Raffaella Tonini

The Intracellular Chloride Ion Channel Protein CLIC1 Undergoes a Redox-controlled Structural Transition*

Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24–Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.

The nuclear chloride ion channel NCC27 is involved in regulation of the cell cycle

1 NCC27 is a nuclear chloride ion channel, identified in the PMA‐activated U937 human monocyte cell line. NCC27 mRNA is expressed in virtually all cells and tissues and the gene encoding NCC27 is also highly conserved. Because of these factors, we have examined the hypothesis that NCC27 is involved in cell cycle regulation. 2 Electrophysiological studies in Chinese hamster ovary (CHO‐K1) cells indicated that NCC27 chloride conductance varied according to the stage of the cell cycle, being expressed only on the plasma membrane of cells in G2/M phase. 3 We also demonstrate that Cl− ion channel blockers known to block NCC27 led to arrest of CHO‐K1 cells in the G2/M stage of the cell cycle, the same stage at which this ion channel is selectively expressed on the plasma membrane. 4 These data strongly support the hypothesis that NCC27 is involved, in some as yet undetermined manner, in regulation of the cell cycle.

Functional characterization of the NCC27 nuclear protein in stable transfected CHO-K1 cells

NCC27 belongs to a family of small, highly conserved, organellar ion channel proteins. It is constitutively expressed by native CHO‐K1 and dominantly localized to the nucleus and nuclear membrane. When CHO‐K1 cells are transfected with NCC27‐expressing constructs, synthesized proteins spill over into the cytoplasm and ion channel activity can then be detected on the plasma as well as nuclear membrane. This provided a unique opportunity to directly compare electrophysiological characteristics of the one cloned channel, both on the nuclear and cytoplasmic membranes. At the same time, as NCC27 is unusually small for an ion channel protein, we wished to directly determine whether it is a membrane‐resident channel in its own right. In CHO‐K1 cells transfected with epitope‐tagged NCC27 constructs, we have demonstrated that the NCC27 conductance is chloride dependent and that the electrophysiological characteristics of the channels are essentially identical whether expressed on plasma or nuclear membranes. In addition, we show that a monoclonal antibody directed at an epitope tag added to NCC27 rapidly inhibits the ability of the expressed protein to conduct chloride, but only when the antibody has access to the tag epitope. By selectively tagging either the amino or carboxyl terminus of NCC27 and varying the side of the membrane from which we record channel activity, we have demonstrated conclusively that NCC27 is a transmembrane protein that directly forms part of the ion channel and, further, that the amino terminus projects outward and the carboxyl terminus inward. We conclude that despite its relatively small size, NCC27 must form an integral part of an ion channel complex.—Tonini, R., Ferroni, A., Valenzuela, S. M., Warton, K., Campbell, T. J., Breit, S. N., Mazzanti, M. Functional characterization of the NCC27 nuclear protein in stable transfected CHO‐K1 cells. FASEB J. 14, 1171–1178 (2000)

USING ATOMIC FORCE MICROSCOPY TO INVESTIGATE PATCH-CLAMPED NUCLEAR MEMBRANE

Nuclear patch clamp is an emerging research field that aims to disclose the electrical phenomena underlying macromolecular transport across the nuclear envelope (NE), its properties as an ion barrier and its function as an intracellular calcium store. The authors combined the patch clamp technique with atomic force microscopy (AFM) to investigate the structure—function relationship of NE. In principle, patch clamp currents, recorded from the NE can indicate the activity of the nuclear pore complexes (NPCs) and/or of ion channels in the two biomembranes that compose the NE. However, the role of the NPCs is still unclear because the observed NE current in patch clamp experiments is lower than expected from the known density of the NPCs. Therefore, AFM was applied to link patch clamp currents to structure. The membrane patch was excised from the nuclear envelope and, after electrical evaluation, transferred from the patch pipette to a substrate. We could identify the native nuclear membrane patches with AFM at a lateral and a vertical resolution of 3nm and 0.1nm, respectively. It was shown that complete NE together with NPCs can be excised from the nucleus after their functional identification in patch clamp experiments. However, we also show that membranes of the endoplasmic reticulum can contaminate the tip of the patch pipette during nuclear patch clamp experiments. This possibility must be considered carefully in nuclear patch clamp experiments.

