Kühn FJ , Greeff NG .

	Figure 1. Effects of S4D4 point mutations on macroscopic Na+ currents. (A) Na+ currents from WT-, R4H-, R4/5H-, and R5H-injected Xenopus oocytes elicited by step depolarizations from −80 to 80 mV, holding potential −100 mV. A single trace displaying an outward gating current followed by an inward ionic current near sodium reversal potential is indicated in R4/5H. (B) Corresponding current–voltage relationship of peak Na+ currents, normalized to the largest inward current. (C) Steady-state inactivation at 0 mV induced by a 100-ms prepulse at voltages between −120 and 20 mV, increments of 10 mV, holding potential −100 mV. The curves are fitted to a standard Boltzmann distribution with slopes and V0.5 given in the text. Values are mean ± SEM of n > 3 cells. (D) Semilogarithmic plot of time constant and voltage of recovery from fast inactivation. Pulse protocol: a 80-ms test pulse to 0 mV was preceded by a 100-ms pulse to 0 mV and variable recovery periods at four different holding potentials (−80, −100, −120, and −140 mV). Values are mean recovery time constants in milliseconds ± SEM of n = 1–7 cells; WT, 1.8 ± 0.6 (−140 mV), 3.9 ± 1.8 (−120 mV), 7.9 ± 1.5 (−100 mV), 24.8 ± 6.0 (−80 mV); R4H, 30.8 ± 9.6 (−140 mV), 89.4 ± 19.3 (−120 mV), 264 ± 59 (−100 mV), 347 ± 55.1 (−80 mV); R4/5H, 5.4 ± 0.5 (−140 mV), 9.1 ± 0.9 (−120 mV), 15.1 ± 0.8 (−100 mV), 26.7 ± 1.8 (−80 mV); R5H, 2.6 (−140mV), 3.7 ± 0.2 (−120 mV), 7.4 ± 0.6 (−100 mV), 15.4 ± 0.9 (−80 mV).
	Figure 9. Scheme of possible interactions of the central arginines of S4D4 with a putative counter-charge that could explain the different inactivation behavior of the mutants. Illustrated is the central section of S4D4 containing the highly conserved central arginines R3–R6; the hypothetical negative counter-charge is indicated. WT and R5H are shown in the open state and R4H and R4/5H are shown in the inactivated state. Compared with WT, the O → I transition is considerably slowed in R5H (indicated by a thin arrow) and moderately slowed in R4H (not illustrated). This is due to the electrostatic asymmetry resulting from the interaction of the negative counter-charge with the neighbored arginines and histidines in the single mutants. On the other hand, the I2 → C2 transition (recovery from inactivation) is for the same reason drastically slowed in R4H (indicated by a thin arrow) and hardly affected in R5H (not illustrated). In contrast, WT and R4/5H display more symmetrical electrostatics, but due to the considerably altered structure in the double mutant, the mobility of S4D4 is slowed in both directions.
	Figure 2. Effects of S4D4 point mutations on kinetics and on voltage dependence of macroscopic Na+ currents. (A) Comparison of normalized single current traces of WT (solid line) and mutant (dotted line) channels, elicited by a depolarizing pulse to −5 mV from a −100-mV holding potential. The settling times of the corresponding capacitance transients are almost identical (data not shown). (B) Semilogarithmic plots of single (mutants) or double (WT inactivation) exponential fits, as indicated, from representative current traces elicited by depolarizing pulses to −20, −5, and 20 mV; holding potential −100 mV. Single fits accord to the following equation. INa(t) = A * (1 − exp (−t/τm))3*exp(−t/τh) + P * (1 − exp(−t/τm))3. WT: (τm/τh (fast)/τh (slow) in ms) 0.48/0.70/4.20 (−20 mV); 0.28/0.70/5.10 (−5 mV); 0.17/0.51/3.10 (20 mV); R4H: (τm/τh in ms) 0.41/2.90 (−20 mV); 0.31/2.50 (−5 mV); 0.23/1.80 (20 mV); R4/5H: (τm/τh in ms) 0.45/4.30 (−20 mV); 0.24/3.80 (−5 mV); 0.15/2.90 (20 mV); R5H: (τm/τh in ms) 0.45/5.50 (−20 mV); 0.25/4.90 (−5 mV); 0.16/3.10 (20 mV).
