Na current in membrane blebs: Implications for channel mobility and patch clamp recording

Abstract

When suction was applied to loose patch clamp pipettes while recording from enzymatically dissociated muscle fibers, large membrane blebs formed within the pipettes. We initiated a study of these suction-induced blebs because ion channels in the blebs would complicate or possibly invalidate loose patch voltage clamp measurements of membrane current density. The low lateral mobility (Stühmer and Almers, 1982) and steep gradients of Na channels at the endplate and tendon (Caldwell et al., 1986) imply tight binding of Na channels to cytoskeletal elements and led us to expect few, if any, Na channels in the blebs. Bleb formation produced an increase in membrane capacitance, as expected from the increase in membrane area. Bleb formation also increased the Na current, indicating that the blebs contained Na channels. Assuming that the increased capacitance and Na current were due to lipid and Na channels moving from membrane outside the pipette, ejection of the bleb from the pipette was expected to bring the capacitance and Na current back to their original values. Capacitance did return to its original value, but Na current was lower than expected. The decrease in Na current is explained by Na channels moving from the patch membrane into the bleb. Normalization of bleb and patch Na current to their respective capacitances revealed that bleb membrane had a Na channel density almost 50% that of normal surface membrane. Thus, bleb membrane is neither devoid of proteins nor truly representative of the normal surface membrane from which it arose. It is enriched in membrane lipids and is relatively protein poor. Two conclusions can be drawn. (1) Measurements of current density made with the loose patch voltage clamp may appear highly variable and be inaccurate if bleb formation occurs and is ignored. (2) A large fraction of Na channels in adult mouse muscle may be mobile. A model is presented to reconcile the apparent contradiction between the existence of large gradients of Na channel density at the endplate and tendon and the presence of mobile channels.