Modulation of the Inward Rectifier Potassium Channel IRK1 by the Ras Signaling Pathway

In this study, we investigated the role of Ras and the mitogen-activated protein kinase (MAPK) pathway in the modulation of the inward rectifier potassium channel IRK1. We show that although expression of IRK1 in HEK 293 cells leads to the appearance of a potassium current with strong inward rectifying properties, coexpression of the constitutively active form of Ras (Ras-L61) results in a significant reduction of the mean current density without altering the biophysical properties of the channel. The inhibitory effect of Ras-L61 is not due to a decreased expression of IRK1 since Northern analysis indicates that IRK1 mRNA level is not affected by Ras-L61 co-expression. Moreover, the inhibition can be relieved by treatment with the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor PD98059. Confocal microscopy analysis of cells transfected with the fusion construct green fluorescent protein-IRK1 shows that the channel is mainly localized at the plasma membrane. Coexpression of Ras-L61 delocalizes fluorescence to the cytoplasm, whereas treatment with PD98059 partially restores the membrane localization. In conclusion, our data indicate that the Ras-MAPK pathway modulates IRK1 current by affecting the subcellular localization of the channel. This suggests a role for Ras signaling in regulating the intracellular trafficking of this channel.

Expression of Ras-GRF in the SK-N-BE neuroblastoma accelerates retinoic-acid-induced neuronal differentiation and increases the functional expression of the IRK1 potassium channel

Ras‐GRF, a neuron‐specific Ras exchange factor of the central nervous system, was transfected in the SK‐N‐BE neuroblastoma cell line and stable clones were obtained. When exposed to retinoic acid, these clones showed a remarkable enhancement of Ras‐GRF expression with a concomitant high increase in the level of active (GTP‐bound) Ras already after 24 h of treatment. In the presence of retinoic acid, the transfected cells stopped growing and acquired a differentiated neuronal‐like phenotype more rapidly than the parental ones. Cells expressing Ras‐GRF also exhibited a more hyperpolarized membrane potential. Moreover, treatment with retinoic acid led to the appearance of an inward rectifying potassium channel with electrophysiological properties similar to IRK1. This current was present in a large number of cells expressing Ras‐GRF, while only a small percentage of parental cells exhibited this current. However, Northern analysis with a murine cDNA probe indicated that IRK1 mRNA was induced by retinoic acid at a similar level in both kinds of cells. Brief treatment with a specific inhibitor of the mitogen‐activated protein kinase (MAPK) pathway reduced the number of transfected cells showing IRK1 activity. These findings suggest that activation of the Ras pathway accelerates neuronal differentiation of this cell line. In addition, our results suggest that Ras‐GRF and/or Ras‐pathway may have a modulatory effect on IRK1 channel activity.

Involvement of CDC25mm/Ras-GRF1-dependent signaling in the control of neuronal excitability

Ras-GRF1 is a neuron-specific guanine nucleotide exchange factor for Ras proteins. Mice lacking Ras-GRF1 (-/-) are severely impaired in amygdala-dependent long-term synaptic plasticity and show higher basal synaptic activity at both amygdala and hippocampal synapses (Brambilla et al., 1997). In the present study we investigated the effects of Ras-GRF1 deletion on hippocampal neuronal excitability. Electrophysiological analysis of both primary cultured neurons and adult hippocampal slices indicated that Ras-GRF1-/- mice displayed neuronal hyperexcitability. Ras-GRF1-/- hippocampal neurons showed increased spontaneous activity and depolarized resting membrane potential, together with a higher firing rate in response to injected current. Changes in the intrinsic excitability of Ras-GRF1-/- neurons can entail these phenomena, suggesting that Ras-GRF1 deficiency might alter the balance between ionic conductances. In addition, we showed that mice lacking Ras-GRF1 displayed a higher seizure susceptibility following acute administration of convulsant drugs. Taken together, these results demonstrated a role for Ras-GRF1 in neuronal excitability.

