Interaction of the antiepileptic drug lamotrigine with recombinant rat brain type IIA Na+ channels and with native Na+ channels in rat hippocampal neurones

Actions of the new antiepileptic drug lamotrigine (LTG, Lamictal) were characterised using recombinant rat brain type IIA Na⁺ channels expressed in Chinese hamster ovary (CHO) cells and native Na⁺ channels in rat hippocampal pyramidal neurones, using whole-cell recording and intracellular recording techniques. In CHO cells, LTG caused a tonic inhibition of Na⁺ currents in a concentration-dependent and voltage-dependent manner. The half-maximal inhibitory concentration (IC₅₀) of approximately 500 M was obtained at a holding potential (V _h) of –90 mV compared with an IC₅₀ of 100 M at a V _h of –60 mV. LTG (50 M) caused a 10–mV negative shift in the slow, steady-state inactivation curve and delayed considerably the recovery from inactivation, but had no significant effects on the voltage dependence of activation or fast inactivation, suggesting that LTG acts mainly on the slow inactivated state. The affinity for the inactivated channels was estimated at 12 M. The tonic inhibition was augmented by a use-dependent action in which a further inhibition by the drug developed during rapid repetitive stimulation using a train of 20-ms duration pulses (11 Hz). These results were consistent with the drug action being on firing properties of pyramidal neurones. Only in those epileptiform bursts which caused cumulative inactivation of Na⁺ spikes did LTG produce a potent inhibition. Our data suggest that the inactivated channel is a primary target for LTG action at therapeutic concentrations.     

The effects of nucleotides and potassium channel openers on the SUR2A/Kir6.2 complex K+ channel expressed in a mammalian cell line, HEK293T cells

 The effects of potassium channel opening drugs and intracellular nucleotides on the ATP-sensitive K+ (K_ATP) channel composed of SUR2A and Kir6.2 in HEK293T cells were examined using the patch-clamp technique. The SUR2A/Kir6.2 channel was activated effectively by pinacidil, marginally by nicorandil but not by diazoxide. The pinacidil-activated channel currents were inhibited by glibenclamide with a K _i value of 160 nM. Upon formation of inside-out (I-O) patches, spontaneous openings of the channels appeared, which were inhibited by intracellular ATP (ATP_i) equipotently in the presence and in the absence of intracellular Mg²⁺ (Mg²⁺ _i). The channel activity ran-down gradually in I-O patches. The run-down channels could be reactivated by ATP_i only in the presence of Mg²⁺ _i. Uridine 5’-diphosphate (UDP) antagonized the ATP_i-mediated inhibition of the channel activity before run-down. After run-down, UDP activated the channel without antagonizing ATP_i-mediated channel inhibition. Thus, the SUR2A/Kir6.2 reproduced the major properties of the native cardiac K_ATP channel well in terms of nucleotide regulation and pharmacology, and therefore can be a useful tool with which to elucidate the molecular mechanisms characterizing the K_ATP channel. Received: 24 October 1997 / Received after revision and accepted: 4 December 1997     

Electrophysiological and pharmacological properties of GluR1, a subunit of a glutamate receptor-channel expressed in Xenopus oocytes.

A cDNA clone encoding an excitatory amino acid receptor was isolated from a rat brain cDNA library by Hollmann et al. (Nature, 342 (1989) 643-648). In Xenopus oocytes, this clone, GluR1, expressed a functional receptor-channel activated by kainate (KA), domoate (D), glutamate and quisqualate (QA). The apparent affinity (EC50) for QA (0.1 microM) was higher than that for KA (50 microM). The maximal response to QA was about 1/10 of that to KA. QA inhibited the KA induced current. The N-methyl-D-aspartate (non-NMDA) receptor antagonist 6,7-dinitroquinoxaline-2,3 dione (DNQX) competitively blocked the effects of both agonists. Currents induced by KA, QA and D in oocytes expressing GluR1 showed identical voltage sensitivities. GluR1 and KA receptor-channels expressed from rat striatum poly(A)+ RNA showed the same ionic selectivity, being permeable mostly to Na+ and K+. The current-voltage relationships of GluR1 showed a strong inward rectification, whereas those of KA receptor-channels expressed from poly(A)+ RNA from various rat brain regions were more linear.     

