Sodium conductance in cultured myenteric AH-type neurons from guinea-pig small intestine

Abstract

Whole-cell patch clamp methods were used to investigate sodium conductance in after-hyperpolarization-type (AH) enteric neurons in culture after dissociation from the myenteric plexus of guinea-pig small intestine. Inward current carried by Na + ( I Na) was identified and its current–voltage characteristics were compared with those for inward Ca 2+ current ( I Ca). The I Na current was a rapidly inactivating current relative to I Ca. Application of tetrodotoxin (TTX) blocked I Na with an EC 50 of 10.7 nM. Activation curves for I Na showed a rapid decrease in time to peak for test potentials from holding potentials of −80 mV to between −40 and −10 mV. Voltage-dependence of steady-state inactivation curves for I Na was fit to the Boltzmann equation with potential for half-inactivation ( V 1/2)=−55.6 mV and slope factor ( k)=6.4 mV. Steady-state inactivation for I Ca fit the Boltzmann equation with a V 1/2=−38.9 mV and k=14.4 mV. Kinetics for inactivation of I Na were voltage dependent at potentials between −70 and −30 mV and accelerated and became less voltage-dependent at more positive potentials. The time constant ( τ) for inactivation at −70 mV was τ=161±23 ms and decreased to τ=2.3±0.2 ms at −30 mV. Rapid acceleration of inactivation occurred between −50 and −40 mV. This was also the range where activation began. Recovery from inactivation with the membrane potential clamped at −100 or −80 mV was rapid and fit by a single exponential with τ=7.3±1.1 ms for −100 mV and 21.5±5.1 ms for −80 mV. The results suggest that AH-type enteric neurons have only one type of Na + channel that behaves like the “classical” voltage-gated tetrodotoxin-sensitive fast channel. The findings support the hypothesis that I Na current is an important factor in determination of excitability and firing behavior in AH neurons. I Na and I Ca together determine the properties of the rising phase of the spike and thereby contribute to global determinants of excitability as the neurons are exposed to multiple depolarizing and hyperpolarizing stimuli from synaptic inputs and mediators released from enteroparacrine cells.