	Figure 3. Gating current recordings in WT and mutant sodium channels. (A–D) Sodium ionic (INa) and gating (Ig) currents recorded from the whole cell membrane of Xenopus oocytes using a TEVC after blocking most (B and C) or all (D) of the ionic current with TTX. (A) Pulse protocol; the traces were elicited by 13 ms depolarizing test pulses to membrane potentials of −60 to 60 mV increasing in steps of 10 mV without (B) or with (C) a 20-ms prepulse to 0 mV from a holding potential of −100 mV, interval 1 ms at −100 mV, temperature 15°C. The OFF-gating current of the prepulse and the non-immobilized fraction of the test pulse ON-gating current (Ig,n) are indicated in C. Notice the elimination of INa and the partial immobilization (∼50%) of total gating current (Ig) due to the inactivating pulse. The corresponding capacitance current (Ic) reflects the actual clamp speed. (D) Total ON-gating currents of WT and mutant channels activated by step depolarizations in 20-mV increments from −80 to 80 mV from a holding potential of −100 mV, recorded in presence of 2 μM TTX, pulse duration 13 ms.
	Figure 4. Charge–voltage (Q/V) distributions in WT and mutant sodium channels. Open symbols are data obtained in the presence of a 20-ms inactivating prepulse to 0 mV (1-ms interval at −100 mV). Filled symbols are data without prepulse; test pulses were 13 ms long. The gating charge (Qg) represents the time integral of the corresponding gating current. The individual Q/V curves were fitted by a Boltzmann distribution as described in Table . The degree of immobilization at Qmax derived as mean value ± SD from Table is indicated in each diagram.
	Figure 5. Recovery from fast inactivation of macroscopic ionic and gating currents. (A) Recovery of WT gating current (Ig, in the presence of 2 μM TTX) and ionic current (INa, in the absence of TTX) obtained from different oocytes. Pulse protocol: a 100-ms prepulse to 0 mV from a holding potential of −100 mV was followed by a recovery period of variable duration (2–90 ms) at −100 mV. Test pulses have a duration of 80 ms (ionic current) or 13 ms (gating current) and responses are superimposed for all recovery periods. For the calculation of the recovery time course, the current traces with prepulse were routinely normalized to the current traces without prepulse in order to compensate the slight decrease of the current amplitude during pulse series. The series of increasing recovery times, starting from 2 ms, where INa is almost totally inactivated, was preceded by the longest recovery time where the plateau of recovery is observed (first and last pulses are indicated in INa recordings). (B) Gating current recovery of WT and mutant sodium channels at −100 mV. Pulse protocol was as described above. The time intervals for recovery were between 10 and 1,250 ms for R4H and between 2 and 90 ms for WT and the other mutant channels. The recovering gating charges were fitted to a single exponential with corresponding recovery time constants (τR) as indicated. Notice the different levels of the nonimmobilized gating current fraction at the onset of recovery, reflecting the different degrees of immobilization.
	Figure 6. Comparison of the recovery of ionic current and gating charge of WT and mutant sodium channels at different potentials. Sodium current recovery (left panel) and gating charge recovery (right panel) at recovery potentials of −80, −100, and −120 mV in WT-, R4H-, R4/5H-, and R5H-sodium channels. The data of each subdiagram were recorded from different oocytes and normalized as described in Fig. 5. Notice the different starting points of gating current recovery in R4H that strictly depend on the effective recovery potential (indicated by arrows). Corresponding sodium current (INa) and gating charge (Qg) recovery time constants obtained from single exponential fits are as follows (INa/Qg in ms): WT, 22.8/54.9 (−80 mV), 7.6/16.7 (−100 mV), 2.1/6.4 (−120 mV); R4H, 359/113 (−80 mV), 228/269 (−100 mV), 59.7/349 (−120 mV); R4/5H, 25.4/39.7 (−80 mV), 14.9/20.6 (−100 mV), 8.4/9.5 (−120 mV); R5H, 16.3/12.6 (−80 mV), 8.0/11.6 (−100 mV), 3.8/6.9 (−120 mV).