Identification of a novel voltage-gated Na+ channel rNav1.5a in the rat hippocampal progenitor stem cell line HiB5

A conditionally immortalised cell line, HiB5, derived from embryonic hippocampal precursor cells expressed a voltage-gated Na+ channel with electrophysiological characteristics shifted to more negative voltages and a lower sensitivity to tetrodotoxin [concentration required for half-maximal inhibition (IC50) 0.9 microM] compared with endogenous neuronal Na+ channels. The channel activation and steady-state inactivation occurred at very negative potentials with the threshold for activation at -55 mV and half-maximal inactivation at -78.7 mV. The channel was blocked by lamotrigine and sipatrigine voltage and state dependently, with potencies 5-20 times higher (IC50 12 and 1.8 microM at -80 mV respectively) than the corresponding block of endogenous Na+ channels from neurones and cloned rNa(v)1.2a (rBIIA) alpha-subunits. Both compounds slowed the channel's recovery from inactivation. Whereas lamotrigine and sipatrigine had similar effects on the fast inactivated state, the effect of sipatrigine on the slow inactivation state was more pronounced, rendering this compound overall the more effective. The molecular subtype mainly expressed by HiB5 cells was identified using RT-PCR and was a novel splice variant of rNa(v)1.5 (rNa(v)1.5a). It differs from the known rNa(v)1.5 version in that it lacks 53 amino acids in the intracellular loop between domains II and III. rNa(v)1.5a was also detected in mRNA derived from rat whole brain.     

Contribution of N- and C-terminal channel domains to Kv channel interacting proteins in a mammalian cell line

Association of Shal gene-related voltage-gated potassium (Kv4) channels with cytoplasmic Kv channel interacting proteins (KChIPs) influences inactivation gating and surface expression. We investigated both functional and biochemical consequences of mutations in cytoplasmic N and C-terminal Kv4.2 domains to characterize structural determinants for KChIP interaction. We performed a lysine-scanning mutagenesis within the proximal 40 amino acid portion and a structure-based mutagenesis in the tetramerization 1 (T1) domain of Kv4.2. In addition, the cytoplasmic Kv4.2 C-terminus was truncated at various positions. Wild-type and mutant Kv4.2 channels were coexpressed with KChIP2 isoforms in mammalian cell lines. The KChIP2-induced modulation of Kv4.2 currents was studied with whole-cell patch clamp and the binding of KChIP2 isoforms to Kv4.2 channels with coimmunoprecipitation experiments. Our results define one major interaction site for KChIPs, including amino acids in the proximal N-terminus between residues 11 and 23, where binding and functional modulation are essentially equivalent. A further interaction site includes residues in the T1 domain. Notably, C-terminal deletions also had marked effects on KChIP2-dependent gating modulation and KChIP2 binding, revealing a previously unknown involvement of domains within the cytoplasmic Kv4.2 C-terminus in KChIP interaction. Less coincidence of binding and functional modulation indicates a more loose 'anchoring' at T1- and C-terminal interaction sites. Our results refine and extend previously proposed structural models for Kv4.2/KChIP complex formation.     

慢性房颤患者心房肌细胞钠通道的变化

目的 研究慢性房颤病人心房肌细胞钠通道电流特征及其基因表达.方法 膜片钳全细胞技术记录风湿性心脏病慢性房颤者和窦性心律者心房肌细胞钠通道电流;半定量反转录聚合酶链式反应测量心房肌细胞钠通道α亚单位基因(SCNSA)mRNA的表达.结果 (1)与窦性心律相比,慢性房颤病人钠通道电流密度和恢复动力学无改变,钠通道电压依赖性失活曲线向更正的方向偏移;(2)慢性房颤病人基因表达无改变.结论 慢性房颤病人心房传导速度的减慢不是由于钠通道电流的减小所致.     

Discovery of a stable cell line antigen in human internal organs

A cell membrane antigen was found in stable cell lines of epithelial origin, identical to an antigen in homogenates of the human gastric mucosa. The antigen was detected by means of antiserum against extracted membrane antigens of HeP-2 cells, absorbed by a mixture of homogenates of human lung, liver, and papillomas of the human larynx and breast. The antigen described was found in some of the adenocarcinomas of the stomach that were tested. It differs from Gold's carcinoembryonic antigen and from the secretory component of IgA, it is not the structural antigen of the D-type primate oncornavirus produced by HeP-2 cells and, evidently, it is not coded by the genome of that virus.Department of Etiology and Diagnosis of Tumors, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR G. V. Vygodchikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 12, pp. 690–693, December, 1977.     

Influence of voltage and extracellular Na+ on amiloride block and transport kinetics of rat epithelial Na+ channel expressed in Xenopus oocytes

