Serotonin-induced changes in the activity of single Ca2+ channels inHelix pomatia neurons

The influence of the neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) on single Ca²⁺ channel activity was studied on unidentified neurons of the snailHelix pomatia. Only one type of Ca²⁺ channels with the unitary conductance of 5 pS was identified using 100 mM Ca²⁺ in the patch pippette under patch-clamp in a cell-attached configuration. The amplitude histogram showed only one peak with the mean value of 0.5 pA at the testing potential of –30 mV. The distribution of channel open times monotonically declined with the mean time constant of 0.2 msec. The distribution of channel closed times could be fitted by a double-exponential curve with time constants of 1 and 12 msec. The study of the effect of 5-HT on Ca²⁺ single channel activity showed that 5-HT influenced the channel molecule indirectly, as the transmitter could exert its effect by being added to the bath solution, which did not come into contact with the tested membrane fragment under the micropipette tip. 5-HT prolonged the mean channel open time (up to 0.3 msec) and proportionally decreased both channel closed time constants to 0.4 and 6.0 msec, respectively. A conclusion is made that enhancement of Ca²⁺ macrocurrent by 5-HT is determined by three factors: (i) changes in kinetics of aiready existing channels, (ii) an increase in the number of active channels of the same type, and (iii) an increase in probability of a channel being open. At the same time, the unitary channel conductance was not affected by the transmitter.Neirofiziologiya/Neurophysiology, Vol. 28, No. 2/3, pp. 132–140, March–June, 1996.     

Electrical connection between two bursting neurons inHelix pomatia

In some preparations of the CNS ofHelix pomatia, two neurons with bursting activity may be present in the right parietal ganglion, where usually there is only one bursting neuron RPal. If electrical activity of these neurons is recorded simultaneously, fluctuations of membrane potential are almost completely synchronized. Artificial depolarization and hyperpolarization of the membrane of one neuron caused depolarization or hyperpolarization of the other neuron. During long-term recording of the activity of both neurons synchronous modulation of their bursting activity was observed. Modulating factor (a peptide fraction obtained from the water-soluble part of snail brain homogenate) led to potentiation of the bursting activity of both neurons. It is concluded from the results of these experiments that two bursting RPal neurons, connected electrically with one another, may exist in the snail nervous system. In cases when the parameters of pacemaker activity of these two neurons are closely similar, electrical connection guarantees synchronization of their bursting activity and ensures a common frequency of changes in their membrane potential.     

Action of lead on glutamate-activated chloride currents inHelix pomatia L. neurons

Summary 1. In molluscan neurons glutamate may, on different neurons, evoke either excitation or inhibition. We studied neurons ofHelix pomatia which have hyperpolarizing responses to glutamate and determined the effects of lead on these responses.2. In voltage clamp experiments, the reversal potentials of these glutamate responses indicate that they are due to a conductance increase to chloride ions. Further evidence for this conclusion was obtained by the demonstration that responses to glutamate remained unaffected in experiments with intracellular dialysis with K-free saline in the presence of Na- and K-free extracellular media. In these circumstances, there is effectively no other ion than chloride to carry the current. In isolated neurons the glutamate-evoked chloride current is concentration dependent between 25 and 2500 µM. The current rises over 200 msec and declines in the continued presence of glutamate over a period of about 3 sec.3. Lead (0.5–1.0 µM) potentiated the glutamate-evoked chloride current provided that the channels were not maximally activated. The potentiation was greater if lead was added 30–60 sec before glutamate application.4. These results suggest that potentiation of transmitter-evoked responses by lead must be considered as yet another possible site of action of lead on neurons, and thus this effect must be considered as a part of the mechanism responsible for the neurotoxicity of this heavy metal.     

Properties of transient K+ currents and underlying single K+ channels in rat olfactory receptor neurons

