Voltage-dependent activation in purified reconstituted sodium channels from rabbit T-tubular membranes

We have examined the voltage-dependent gating of batrachotoxin-modified sodium channels purified from rabbit T-tubular membranes in two ways. First, purified channels were reconstituted into planar bilayers and single-channel properties were measured. Batrachotoxin-activated channels showed steep voltage-dependent activation with half-maximal opening probabilities at potentials between -95 and -116 mV. The single-channel conductance (500 mM Na+ cis, 200 mM Na+ trans) averaged 20 pS and was independent of membrane potential. Channels usually inserted with their extracellular faces on the trans side of the bilayer; addition of tetrodotoxin to the cis side had no effect, whereas addition to the trans side blocked greater than 95% of channel openings at -77 mV. A second approach was used to establish that this voltage dependence was a characteristic of the entire population of purified channels and not just those few channels observed in planar bilayers. Channels reconstituted into egg phosphatidylcholine vesicles were functionally oriented by inclusion of internal saxitoxin; vesicle membrane potentials were then generated by K+ gradients in the presence of valinomycin. After batrachotoxin activation, Vm was altered by shifts of K+o. All of the specific 22Na+ influx activated by batrachotoxin and blocked by saxitoxin was found to be voltage sensitive, activating between predicted membrane potentials of -100 and -50 mV. The single-channel properties of the purified T-tubular sodium channel correspond closely to those seen with native sodium channels from rat sarcolemma. The voltage-dependent activation of the batrachotoxin-modified reconstituted channel is the same as that seen with native channels in situ or in bilayers after exposure to this toxin. Most importantly, this voltage-dependent gating is a property of all of the purified channels capable of specific pharmacological activation.