We expressed the three subunits of the epithelial amiloride-sensitive Na(+) channel (ENaC) from rat distal colon heterologously in oocytes of Xenopus laevis and analysed blocker-induced fluctuations in current using conventional dual-microelectrode voltage-clamp. To minimize Na(+) accumulation we performed all experiments in low-Na(+) solutions (15 mM). Noise analysis revealed that control or ENaC-injected oocytes did not exhibit spontaneous relaxation noise. However, in ENaC-expressing oocytes, amiloride induced a distinct Lorentzian component in the power density spectra. With three amiloride concentrations and a linear analysis of the respective changes in the corner frequency f(c) (2 pi f(c) plot) we determined the rate constants k(on) and k(off) for the amiloride-ENaC interaction. At a clamp potential (V(m)) of -60 mV k(on) was 80.8 +/- 5.1 microM(-1) s(-1) and k(off) 15.4 +/- 4.2 s(-1). The half-maximal blocker concentration (K(mic,ami)) was 0.19 microM (V(m)=-60 mV). While k(on) was voltage-independent in the range -50 to -100 mV, k(off) and K(mic,ami) decreased significantly with increasing membrane hyperpolarization, resulting in an increased affinity of amiloride for its binding site on ENaC. Increasing extracellular [Na(+)] ([Na(+)](o)) led to saturation of ENaC. Subsequent noise analysis revealed that single-channel current increased non-linearly with [Na(+)](o) and that saturation was not due to a reduction in the number of open channels. The apparent affinity of Na(+) for its binding site on the channel was voltage dependent and increased with hyperpolarization. Noise analysis revealed that k(on) and k(off) for amiloride decreased with increasing [Na(+)](o), while the affinity of the amiloride-binding site did not change. These findings show that the affinity of rat intestinal ENaC for amiloride is voltage dependent and is influenced non-competitively by [Na(+)](o), indicating that Na(+) and amiloride do not compete for the same binding site at the channel.     

Dual effect of temperature on the human epithelial Na+ channel

The amiloride-sensitive epithelial sodium channel (ENaC) is the rate-limiting step for sodium reabsorption in the distal segments of the nephron, in the colon and in the airways. Its activity is regulated by intracellular and extracellular factors but the mechanisms of this regulation are not yet completely understood. Recently, we have shown that the fast regulation of ENaC by the extracellular [Na⁺], a phenomenon termed self-inhibition, is temperature dependent. In the present study we examined the effects of temperature on the single-channel properties of ENaC. Single-channel recordings from excised patches showed that the channel open probability (P _o, estimated from the number of open channels N·P _o, where N is the total number of channels) increased on average two- to threefold while the single-channel conductance decreased by about half when the temperature of the perfusion solution was lowered from ~30 to ~15 °C. The effects of temperature on the single-channel conductance and P _o explain the changes of the macroscopic current that can be observed upon temperature changes and, in particular, the paradoxical effect of temperature on the current carried by ENaC.     

Characterization of hormone-stimulated Na+ transport in a high-resistance clone of the MDCK cell line

The Madin-Darby canine kidney (MDCK) cell line forms an epithelial monolayer which expresses many of the morphological and functional properties of the renal collecting duct. The C7 subclone of the parent line forms an epithelium which expresses many of the characteristics of principal cells. The MDCK-C7 subclone forms a high-resistance epithelium that is capable of vectorial ion transport. We have found that this epithelium responds to aldosterone, antidiuretic hormone (ADH) and insulin like growth factor 1 (IGF1) with increases in amiloride-sensitive Na⁺ transport. The responses to aldosterone and ADH follow time-courses that are consistent with the action of these hormones in vivo. This is the first demonstration of IGF1-induced Na⁺ reabsorption in a mammalian model system. Interestingly, a maximal response to any one of these natriferic factors does not inhibit a subsequent response to another hormone. These studies indicate that the C7 subclone retains many of the natriferic responses of the native principal cells and is an ideal model for studying hormonal modulation of Na⁺ transport. Received: 12 January 1996/Accepted: 19 March 1996     

Serine-401 as a batrachotoxin- and local anesthetic-sensing residue in the human cardiac Na+ channel

Sequence alignment of four S6 segments in the human cardiac Na⁺ channel suggests that serine-401 (hNav1.5-S401) at D1S6 along with asparagine-927 (N927) at D2S6, serine-1458 (S1458) at D3S6, and phenylalanine-1760 (F1760) at D4S6 may jointly form a pore-facing S₄₀₁N₉₂₇S₁₄₅₈F₁₇₆₀ ring. Importantly, this pore-facing structure is adjacent to the putative gating-hinge (G₄₀₀G₉₂₆G₁₄₅₇S₁₇₅₉) and close to the selectivity filter. Within this SNSF ring, only S401 has not yet been identified as a batrachotoxin (BTX) sensing residue. We therefore created S401 mutants with 12 substitutions (S401C,W,P,A,K,F,R,E,L,N,D,G) and assayed their BTX sensitivity. All S401 mutants expressed Na⁺ currents but often with altered gating characteristics. Ten mutants were found sensitive to 5 μM BTX, which eliminated Na⁺ channel fast inactivation after repetitive pulses. However, S401K and S401R became BTX resistant. In addition, the block of open and inactivated hNav1.5-S401K Na⁺ channels by local anesthetic bupivacaine was reduced by ∼8–10-fold, but not the block of resting Na⁺ channels. Qualitatively, these ligand-sensing phenotypes of hNav1.5-S401K channels resemble those of S1458K and F1760K channels reported earlier. Together, our results support that residue hNav1.5-S401 at D1S6 is facing the inner cavity and is in close proximity to the receptor sites for BTX and for local anesthetics.     