The transient potassium current, IK(t), of enzymatically dissociated rat olfactory receptor neurons was studied using patch-clamp techniques. Upon depolarization from negative holding potentials, IK(t) activated rapidly and then inactivated with a time course described by the sum of two exponential components with time constants of 22.4 and 143 ms. Single-channel analysis revealed a further small component with a time constant of several seconds. Steady-state inactivation was complete at -20 mV and completely removed at -80 mV (midpoint -45 mV). Activation was significant at -40 mV and appeared to reach a maximum conductance at +40 mV (midpoint -13 mV). Deactivation was described by the sum of two voltage-dependent exponential components. Recovery from inactivation was extraordinarily slow (50 s at -100 mV) and the underlying processes appeared complex. IK(t) was reduced by 4-aminopyridine and tetraethylammonium applied externally. Increasing the external K+ concentration ([K+]o) from 5 to 25 mM partially removed IK(t) inactivation, usually without affecting activation kinetics. The elevated [K+]o also hyperpolarized the steady-state inactivation curve by 9 mV and significantly depolarized the voltage dependence of activation. Single transient K+ channels, with conductances of 17 and 26 pS, were observed in excised patches and often appeared to be localized into large clusters. These channels were similar to IK(t) in their kinetic, pharmacological, and voltage-dependent properties and their inactivation was also subject to modulation by [K+]o. The properties of IK(t) imply a role in action potential repolarization and suggest it may also be important in modulating spike parameters during neuronal burst firing. A simple method is also presented to correct for errors in the measurement of whole-cell resistance (Ro) that can result when patch-clamping very small cells. The analysis revealed a mean corrected Ro of 26 G omega for these cells.     

Mechanisms of up-regulation of single calcium channels by serotonin in Helix pomatia neurons

Action of serotonin (5-HT) on single Ca(2+) channel activity was studied in identified neurons of snail Helix pomatia. Only one type of Ca(2+) channels of 5 pS unitary conductance was determined under patch-clamp cell-attached mode. Kinetic analysis have shown a monotonically declining distribution of channel open times (OT) with mean time constant of 0.2 ms. The distribution of channel closed times (CT) could be fitted by double-exponential curve with time constants 1 and 12 ms. We established that 5-HT acts on Ca(2+) channel activity indirectly via cytoplasm. 5-HT prolonged the OT (up to 0.3 ms) and shortened the CT proportionally for both constants to 0.4 and 6 ms correspondingly. A conclusion is made that enhancement of Ca(2+) macro-current by 5-HT is determined by kinetic changes, increase of the number of active channels, and increase of the probability of OT. At the same time the transmitter did not affect the unitary channel conductance.     

Calcium and voltage dependence of single Ca2+-activated K+ channels from cultured hippocampal neurons of rat

Calcium and voltage dependence of the Ca2+-activated K+ channel, K(Ca), was studied at the single-channel level in cultured hippocampal neurons from rat. The K(Ca) channel has approx. 220 pS conductance in symmetrical 150 mM K+, and is gated both by voltage and by Ca2+ ions. For a fixed Ca2+ concentration at the inner membrane surface, [Ca]i, channel open probability, Po, increases e-fold for 14 mV positive change in membrane potential. At a fixed membrane potential (0 mV), channel activity is first observed at [Ca]i = 10(-6) M, and increases with Ca2+ concentration approximating an absorption isotherm with power 1.4. The [Ca]i required to half activate (Po = 0.5) the channel is 4.10(-6) M. When compared to other preparations, the K(Ca) channel from hippocampal neurons reported here shows the lowest Ca2+ sensitivity and the highest voltage sensitivity. These findings are interpreted in evolutionary terms.     

Tandem-pore K+ channels mediate inhibition of orexin neurons by glucose

Glucose-inhibited neurons orchestrate behavior and metabolism according to body energy levels, but how glucose inhibits these cells is unknown. We studied glucose inhibition of orexin/hypocretin neurons, which promote wakefulness (their loss causes narcolepsy) and also regulate metabolism and reward. Here we demonstrate that their inhibition by glucose is mediated by ion channels not previously implicated in central or peripheral glucose sensing: tandem-pore K(+) (K(2P)) channels. Importantly, we show that this electrical mechanism is sufficiently sensitive to encode variations in glucose levels reflecting those occurring physiologically between normal meals. Moreover, we provide evidence that glucose acts at an extracellular site on orexin neurons, and this information is transmitted to the channels by an intracellular intermediary that is not ATP, Ca(2+), or glucose itself. These results reveal an unexpected energy-sensing pathway in neurons that regulate states of consciousness and energy balance.     

Slowly activating K+ channels in rat olfactory receptor neurons.

