Reconstitution of ionic channels from inner and outer membranes of mammalian cardiac nuclei.
Recent reports suggest that the nuclear envelope possesses specific ion transport mechanisms that regulate the electrolyte concentrations within the nucleoplasm and perinuclear space. In this work, intact nuclei were isolated from sheep cardiac cells. After chromatin digestion, the nuclear envelopes were sonicated and four nuclear vesicle populations were separated by sucrose step gradients (SF1-SF4). These fractions were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their protein content was analyzed by Western blot, using lamin and SEC 61 antibodies. The lamins, which are associated with the inner nuclear membrane, were present in three fractions, SF2, SF3, and SF4, with a lower amount in SF2. The SEC 61 protein, a marker of the rough endoplasmic reticulum, was detected in small amounts in SF1 and SF2. Upon fusion of vesicles into bilayers, the activities of nuclear ionic channels were recorded in 50 mM trans/250 mM cis KCl or CsCl, pH 7.2. Two types of Cl- selective channels were recorded: a large conducting 150-180-pS channel displaying substates, and a low conducting channel of 30 pS. They were both spontaneously active into bilayers, and their open probability was poorly voltage dependent at negative voltages. Retinoic acid (10(-8) M) increases the po of the large Cl- conducting channel, whereas ATP modifies the kinetics of the low conductance anion selective channel. Our data also suggest that this anionic channel is mainly present in the SF3 and SF4 population. The presence of a 181 +/- 10 pS cation-selective channel was consistently observed in the SF2 population. The behavior of this channel was voltage dependent in the voltage range -80 to +60 mV. Furthermore, we report for the first time the activity of a channel exclusively present in the SF3 and SF4 fractions, shown to contain mainly inner membrane vesicles. This cation selective channel displays a 75-pS conductance in 50 mM trans/250 mM cis K-gluconate. It is concluded that the bilayer reconstitution technique is an attractive approach to studying the electrophysiological properties of the inner and outer membranes of the nuclear envelope.
Rousseau
E
E
Department of Physiology and Biophysics, University of Sherbrooke, Québec J1H 5N4, Canada. e.rousse@courrier.usherb.ca
Michaud
C
C
Lefebvre
D
D
Proteau
S
S
Decrouy
A
A
D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D004594:Electrophysiology; D066298:In Vitro Techniques; D007473:Ion Channels / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D017136:Ion Transport; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D008564:Membrane Potentials; D008854:Microscopy, Electron; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D009685:Nuclear Envelope / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D012756:Sheep; D014212:Tretinoin / Q000494:pharmacology
D007473:Ion Channels; D008051:Lipid Bilayers; D014212:Tretinoin