USING ATOMIC-FORCE MICROSCOPY TO INVESTIGATE PATCH-CLAMPED NUCLEAR-MEMBRANE

Nuclear patch clamp is an emerging research field that aims to disclos e the electrical phenomena underlying macromolecular transport across the nuclear envelope (NE), its properties as an ion barrier and its fu nction as an intracellular calcium store. The authors combined the pat ch clamp technique with atomic force microscopy (AFM) to investigate t he structure-function relationship of NE. In principle, patch clamp cu rrents, recorded from the NE can indicate the activity of the nuclear pore complexes (NPCs) and/or of ion channels in the two biomembranes t hat compose the NE. However, the role of the NPCs is still unclear bec ause the observed NE current in patch clamp experiments is lower than expected from the known density of the NPCs. Therefore, AFM was applie d to link patch clamp currents to structure. The membrane patch was ex cised from the nuclear envelope and, after electrical evaluation, tran sferred from the patch pipette to a substrate. We could identify the n ative nuclear membrane patches with AFM at a lateral and a vertical re solution of 3 nm and 0.1 nm, respectively. It was shown that complete NE together with NPCs can be excised from the nucleus after their func tional identification in patch clamp experiments. However, we also sho w that membranes of the endoplasmic reticulum can contaminate the tip of the patch pipette during nuclear patch clamp experiments. This poss ibility must be considered carefully in nuclear patch clamp experiment s. (C) 1997 Academic Press.