A system for the automated data-acquisition of fast transient signals in excitable membranes

1993

Biophysical Journal

Flow of small ions across the nuclear envelope (NE) is thought to occur without restriction through large diameter nuclear pore complexes (NPCs). However, investigations with electron and fluorescence microscopy, and with patch-clamp and microelectrode electrophysiology, suggest that in many animal and plant cell types small ions move through a barrier having the signature of large conductance nuclear ion channels (NICs). As nucleocytoplasmic transport and gene activity are regulated by cytoplasmic signals and as it has recently been shown by this investigator that cardiac NICs are sensitive to cAMP-dependent processes (1), it was considered relevant to further investigate the effects of various cytosolic signals on NIC activity. Ion species substitution demonstrated that K+ is the major species responsible for NIC currents. The Na-channel blocker tetrodotoxin (TTX, 100 microM) and the Ca-channel blocker diltiazem (100 microM) had no effect, indicating no relation of NICs to Na- or Ca-channels in transit to the cell surface membrane. Zn2+ (100 microM) blocked NIC activity, suggesting a dual role in nucleocytoplasmic transport and gene function. GTP did not produce measurable effect. However, its nonhydrolyzable analogue GTP-gamma-S (10 microM) suppressed NIC activity, suggesting a role for GTP hydrolysis in NIC function. Deoxynucleotides (dNTPs, 200 microM) produced a transient increase in NIC activity, pointing to a modulation of NIC function by nucleic acid substrates. These results indicate a role for NICs in mediating: (a) control of gene activity by transduction and other cytosolic signals, and (b) nuclear demands and response to such signals.

Section: Patch-clampmentioning

confidence: 99%

Section: Patch-clampmentioning

confidence: 99%

Restricted ion flow at the nuclear envelope of cardiac myocytes

1993

Biophysical Journal

“…It also takes advance of the author's experience in the design and construction of electrophysiological electronics since 1977 (Bustamante and McDonald 1983;Bustamante 1988). A great deal of hindsight, based on discussions with colleagues and on comments from anonymous referees of my published and unpublished manuscripts has helped to shape the organization and content of this paper.…”

Section: Brief Historymentioning

confidence: 95%

Current concepts in nuclear pore electrophysiologyThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

Can. J. Physiol. Pharmacol.

2006

Over 4 decades ago, microelectrode studies of in situ nuclei showed that, under certain conditions, the nuclear envelope (NE) behaves as a barrier opposing the nucleocytoplasmic flow of physiological ions. As the nuclear pore complexes (NPCs) of the NE are the only pathways for direct nucleocytoplasmic flow, those experiments implied that the NPCs are capable of restricting ion flow. These early studies validated electrophysiology as a useful approach to quantify some of the mechanisms by which NPCs mediate gene activity and expression. Since electron microscopy (EM) and other non-electrophysiological investigations, showed that the NPC lumen is a nanochannel, the opinion prevailed that the NPC could not oppose the flow of ions and, therefore, that electrophysiological observations resulted from technical artifacts. Consequently, the initial enthusiasm with nuclear electrophysiology faded out in less than a decade. In 1990, nuclear electrophysiology was revisited with patch-clamp, the most powerful electrophysiological technique to date. Patch-clamp has consistently demonstrated that the NE has intrinsic ion channel activity. Direct demonstrations of the NPC on-off ion channel gating behavior were published for artificial conditions in 1995 and for intact living nuclei in 2002. This on-off switching/gating behavior can be interpreted in terms of a metastable energy barrier. In the hope of advancing nuclear electrophysiology, and to complement the other papers contained in this special issue of the journal, here I review some of the main technical, experimental, and theoretical issues of the field, with special focus on NPCs.

Nuclear ion channels in cardiac myocytes

1992

Pfl�gers Arch

The paradigm that nucleocytoplasmic transport of ions occurs without a diffusional barrier has been challenged by the recent demonstration with patch-clamp techniques of the existence of ion channels in the nuclear envelope of murine zygotes and hepatocytes. This report demonstrates the existence of nuclear ion channels (NIC) in murine ventricular cardiac myocytes. NIC conductance (gamma), calculated from current histogram peaks, was 106-532 pS at 22-36 degrees C. In nucleus-attached patches, replacement of cytoplasmic K+ with Na+ reduced NIC activity within 30 s, suggesting that intranuclear-delimited mechanisms mediate this phenomenon. In excised, inside-out patches K+ was as permeable as Na+ through NIC. NIC activity was observed in 0-4 mM Mg2+ and/or ATP2-, with or without 0-1 mM Ca2+, indicating a minor direct role of these ions. However, in non-responsive excised inside-out patches, NIC activity appeared when the catalytic subunit of the cAMP-dependent protein kinase was applied to the nucleoplasmic side of the patch, in the presence of Mg2+ and ATP2-, indicating an important role for phosphorylation-dependent process(es) in NIC function--an observation supported by the depressing effects of protein kinase inhibitor on responsive NIC. The concept that nucleopore complexes are solely responsible for nucleocytoplamic transport leads to the speculation that these structures are the physical substrate for NIC.