Properties of neuronal α7 mutant nicotinic acetylcholine receptors gated by bicuculline

We have shown previously that mutating to threonine the leucine residue in the M2 domain of the alpha7 nicotinic acetylcholine receptor (human L248T, L248T; chick L247T, L247T) converts bicuculline (BIC) from an antagonist into an agonist. In this work we studied the functional properties of the BIC-activated channels and report that, in Xenopus oocytes injected with L248T subunit cDNA, BIC activates single-channel currents that have similar conductances, but shorter mean burst duration, than the channels activated by ACh. In contrast, both the conductance and kinetics of the channels activated by either ACh or BIC are substantially the same in oocytes expressing L247T receptors. We have also shown previously that if Cys 189 and 190, which are thought to be at or near the transmitter binding site, are additionally mutated to Ser, the new mutant receptor (L247T-C189S-C190S) has a reduced affinity for ACh. We now find that the EC(50) in the BIC dose-current response relation, as well the characteristics of the channels activated by BIC, are similar in oocytes expressing either L247T or L247T-C189S-C190S receptors. On the other hand, ACh activation of L247T-C189S-C190S receptors gates channels whose mean open time and burst duration are much shorter than those of ACh-gated L247T-channels. Therefore, the gating kinetics of both L248T and L247R-C189S-C190S receptor-channels change when BIC is replaced by ACh; and we conclude that both ACh and BIC activate mutant alpha7 receptors with different patterns of activation.

Unliganded human mutant α7 nicotinic receptors are modulated by Ca2+ and trace levels of Zn2+

A large body of evidence indicates that ligand-gated channels may open spontaneously, exhibiting a basal activity in the absence of the neurotransmitter. In the present work, we were interested in studying the Ca2+-induced modulation of the basal channel activity of unliganded human L248Tα7 receptors expressed in Xenopus oocytes. While the basal channel activity was blocked by either the nicotinic antagonist methyllycaconitine or the superfusion with a Ca2+-free medium, it was enhanced by increasing external Ca2+ concentrations. External Ca2+ significantly influenced the channel properties lengthening the channel duration and reducing the channel conductance, in a dose dependent manner. Furthermore, the basal channel activity in standard medium was blocked by N,N,N′,N′-tetrakis-2-pyridylmethyl-ethylenediamine, the chelator of divalent cations with very high affinity for Zn2+, and was induced by Zn2+ when Ca2+ was present in the external medium. We conclude that basal activity of α7 mutant receptor-channels is caused by divalent cation contaminants present in the external medium, namely Zn2+; is positively modulated by the external Ca2+; and is inhibited when Ca2+ is absent from the medium. The patho-physiological consequences of these findings are discussed.

Ion Channel Measurement on the Cell Nucleus

The purpose of a biological membrane is to separate two different environments. The lipid bilayer can function as a semipermeable barrier against free diffusion using protein structures, ionic channels and transporters which regulate the movement of molecules through the membrane. This is known to be the case for the plasma membrane and for most membranes of intracellular organelles. The nucleus is a special intracellular component, not only because it contains the genetic material, but also because it is delimited by a double membrane, probably originating from the endoplasmic reticulum. Nucleocytoplasmic communication is achieved by unique protein complexes, the nuclear pores, which span the double membrane and modulate nuclear envelope. permeability. During regular cell functioning the nuclear envelope represents a barrier only for solutes above 40 kDa; these must have a consensus sequence, a sort of molecular key, to cross the envelope through the pores. Small molecules and ions are freely diffusible in and out of the nucleus (Csermely et al. 1995), although under certain conditions the nuclear envelope is able to reduce its passive permeability. Loewenstein and colleagues demonstrated that the resistance of the envelope increases upon hormone stimulation (Ito and Loewenstein 1965). The same. group, and later others, showed the presence of a nuclear resting potential (Kanno and Loewenstein 1963; Mazzanti et al. 1990; Matzke et al. 1990; Dale et al. 1994) that could be due to the presence of an ion selective membrane. More recently in situ patch-clamp experiments on Xenopus oocyte nuclei demonstrated that ATP is essential to observe large ionic conductances in the envelope (Mazzanti et al. 1994). However, there are still many conflicting results and opinions regarding nuclear Ca2+ permeability (Nicotera et al. 1989; Gerasimenko et al. 1994; Al-Mohanna et al. 1994; Stehno-Bittel et al. 1995).