments, reduced g(K) by about 90%. A concentration-effect relation for TEA in its action on the delayed rectifier could be fitted by a curve for a drug-receptor complex assuming one molecule of TEA to combine reversibly with one receptor, and a dissociation constant of 8 x 10(-3)M.4. TEA tended to shift the threshold for delayed rectification to slightly more negative membrane potentials. TEA caused a similar shift in the relation between n(infinity) and membrane potential, but did not much alter the form of the relation.5. The relation between n(infinity) and membrane potential and between tau(n) (-1) and membrane potential were well fitted by the model of Adrian, Chandler & Hodgkin (1970a) assuming that the Q(10) was 2.5.6. TEA slowed the rate of onset of the delayed potassium currents, decreasing tau(n) (-1) (the reciprocal of the time constant of the fourth power function which described the current's development) by about 80%.7. The inactivation of the delayed current with time was shown to follow a complex time course. A fast phase decays with a time constant of 270 msec and a slow phase with a time constant of 2.3 sec at a membrane potential of + 10 mV.8. The fast phase of the delayed current is much more susceptible to the action of TEA than the slow phase, and these are interpreted in terms of different potassium channels. TEA has little effect on the time constant with which either the fast current or the slow current inactivates.     

Two distinct calcium-activated potassium currents in larval muscle fibres ofDrosophila melanogaster

The non-synaptic membrane currents of muscle fibres have been studied in late embryogenesis ofDrosophila melanogaster using the voltage-clamp technique in wild-type andShaker mutant third instar larvae. Five currents were found in the wild type muscle membrane at this embryonic stage: one fast inward Ca current (I_Ca), two fast outward K currents (I_A and I_Acd) and two slow outward K currents (I_K and I_C). I_Acd and I_C are Ca-dependent.Several procedures were used to separate I_Acd from I_A: I_Acd is present inShaker mutants which are characterized by the absence of I_A (Salkoff and Wyman 1981); I_Acd, but not I_A, is suppressed by Co²⁺ (10 mM) or La³⁺ (1 mM); I_Acd shows steady-state inactivation at more positive potentials than I_A; I_Acd, unlike I_A, is 3,4-diaminopyridine (3,4-DAP) resistant. Furthermore, tetraethylammonium (TEA, 20 mM) which is known to be uneffective on I_A, blocks I_Acd. I_Acd could not be triggered by using strontium or barium as calcium substitutes. By partial substitution of Ca by Ba or Sr ions, it was found that Ba, but not Sr, blocks the I_Acd channel.A non-inactivating, TEA sensitive, Ca-dependent K current (I_C), which gave N-shaped I-V plots, could be separated from I_K by using Ca-channel blockers. I_C and I_K activate at membrane potentials of about –25 mV and –10 mV, respectively.The participation of I_Acd and I_C to membrane electrophysiology is discussed.     

Kinetics of acetylcholine activated ion channels in chick ciliary ganglion neurones grown in tissue culture

The acetylcholine activated conductance of chick ciliary ganglion neurones grown in tissue culture was studied by the patch clamp method. Single channel currents at 30°C had a conductance of 38–42 pS, a reversal potential near +10 mV and an average open lifetime of 1.08 ms (range 0.74–1.54 ms) at the resting potential. The presence of a single component in the distributions of amplitudes and open lifetimes, and also in the noise spectrum of voltage clamp currents, suggests that acetylcholine channels have uniform characteristics in these cells. Evidence of a desensitised state of the receptor was obtained from the distribution of gap intervals and the decline of voltage clamp current. These properties are similar to those of acetylcholine channels at the vertebrate neuromuscular junction. However, two important differences were found. (a) The acetylcholine concentrations used here were 10–25 times higher than those required to produce a similar degree of channel activation at the endplate. (b) When the membrane was hyperpolarised the mean open lifetime of the channel showed no change or a slight reduction.     