Electrophysiological characterization of the tetrodotoxin-resistant Na+ channel, Nav1.9, in mouse dorsal root ganglion neurons

Small dorsal root ganglion neurons express preferentially the Na⁺ channel isoform Na_V1.9 that mediates a tetrodotoxin-resistant (TTX-R) Na⁺ current. We investigated properties of the Na⁺ current mediated by Na_V1.9 (I_NaN) using the whole-cell, patch-clamp recording technique. To isolate I_NaN from heterogeneous TTX-R Na⁺ currents that also contain another type of TTX-R Na⁺ current mediated by Na_V1.8, we used Na_V1.8-null mutant mice. When F^– was used as an internal anion in the patch pipette solution, both the activation and inactivation kinetics for I_NaN shifted in the hyperpolarizing direction with time. Such a time-dependent shift of the kinetics was not observed when Cl^– was used as an internal anion. Functional expression of I_NaN declined with time after cell dissociation and recovered during culture, implying that Na_V1.9 may be regulated dynamically by trophic factors or depend on subtle environmental factors for its survival. During whole-cell recordings, the peak amplitude of I_NaN increased dramatically after a variable delay, as if inactive or silent channels had been kindled. Such an unusual increase of the amplitude could be prevented by adding ATP to the pipette solution or by recording with the nystatin-perforated patch-clamp technique, suggesting that the rupture of patch membrane affected the behaviour of Na_V1.9. These peculiar properties of I_NaN may provide an insight into the plasticity of Na⁺ channels that are related to pathological functions of Na⁺ channels accompanying abnormal pain states.     

Voltage-dependent gating of veratridine-modified RIIA Na+ channel α subunit expressed heterologously in CHO cells

The voltage-dependent kinetics of veratridine-modified RIIA Na⁺ channel α subunit expressed heterologously in CHO cells were studied using the whole-cell patch-clamp technique. The activation and deactivation kinetics are well described by double exponential functions but poorly by a monoexponential function. Unlike the slow component, the fast time constant and associated amplitude factor depended steeply on the potential. The steady-state activation of veratridine-modified channels is described by a Boltzmann function with a V _1/2 of –131.9 mV and a slope of 9.41 mV. A two-state model is proposed for the fast component that explains the kinetics of veratridine’s mechanism of action. Received: 11 December 1998 / Received after revision: 23 March 1999 / Accepted: 7 April 1999     

Electrophysiological and pharmacological properties of GABAergic cells in the dorsal raphe nucleus

The dorsal raphe nucleus (DRN) is the origin of the central serotonin [5-hydroxytryptamine (5-HT)] system and plays an important role in the regulation of many physiological functions such as sleep/arousal, food intake and mood. In order to understand the regulatory mechanisms of 5-HT system, characterization of the types of neurons is necessary. We performed electrophysiological recordings in acute slices of glutamate decarboxylase 67–green fluorescent protein knock-in mice. We utilized this mouse to identify visually GABAergic cells. Especially, we examined postsynaptic responses mediated by 5-HT receptors between GABAergic and serotonergic cells in the DRN. Various current responses were elicited by 5-HT and 5-HT_1A or 5-HT_2A/2C receptor agonists in GABAergic cells. These results suggested that multiple 5-HT receptor subtypes overlap on GABAergic cells, and their combination might control 5-HT cells. Understanding the postsynaptic 5-HT feedback mechanisms may help to elucidate the 5-HT neurotransmitter system and develop novel therapeutic approaches.     

Events preceding stable integration of SV40 genomes in a human cell line

We have examined the organization of integrated SV40 sequences in an uncloned population of a transformed human fibroblast cell line. Somatic cell hybrids between mouse B82 cells and human GM847 cells were examined for SV40 T-antigen expression and individual human chromosome presence. This analysis revealed that a functional SV40 genome is located on human chromosome 7. Restriction endonuclease digestion followed by blot hybridization of the parental human cell line revealed that it contains mutliple normal and defective SV40 copies integrated into the host genome in tandem. A similar analysis of several T-ag⁺ hybrid cell lines indicated that the integrated viral sequences in different hybrid cell lines (thus in different cells of the original population) are very closely related but not always identical. Analysis of subclones of GM847 also revealed such differences. Based upon these results, we postulate that following the initial integration event, viral as well as the flanking host DNA sequences become unstable and are subject to deletions and rearrangements. This short-lived structural instability is followed by highly stable integration of SV40 which is maintained in these cells or their hybrid derivatives for at least hundreds of cell generations.