Patch-clamp techniques were used to investigate slowly activating, Ca(2+)-insensitive K+ channels of isolated rat olfactory receptor neurons. These channels had a unitary conductance of 135 pS and were only found in a small proportion (less than 5%) of membrane patches. Upon depolarization to voltages more positive than -50 mV, the channels activated gradually over a period of at least 10 s. When hyperpolarized to negative voltages, channel activity deactivated in a slow but voltage-dependent manner. These channels may underlie a slowly activating K+ current that is observed in approximately 30% of whole-cell recordings. Similar single channels have been reported in smooth muscle cells, but this is the first demonstration of these channels in any type of neuron. The channels may contribute to the spike frequency adaptation and post-stimulus hyperpolarization that are observed during the excitatory response to odorants. They may also contribute to cell repolarization following large odorant-stimulated receptor currents.     

Effect of high intracellular calcium ion concentration on the transmembrane current induced by iontophoretic injection of cAMP inHelix pomatia neurons

The effect of increasing the intracellular calcium ion concentration by various methods (iontophoretic injection into the cytoplasm, generation of a burst of action potentials, addition of uncouplers of oxidative phosphorylation to the external solution, causing release of calcium from mitochondria) on the inward current induced by injection of cAMP into the neuron (the cAMP current) was investigated on the neuron membrane ofHelix pomatia under voltage clamp conditions. In all cases an increase in the intracellular calcium ion concentration was found to lead to an increase in amplitude, and in many cases duration, of the cAMP current. It is suggested that membrane structures responsible for appearance of the cAMP current have two phosphorylation centers: cAMP-dependent and calcium-calmodulin-dependent. The possible role of this process in signal integration at the intraneuronal level is discussed.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 17, No. 1, pp. 78–84, January–February, 1985.     

Physiological role of dendrotoxin-sensitive K+ channels in the rat cerebellar Purkinje neurons

To understand the contribution of potassium (K+) channels, particularly alpha-dendrotoxin (D-type)-sensitive K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits), to the generation of neuronal spike output we must have detailed information of the functional role of these channels in the neuronal membrane. Conventional intracellular recording methods in current clamp mode were used to identify the role of alpha-dendrotoxin (alpha-DTX)-sensitive K+ channel currents in shaping the spike output and modulation of neuronal properties of cerebellar Purkinje neurons (PCs) in slices. Addition of alpha-DTX revealed that D-type K+ channels play an important role in the shaping of Purkinje neuronal firing behavior. Repetitive firing capability of PCs was increased following exposure to artificial cerebrospinal fluid (aCSF) containing alpha-DTX, so that in response to the injection of 0.6 nA depolarizing current pulse of 600 ms, the number of action potentials insignificantly increased from 15 in the presence of 4-AP to 29 action potentials per second after application of DTX following pretreatment with 4-AP. These results indicate that D-type K+ channels (Kv.1, Kv1.2 or Kv1.6 subunits) may contribute to the spike frequency adaptation in PCs. Our findings suggest that the activation of voltage-dependent K+ channels (D and A types) markedly affect the firing pattern of PCs.     

Trifluoperazine enhancement of Ca2+-dependent inactivation of L-type Ca2+ currents inHelix aspersa neurons

The effects of trifluoperazine hydrochloride (TFP), a calmodulin antagonist, on L-type Ca²⁺ currents (L-type ICa²⁺) and their Ca²⁺-dependent inactivation, were studied in identifiedHelix aspersa neurons, using two microelectrode voltage clamp. Changes in [Ca²⁺]_i were measured in unclamped fura-2 loaded neurons. Bath applied TFP produced a reversible and dose-dependent reduction in amplitude of L-type ICa²⁺ (IC₅₀=28 μM). Using a double-pulse protocol, we found that TFP enhances the efficacy of Ca²⁺-dependent inactivation of L-type ICa²⁺. Trifluoperazine sulfoxide (50 μM), a TFP derivative with low calmodulin-antagonist activity, did not have any effects on either amplitude or inactivation of L-type ICa²⁺. TFP (20 μM) increased basal [Ca²⁺]_i from 147±37 nM to 650±40nM (N=7). The increase in [Ca²⁺]_i was prevented by removal of external Ca²⁺ and curtailed by depletion of caffeine-sensitive intracellular Ca²⁺ stores. Since TFP may also block protein kinase C (PKC), we tested the effect of a PKC activator (12-O-tetradecanoyl-phorbol-13-acetate) on L-type Ca²⁺ currents. This compound produced an increase in L-type ICa²⁺ without enhancing Ca²⁺-dependent inactivation. The results show that 1) TFP reduces L-type ICa²⁺ while enhancing the efficacy of Ca²⁺-dependent inactivation. 2) TFP produces an increase in basal [Ca²⁺]_i which may contribute to the enhancement of Ca²⁺-dependent inactivation. 3) PKC up-regulates L-type ICa²⁺ without altering the efficacy of Ca²⁺ dependent inactivation. 4) The TFP effects cannot be attributed to its action as PKC blocker.     

