Development and subsequent neural tube effects on the excitability of cultured Xenopus myocytes

Abstract

We examined both the development of electrical excitability in cultured Xenopus muscle over a period of 7 days, and the effects of neural tube on the muscle action potential. During muscle development, delayed and anomalous rectification were present in most cases within 24 hr. The action potential was dependent on Na at all times examined, and the rate of rise of the action potential (Vmax) increased substantially (seven-fold) from the first 2 days to 6 to 7 days in vitro, reflecting an increase in Na current density. In order to determine the mechanism for the increase in Vmax, we examined single-channel Na currents using the gigaseal technique. Single-channel conductance (gamma) did not increase substantially when measured using the patch clamp technique: gamma = 24 pS at 1 to 2 days, and gamma = 28 pS at 4 to 6 days. The channel open time at 14 degrees C was 0.6 msec for 1- to 2-day-old cells and 0.5 msec in 4- to 6-day-old cells at a step potential 40 mV from rest. The time constant for current decay as well as the time-to-peak current also did not change over time. Thus, channel kinetics appear unchanged. The maximum inward current from summed records was statistically greater for older cells, and the frequency of patches displaying single-channel events increased from 75 to 98%. Thus, we conclude that during development in vitro, Na current density increases as a result of an increase in channel density without detectable alterations in single-channel properties. Neural tube addition led to a further increase in Vmax (two-fold), even in muscle cells with no apparent nerve contact. Single channel analysis of cells in coculture revealed gamma to be 28 pS in three cells displaying a single amplitude peak for individual Na currents. In the majority of cases (9/12), however, there appeared to be two classes of Na channels present which were difficult to separate. The larger conductance channel likely corresponds to the 28 pS class. The smaller channels, when present, did not contribute substantially to the population of events comprising the amplitude histogram. Other single-channel kinetic parameters also did not change. We, therefore, conclude that neural tube addition does not effect activation or inactivation kinetics but likely causes a further increase in channel density and possibly the induction of a second type of Na channel.