Localization of sodium and potassium currents at sites of nerve-muscle contact in embryonic Xenopus muscle cells in culture

Abstract

Macro-patch current recordings were used to determine the spatial distribution of INa, inactivating (IIK) and non-inactivating (IK) outward potassium currents on nerve-contacted Xenopus muscle cells grown in culture. Sites of nerve contact were identified under 400x magnification and macroscopic currents recorded from cell-attached membrane patches ranging in diameter from 2 microm to 6 microm. Membrane patch (n=133) recordings revealed no detectable current, or INa and/or IIK, or just IK. More than 85% of the membrane patches from which INa (n=35) could be recorded occurred within 40 microm of the position of nerve contact. In addition, more than 90% of the membrane patches which contained INa also contained IIK. The density of IIK in these membrane patches that also contained INa was significantly higher than the density of IIK or IK in membrane patches without INa. Sites of ACh receptor localization at nerve-muscle contacts, identified by labelling with rhodamine-alpha-bungarotoxin, were also found to contain both INa (4/5 cells) and IIK (5/5 cells). These results suggest that in Xenopus muscle cells in culture the channels mediating IIK along with sodium channels are clustered at sites of nerve contact and synaptic specializations.

Sodium channel distribution on uninnervated and innervated embryonic skeletal myotubes

“…The accumulation of Na ϩ channels at the NMJ in both species is roughly coincident with neuromuscular synaptic pruning in each species (Brown et al, 1976;Phillips and Bennett, 1987), as well as increases in coordinated animal movement. Fry and Moody-Corbett (1999) reported increases in Na ϩ current densities at cultured Xenopus myocytes in contact with a single neurite after 1-3 days in coculture, thus suggesting a much shorter delay between AChR and Na ϩ channel accumulation. However, Xenopus embryogenesis is very rapid compared to rodent and avian development, with swimming larvae appearing much earlier than mobile rat pups or chicks in their respective life cycles.…”

“…However, given the different time courses over which these proteins accumulate at the developing NMJ, it is obvious that differences exist within their respective organizing mechanisms. Furthermore, the observation that increases in Na ϩ channel densities at the developing rodent NMJ do not occur until approximately 2 weeks after the appearance of AChRs (Lupa et al, 1993) while increases in Na ϩ channels may appear at the developing Xenopus NMJ within 24 h of AChR aggregation (Fry and Moody-Corbett, 1999) suggests that species-specific factors determining Na ϩ channel distributions exist. Even less is known about the development of extrajunctional Na ϩ channel distributions.…”

Properties of Xenopus Kv1.10 channels expressed in HEK293 cells

“…As the muscle cells mature in culture, expression of both currents increases (Ribera and Spitzer, 1991;Moody-Corbett and Gilbert, 1992a;Linsdell and Moody, 1995;Chauhan-Patel and Spruce, 1998;Currie and Moody, 1999). Patch clamp experiments using cell attached recording techniques suggest that I IK and I K are mediated by at least two different structural classes of K ϩ channel (Ernsberger and Spitzer, 1995;Fry and Moody-Corbett, 1999). In addition, the inactivating properties of I IK from whole cell current recordings of skeletal muscle in culture suggest that this current may be a composite of more than one channel subtype (Ribera and Spitzer, 1991;Moody-Corbett and Gilbert, 1992b).…”