TRANSFER OF 12 CHARGES IS NEEDED TO OPEN SKELETAL-MUSCLE NA+ CHANNELS

Voltage-dependent Na+ channels are thought to sense membrane potential with fixed charges located within the membrane's electrical field. Me asurement of open probability (P-o) as a function of membrane potentia l gives a quantitative indication of the number of such charges that m ove through the field in opening the channel. The have used single-cha nnel recording to measure skeletal muscle Na+ channel open probability at its most negative extreme, where channels may open as seldom as on ce per minute. To prevent fast inactivation from masking the voltage d ependence of P-o, we have generated a clone of the rat skeletal muscle Na+ channel that is lacking in fast inactivation (IFM1303QQQ). Using this mutant channel expressed in Xenopus oocytes, and the extra resolu tion afforded by single-channel analysis, we have extended the resolut ion of the hyperpolarized tail of the P-o curve by four orders of magn itude, We show that previous measurements, which indicated a minimum o f six effective gating charges, may have been made in a range of P-o v alues that had not yet arrived at its limiting slope. In our preparati on, a minimum of 12 charges must function in the activation gating of the channel. Our results will require reevaluation of kinetic models b ased on six charges, and they have major implications for the interpre tation of S4 mutagenesis studies and structure/function models of the Na+ channel.