	Figure 7. Correlation of the recovery of ionic current and gating charge of WT and R4H at different potentials. (A) Semilogarithmic plot of ionic current (filled symbols) and gating charge (open symbols) recoveries obtained from different oocytes are superimposed for potentials of −140, −120, −100, and −80 mV. Values are mean ± SD of n > 3 cells. Superimposed triangles represent congruent data of ionic current (small, filled triangle) and gating charge recovery (large, open triangle) recorded sequentially in a single oocyte at −100 mV. (B) Simultaneous recording of ionic and gating currents around sodium reversal potential (ENa). Test pulses elicited from +5 to +15 mV in steps of 1 mV, holding potential −100 mV. Extracellular sodium concentration was reduced to 8.8 mM by choline replacement in Modified Barth's Solution. Ionic currents change polarity when crossing ENa, whereas gating currents follow the direction of the electric field. (C) Recordings of gating current and ionic current recovery at sodium reversal potential (ENa) obtained from a single oocyte, T = 8°C. Pulse-protocol: a 20 ms prepulse to 40 mV was followed by a recovery period of variable duration (1–60 ms) at −100 mV and a test pulse to −20 (ionic current) or 34 mV (ENa; gating current), test pulse duration 13 ms, holding potential −100 mV.
	Figure 8. Model of sodium channel fast inactivation controlled by S4D4. (A) Schematic illustration of our interpretations and conclusions displaying the relevant structures of the sodium channel and their movements at closed, open, and inactivated states as well as during recovery from inactivation. The positively charged amino acids of the S4 voltage sensors in domains 2–4 and the analyzed mutations in S4D4 are indicated. R symbolizes the putative receptor site in the S4–S5 linker of domain 4, which binds the docking region (D) of the inactivation loop connecting domains 3 and 4 (L3–4), leading to fast inactivation of the channel. Moreover, the position of L3–4 during inactivation causes the partial immobilization of the voltage sensors (most probably S4D4 and S4D3; see discussion), which is indicated here as blockade of m by B. (B) State diagram with lower level reflecting the voltage-dependent activation pathway from several closed (C) to the open state (O) and further to the open state (OR), which presents the receptor instantly followed by the voltage-independent binding of L3–4. The upper level reflects the transitions between several inactivated states producing the nonimmobilized gating current fraction. For recovery from fast inactivation, hyperpolarization causes the reverse movement of S4D4, which disrupts the connection of the inactivation loop to its receptor and simultaneously causes the partial immobilization of the voltage sensors, thereby permitting the return of the channels into the resting (closed) state.

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed , Xenbase
Aldrich, A reinterpretation of mammalian sodium channel gating based on single channel recording. , Pubmed
Armstrong, Sodium channels and gating currents. 1981, Pubmed
Armstrong, Inactivation of the sodium channel. II. Gating current experiments. 1977, Pubmed
Armstrong, Voltage-gated ion channels and electrical excitability. 1998, Pubmed
Bezanilla, Gating of Shaker K+ channels: II. The components of gating currents and a model of channel activation. 1994, Pubmed , Xenbase
Catterall, Molecular properties of voltage-sensitive sodium channels. 1986, Pubmed
Cha, Voltage sensors in domains III and IV, but not I and II, are immobilized by Na+ channel fast inactivation. 1999, Pubmed , Xenbase
Chahine, Sodium channel mutations in paramyotonia congenita uncouple inactivation from activation. 1994, Pubmed
Chen, A unique role for the S4 segment of domain 4 in the inactivation of sodium channels. 1996, Pubmed , Xenbase
