Transfer of twelve charges is needed to open skeletal muscle Na+ channels.

Abstract

Voltage-dependent Na + channels are thought to sense membrane potential with fixed charges located within the membrane's electrical field. Measurement of open probability (Po) as a function of membrane potential gives a quantitative indication of the number of such charges that move through the field in opening the channel. We have used single-channel recording to measure skeletal muscle Na § channel open probability at its most negative extreme, where channels may open as seldom as once per minute. To prevent fast inactivation from masking the voltage dependence of Po, 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 resolution afforded by single-channel analysis, we have extended the resolution of the hyperpolarized tail of the Po curve by four orders of magnitude. We show that previous measurements, which indicated a minimum of six effective gating charges, may have been made in a range of Po values that had not yet arrived at its limiting slope. In our preparation, a minimum of 12 charges must function in the activation gating of the channel. Our results will require reevaluation of kinetic models based on six charges, and they have major implications for the interpretation of $4 mutagenesis studies and structure/function models of the Na § channel.

Disulfide locking a sodium channel voltage sensor reveals ion pair formation during activation

“…Otherwise, all other experiments were recorded from cells seeded in tissue culture dishes. The extracellular solution contained (in mM) NaCl (150), CaCl2 (1.5), MgCl2 (1), KCl (2), glucose (10), and Hepes (10) (pH 7.4), and the intracellular (pipette) solution contained (in mM) CsF (105), EGTA (10), NaCl (35), Mg-ATP (4), and Hepes (10) (pH 7.5). ␤ME was added to the extracellular solution to a final concentration of 1 mM within 1 h of use.…”

“…Gating is rapid, reversible, and steeply voltage-dependent, suggesting that charged gating particles associated with the channels move across the membrane in response to the electric field (1). Capacitative ''gating currents'' caused by these charge movements (2,3) indicate that approximately 3-4 positive charges per voltage-sensing module move outward during gating of sodium or potassium channels (4)(5)(6)(7)(8)(9)(10). The primary structure of sodium channels (11) revealed four homologous domains containing six predicted alpha-helical segments (S1-S6) in each.…”

Functional diversity of potassium channel voltage-sensing domains

“…For this estimate to be a good approximation to the gating charge, P o has to be lower than10 ¡4 and preferably between 10 ¡7 ¡10 ¡6 . [79][80][81] Limiting-slope estimates of the charge per channel have been obtained for a number of voltage dependent potassium channels. Table 1 lists the gating charge estimates for those Kv channels for which the conditions for limiting-slope measurements mentioned above have been experimentally justified.…”

Structure, Function, and Modification of the Voltage Sensor in Voltage-Gated Ion Channels

“…At relatively negative voltages (−100 to −60 mV), the open probability changes 10 times every 5 mV for a Na or a K channel [38,77]. This is about 12 times more sensitive than an electronic transistor gated by one elementary charge [78] and depends on the design of the [83,84].…”

“…In Na channels about 12 charges are transferred [77,86], and in Ca channels about 9 charges are transferred [87]. All these studies together suggest that It is generally agreed on that the activation can be divided in two main components: i) independent outward movements of the four S4 helices, followed by ii) a concerted opening step when probably all S4s move together [88][89][90][91][92].…”