Nuclear ion channels in cardiac myocytes

Abstract

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.

Dendrimer-assisted patch-clamp sizing of nuclear pores

“…To ensure that the isolated nuclei remained in a medium with composition close to that of their native environment, we modified the methods previously reported for cardiac myocyte nuclei isolation [6,8,19]. A total of 82 male Swiss-Webster mice (20-22 g) were used.…”

“…Our procedures for patch-clamping isolated nuclei are described elsewhere [6,8,19]. Briefly, fiber-filled glass tubing (TW150F-4, World Precision Instruments, Sarasota, Fla., USA) was used to manufacture the patch-clamp pipettes.…”

“…However, to ensure that no particle tested (i.e., colloidal gold or dendrimer) escaped from the pipette and affected the NPCC, a small negative pressure (−5 mmHg) was applied prior to contact of the NE. As previously reported (e.g., 8,19]), all applied voltages as well as acquisition and processing of the data was carried out with Axon Instruments instrumentation (Foster City, Calif., USA). Voltage values given in this paper are those applied to the glass pipette lumen.…”

“…However, patch-clamp investigations indicate that the NPC behaves like an ion channel (e.g., [6,7], see [4]), with gates opening and closing in a probabilistic manner (e.g., [8]). Patch-clamp demonstrates that, despite the large NPCC diameter, the NPC gate(s) is(are) sufficient to regulate ion flow into and out of the nucleus through changes in its open probability (see [4]).…”

“…A logical continuation of our patch-clamp studies (e.g., [6,8,19,20,21]) would be to attempt to incorporate the EM method of pore sizing with direct measurements of the NPCC electrical conductance, γ, using patch-clamp. As previously explained [10], the central idea of using particles of known diameter to size a membrane channel with electrical methods is similar to the resistive pulse principle of Coulter counters and other technologies.…”