新生大鼠下丘脑神经元L—Ca^2＋通道电流活动特征

目的研究新生大鼠下丘脑神经元L-Ca2+通道单通道特性;Ca2+通道激动剂BayK8644对Ca2+通道单通道特性的影响.方法采用神经元急性分离技术;用膜片钳细胞贴附式记录方式进行研究.结果大鼠下丘脑神经元L-Ca2+通道是一种电导相对较大的Ca2+通道,其电导为(29.5±3.1)pS,平均开放时间(τ0)为0.28ms,平均关闭时间的短关闭时间常数(τc1)为2.91ms,长关闭时间常数(τc2)为53.22ms.此通道几乎不存在时间依赖性失活.BayK8644显著增加通道的开放概率,通道平均开放时间增加为1.61ms.结论下丘脑神经元存在L-Ca2+通道,该通道具有明显电压依赖性,而无显著的时间依赖性.通道特征与文献报道的其它神经元上L-Ca2+通道相似,也有明显不同,显示下丘脑神经元L-Ca2+钙通道的独特性.     

Dose-dependent potentiation and inhibition of single Ca2+-activated K+ channels by flufenamic acid

Using the patch-clamp technique in an inside-out configuration, we studied the action of an antiinflammatory drug, flufenamic acid (FFA), on single large-conductance Ca2+-activated K+ channels in cultured Vero kidney cells. Depending on its concentration, FFA caused either potentiation or inhibition of K(Ca) channel activity of the same channel. Within the concentration ranges of about 5 to 10 microM and of 50 to 500 microM, FFA increased the channel activity; and within the intermediate range of about 10 to 50 microM, FFA inhibited the channels. The effects were only partially reversible. The activating phases were accompanied by an increase in the channel open time and decreases in the channel closed time and slope factor of the Ca2+ concentration-response curve. An apparent dissociation constant of Ca2+ interaction with the channel changed slightly. Possible mechanisms of the FFA effects are discussed.     

Effect of ionizing radiation on the functional state of Helix pomatia neurons. The systems of Na+ and K+ transport

The influence of ionizing radiation (5.16 C/kg) on passive and active Na+ and K+ transfer within the giant neurons of edible snail (Helix pomatia) has been investigated. It has been shown that ionizing radiation increases passive permeability of membranes, inhibits active ion transport, changes the number and the affinity of functionally active Na+,K+-ATPase molecules. The authors discuss the mechanisms of action of ionizing radiation.     

Recording of single K+ channels in the membrane of cytoplasmic drop ofChara australis

Summary The cytoplasmic drop formed of effused cytoplasm fromChara internodes is enclosed by a membrane. Patch clamp experiments have been carried out on this membrane, revealing a K⁺ channel as the most frequently detected ion translocator. The K⁺ channel is saturated at a level of about 20 pA inward and 10 pA outward current. The channel conductance is dependent on the accessability of K⁺ ions, its maximum value amounts to about 165 pS. The discrimination of Na⁺ and Cl^– is significant, permeability ratios P_Na/P_K and P_Cl/P_K were estimated to be 0.01 either. Binding experiments with the fluorescent probe concanavalin A/FITC suggest that the membrane is derived from the tonoplast.Abbreviations E_K K⁺ equilibrium potential - FITC fluorescein isothiocyanat - V_m membrane voltage - V_pip pipette clamp voltage - V_r reversal voltage     

不同温度条件对下丘脑神经元K+单离子通道簇状开放的影响

目的：探讨温度对下丘脑神经元电压依赖性K^ 单离子通道（Kv）簇状开放（Burst opening）的影响。方法：采用膜片钳细胞贴附式技术观察和记录32℃、37℃和39℃时下丘脑神经元Kv的活动。结果：温度升高,单位时间内记录到的Burst开放明显增多,平均开放间期变长。当温度从32℃升至39℃,B1从1．5ms升至8．1ms,B2从6．6ms升至83．2ms,Burst内部开放数目也由1－2个升至8个,从以简单Burst开放（SB）为主转变成以复杂Burst开放（CB）占优势。结论：温度升高,下丘脑神经元上IK通道更多地处于活动状态,这可能有利于下丘脑神经元对体温变化的调节。