Changes in the time course of miniature endplate currents induced by bath-applied acetylcholine

Bath application of 0.5 and 2 microM acetylcholine (ACh) slowed the decay phase of miniature endplate currents (MEPC) recorded in isolated, voltage-clamped and prostigmine-treated frog sartorius muscle. Washout of ACh led to a decrease of the decay time constant of the MEPC to 72 +/- 5% (n = 5) and 51 +/- 3% (n = 6) of initial values, respectively, followed by very slow and incomplete recovery. MEPC amplitude changed slightly and recovered relatively fast. This discrepancy in the recovery rates is suggested to be due to a 'trapping' ability of desensitized receptors which can compete with the free receptors for ACh molecules and prevent repetitive binding. Thus the high affinity of desensitized receptors to ACh may partially compensate the absence of acetylcholinesterase activity.     

Changes in membrane currents in bullfrog atrium produced by acetylcholine.

1. A double sucrose-gap voltage-clamp technique has been used to study the effects of acetylcholine on the membrane currents in atrial trabeculae of the bullfrog, Rana catesbeiana. 2. The second, or slow inward (Ca2+/Na+) current, was found to be markedly reduced by concentrations of acetylcholine greater than approximately 2-0 X 10(-8)M. The resulting decrease in net calcium entry provides a straightforward explanation for the negative inotropic action of acetylcholine in atrial muscle. 3. Measurements of membrane resistance near the resting potential showed that relatively high doses of acetylcholine (approximately 10(-7) M) decrease membrane resistance by about twofold. This effect is shown to be the result of an increase in a time-independent background current which appears to be carried mainly by potassium ions. 4. Using appropriate pharmacological techniques, it has been possible to demonstrate: (i) that the peak slow inward current is reduced to about half its initial value before any significant increase in background current occurs; (ii) that even when a sufficient dose of acetylcholine to produce an increase in background current is used, the background current shows inward-going rectification and cannot account for the observed reduction in the slow inward current. 5. No consistent change was observed in the degree of activation of the time-dependent outward membrane currents after application of concentrations of acetylcholine which produced large decreases in the peak slow inward current. 6. These results are discussed in relation to previous electro-physiological and radioisotope studies of the mechanism of the negative inotropic effect of acetylcholine in cardiac muscle.     

顺铂激活的低分化鼻咽癌细胞氯电流

目的： 研究抗癌药顺铂对低分化鼻咽癌细胞（CNE-2Z）氯通道的激活作用以及该电流的特性。方法：采用膜片钳技术记录顺铂激活的CNE-2Z细胞全细胞电流;离子置换法等方法分析通道的特性。结果：细胞外灌流5 μmol/L的顺铂诱发CNE-2Z细胞产生一个有明显外向优势的电流,电流的翻转电位为(-6.69 ± 0.51) mV,接近氯离子平衡电位(-0.9 mV)。该电流的激活依赖于细胞内ATP。顺铂激活的细胞膜氯通道对阴离子的通透性顺序为：I-≥Br->Cl->gluconate。氯通道阻断剂tamoxifen完全抑制该电流。结论：抗癌药顺铂可以激活一个电流特征与容积激活性氯通道相似的氯通道。     

Steep concentration dependence and fast desensitization of nicotinic channel currents elicited by acetylcholine pulses,studied in adult vertebrate muscle

Skeletal muscles of adult mice and frogs were dissociated enzymatically and prepared for patch-clamping within less than 6 h. Outside-out patches were superfused with repetitive pulses of acetylcholine (ACh) with switching times of about 0.2 ms. Peak responses were reached within 1 ms. In mouse muscle the average channel conductance was 65 pS and the average open time 1 ms (20° C). Between 1 and 10 M ACh, the peak responses increased proportional to the second to third power of the ACh concentration, and less steeply between 10 and 1000 M ACh. The apparent K _m of the dose-response curve was about 100 M. After the peak, channel opening probability declined with time constants decreasing from about 1 s with 1 M ACh to 15–50 ms with 1 mM ACh. After 100 ms desensitization the channel opening had decreased to less than 1/300 peak value. The rate of desensitization increased with rising temperature, with Q ₁₀ values of 1.7–2.5 between 10 and 30° C. The desensitization characteristics of channels from frog muscle were similar to that from mice. With pulses of 100 M ACh the channels opened with a probability of 0.55, the open probability declining with a time constant of about 60 ms and dropping to less than 0.001 after 300 ms. The results support the view that three binding steps of ACh are necessary for opening of the channel. Desensitization in the presence of high ACh concentrations is slower than the decay of synaptic currents.