The β-galactosidase reporter gene, either free or complexed with various cationic vectors, was microinjected into mammalian cells. Cationic lipids but not polyethylenimine or polylysine prevent transgene expression when complexes are injected in the nucleus. Polyethylenimine and to a lesser extent polylysine, but not cationic lipids, enhance transgene expression when complexes are injected into the cytoplasm. This latter effect was independent of the polymer vector/cDNA ionic charge ratio, suggesting that nucleic acid compaction rather than surface charge was critical for efficient nuclear trafficking. Cell division was not required for nuclear entry. Finally, comparative transfection and microinjection experiments with various cell lines confirm that barriers to gene transfer vary with cell type. We conclude that polymers but not cationic lipids promote gene delivery from the cytoplasm to the nucleus and that transgene expression in the nucleus is prevented by complexation with cationic lipids but not with cationic polymers.

There has been considerable debate as to whether adenosine triphosphate (ATP) is compartmentalized within cells and, in particular, whether the ATP concentration directly beneath the plasma membrane, experienced by membrane proteins, is the same as that of the bulk cytoplasm. This issue has been difficult to address because there is no indicator of cytosolic ATP, such as those available for Ca²⁺, capable of resolving the submembrane ATP concentration ([ATP]_sm) in real time within a single cell. We show here that mutant ATP-sensitive K⁺ channels can be used to measure [ATP]_sm by comparing the increase in current amplitude on patch excision with the ATP dose-response curve. In Xenopus oocytes, [ATP]_sm was 4.6 ± 0.3 mm (n = 29) under resting conditions, slightly higher than that measured for the bulk cytoplasm (2.3 mm). In mammalian (COSm6) cells, [ATP]_smwas slightly lower and averaged 1.4 ± 0.1 mm(n = 66). Metabolic poisoning (10 min of 3 mm azide) produced a significant fall in [ATP]_sm in both types of cells: to 1.2 ± 0.1 mm (n = 24) in oocytes and 0.8 ± 0.11 mm for COSm6 cells. We conclude that [ATP]_sm lies in the low millimolar range and that there is no gradient between bulk cytosolic and submembrane [ATP].

Phosphatidylinositol‐4,5‐bisphosphate (PIP2) is a major signaling molecule implicated in the regulation of various ion transporters and channels. Here we show that PIP2 and intracellular MgATP control the activity of the KCNQ1/KCNE1 potassium channel complex. In excised patch–clamp recordings, the KCNQ1/KCNE1 current decreased spontaneously with time. This rundown was markedly slowed by cytosolic application of PIP2 and fully prevented by application of PIP2 plus MgATP. PIP2‐dependent rundown was accompanied by acceleration in the current deactivation kinetics, whereas the MgATP‐dependent rundown was not. Cytosolic application of PIP2 slowed deactivation kinetics and also shifted the voltage dependency of the channel activation toward negative potentials. Complex changes in the current characteristics induced by membrane PIP2 was fully restituted by a model originally elaborated for ATP‐regulated two transmembrane‐domain potassium channels. The model is consistent with stabilization by PIP2 of KCNQ1/KCNE1 channels in the open state. Our data suggest a striking functional homology between a six transmembrane‐domain voltage‐gated channel and a two transmembrane‐domain ATP‐gated channel.