Abstracts

Rationale: The aim of the current study was primarily to quantitate properties of inactivation in a mammalian central neuron, and to examine the relationship between activation and inactivation by enzymatic removal of inactivation with papain. Methods: Hippocampal CA1 neurons(n=100)from female Wistar rats, 2-5 weeks of age were acutely dissociated using a pronase-based protocol. INa(t) was recorded in whole-cell patch clamp configuration using specially fabricated low-resistance patch electrodes, which allowed quite rapid voltage clamp steps (50-100 microseconds). Experiments were performed at room temperature (~22C). Single and double pulse protocols were used to measure the properties of INa(t) inactivation between open and inactivated states, and also between closed states.Results: Monoexponential inactivation of Ina(t) was evident at membrane potentials around -50 mV, but became bi-exponential and more rapid with greater depolarization (fast and slow time constants of 0.5 and 2.5 ms respectively at 0 mV). The amplitude ratio of the fast and slow components approached a limiting value of ~0.7 at potentials up to +40 mV, and the bi-exponential inactivation was remarkably well accounted for by the addition of a second open state in the kinetic description, rather than a second inactivated state. The steady-state inactivation (h∞) relation reached a maximum at -120 mV, with an approximate half-inactivation voltage of -71 mV, complete inactivation at -40 mV and was best fitted by a double Boltzman distribution. Double pulse techniques found that the rate of macroscopic inactivation at moderate depolarisations (>_40 mV) resulted from true transitions of the open channels to the inactivated state, rather than late activation of sodium channels. Similar experiments at potentials negative to INa(t) threshold revealed that some transitions from activatable closed states to inactivated states were surprisingly long (~100’s of ms) which will certainly affect repetitive firing rates. Addition of the enzyme papaine (1mg/ml) to the patch-electrode solution resulted a rapid increase in INa(t) amplitude (~100%), most likely reflecting enzymatic action on the initially closed inactivated states, followed by gradual removal of inactivation the currents activated by depolarisation. The rate of activation of INa(t) actually increased with inactivation removal. The rate of deactivation was grossly unaffected, but notably displayed two time constants of decay at potentials close to threshold after papain treatment. Conclusions: These observations reveal substantial discrepancies between properties of rat CA1 INa(t) and conventional invertebrate data and demonstrate properties that cannot be accounted for by the Hodgkin-Huxley (HH) model. They are consistent with an inactivation process coupled to activation, with a biexponetial time-course most likely resulting from a second open state of the channel. Uncritical use of the HH formalism for this conductance will almost certainly lead to erroneous simulation results.