Conti, Quantal charge redistributions accompanying the structural transitions of sodium channels. 1989, Pubmed , Xenbase
Durell, Atomic scale structure and functional models of voltage-gated potassium channels. 1992, Pubmed
Eaholtz, Restoration of inactivation and block of open sodium channels by an inactivation gate peptide. 1994, Pubmed
Filatov, Inactivation and secondary structure in the D4/S4-5 region of the SkM1 sodium channel. 1998, Pubmed
Greeff, The quantal gating charge of sodium channel inactivation. 1991, Pubmed
HODGKIN, A quantitative description of membrane current and its application to conduction and excitation in nerve. 1952, Pubmed
Ji, Paramyotonia congenita mutations reveal different roles for segments S3 and S4 of domain D4 in hSkM1 sodium channel gating. 1996, Pubmed
Kontis, Sodium channel inactivation is altered by substitution of voltage sensor positive charges. 1997, Pubmed , Xenbase
Kontis, Sodium channel activation gating is affected by substitutions of voltage sensor positive charges in all four domains. 1997, Pubmed , Xenbase
Krafte, Inactivation of cloned Na channels expressed in Xenopus oocytes. 1990, Pubmed , Xenbase
McPhee, A critical role for the S4-S5 intracellular loop in domain IV of the sodium channel alpha-subunit in fast inactivation. 1998, Pubmed , Xenbase
Mitrovic, Role in fast inactivation of conserved amino acids in the IV/S4-S5 loop of the human muscle Na+ channel. 1996, Pubmed
Moran, Sodium ionic and gating currents in mammalian cells. 1990, Pubmed
Noda, Expression of functional sodium channels from cloned cDNA. , Pubmed , Xenbase
Papazian, Alteration of voltage-dependence of Shaker potassium channel by mutations in the S4 sequence. 1991, Pubmed , Xenbase
Papazian, Electrostatic interactions of S4 voltage sensor in Shaker K+ channel. 1995, Pubmed , Xenbase
Patton, A voltage-dependent gating transition induces use-dependent block by tetrodotoxin of rat IIA sodium channels expressed in Xenopus oocytes. 1991, Pubmed , Xenbase
Patton, The adult rat brain beta 1 subunit modifies activation and inactivation gating of multiple sodium channel alpha subunits. 1994, Pubmed , Xenbase
Ptácek, Mutations in an S4 segment of the adult skeletal muscle sodium channel cause paramyotonia congenita. 1992, Pubmed
Roux, Fast inactivation in Shaker K+ channels. Properties of ionic and gating currents. 1998, Pubmed , Xenbase
Ruben, Effects of clamp rise-time on rat brain IIA sodium channels in Xenopus oocytes. 1997, Pubmed , Xenbase
Sancho, Histidine residues at the N- and C-termini of alpha-helices: perturbed pKas and protein stability. 1992, Pubmed
Schreibmayer, Voltage clamping of Xenopus laevis oocytes utilizing agarose-cushion electrodes. 1994, Pubmed , Xenbase
Seoh, Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel. 1996, Pubmed , Xenbase
Sheets, Voltage-dependent open-state inactivation of cardiac sodium channels: gating current studies with Anthopleurin-A toxin. 1995, Pubmed
Shih, High-level expression and detection of ion channels in Xenopus oocytes. 1998, Pubmed , Xenbase
Sigworth, Voltage gating of ion channels. 1994, Pubmed
Stefani, Gating of Shaker K+ channels: I. Ionic and gating currents. 1994, Pubmed , Xenbase
Stühmer, Structural parts involved in activation and inactivation of the sodium channel. 1989, Pubmed , Xenbase
Tang, Role of an S4-S5 linker in sodium channel inactivation probed by mutagenesis and a peptide blocker. 1996, Pubmed
Vassilev, Identification of an intracellular peptide segment involved in sodium channel inactivation. 1988, Pubmed
Vedantham, Slow inactivation does not affect movement of the fast inactivation gate in voltage-gated Na+ channels. 1998, Pubmed , Xenbase
West, A cluster of hydrophobic amino acid residues required for fast Na(+)-channel inactivation. 1992, Pubmed , Xenbase
Yang, Molecular basis of charge movement in voltage-gated sodium channels. 1996, Pubmed
Yang, Evidence for voltage-dependent S4 movement in sodium channels. 1995, Pubmed