Starkus JG et al. (1998), Macroscopic Na+ currents in the "Nonconducting"...

XB-ART-14678

J Gen Physiol 1998 Jul 01;1121:85-93.

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Macroscopic Na+ currents in the "Nonconducting" Shaker potassium channel mutant W434F.

Starkus JG , Kuschel L , Rayner MD , Heinemann SH .

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C-type inactivation in Shaker potassium channels inhibits K+ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na+ and Li+ currents in K+-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na+ and Li+ currents (in K+-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na+ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions.

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Species referenced: Xenopus laevis
Genes referenced: tbx2

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	Figure 1. W434F channels conduct Na+ ions in the absence of internal K+. (A–D) Inside-out macropatch currents from oocytes expressing inactivation-removed ShΔ and W434F channels were evaluated in K+-containing (left) and K+-free Na+ (right) solutions. (A) In physiological solutions, ShΔ channels conduct K+ currents. To permit kinetic comparisons with records in different ionic conditions, the tail current sections of these traces have been magnified 10-fold (inset). (B) The W434F mutant in the presence of K+ ions does not permit ionic current, thus exposing only the ON and OFF gating charge movements. (C) Kinetics of the Na+ tail currents from ShΔ channels in K+-free solutions show a strong dependence on test pulse duration (Starkus et al., 1997). The fast tail currents seen at the shortest test pulse durations convert to slow tail currents as test pulse duration is increased. (D) W434F channels do not undergo marked alterations in kinetics as test pulse duration is increased, as would be expected if these channels are permanently C-type inactivated. The W434F Na+ tail currents are strikingly similar in their kinetics to the ShΔ tail currents after the longest test pulse duration shown here (64 ms). All traces were digitally low-pass filtered at 1 kHz.
	Figure 3. Activation kinetics of Na+ currents through W434F channels. (A) Inside-out current recordings with asymmetrical 115 Na//115 Tris solutions in response to depolarizations ranging from −60 to −10 mV. (B) Current traces elicited by voltage steps ranging from +20 to +100 mV using an inverted Na+ gradient: 115 Tris//115 Na. Holding potential in A and B was −100 mV. (C ) The time course of activation was described by double-exponential data fits (Eq. 2). The resulting time constants of the fast (τf, •) and slow (τs, ○) components are plotted as a function of the test potential. The data points were connected with straight lines. (D) The normalized open-probability after depolarizations for 500 ms was plotted as a function of the test potential. (○) W434F in Na//Na solutions, (□) C-type inactivated ShΔ in symmetrical Na//Na solutions, (▪) noninactivated ShΔ in K//K solutions. All data points are mean ± SD, n = 9–14.
	Figure 4. Kinetics of tail current deactivation in W434F channels. Na+ tail currents from C-type inactivated ShΔ (A) and W434F (B) channels are compared. All data traces are from inside-out patches in asymmetrical 115 Na//115 Tris solutions. Patches were depolarized for 100 ms at +60 mV; the subsequent tail voltages ranged from −90 to −170 mV. (C ) Deactivation time course was described by the sum of two exponential functions. Fits were started 200 μs after the start of the hyperpolarization step to avoid the open-channel currents associated with the rising phase of the voltage clamp. The resulting mean time constants are plotted as a function of the tail voltage. The symbols for the rising phase are (circles) ShΔ and (squares) W434F. The symbols for the falling phase are (downward triangles) ShΔ and (upward triangles) W434F. The data points indicated by filled symbols were used for the estimation of the voltage dependencies according to Eq. 1. The resulting apparent gating charges are for ShΔ: q(rise) = 0.57 e0, q(fall) = 1.15 e0; for W434F: q(rise) = 0.78 e0, q(fall) = 1.04 e0. All data points are mean ± SD, n = 5 patches.
	Figure 5. The rate of gating charge return in W434F channels is affected by ionic conditions. Double-pulse protocols with variable interpulse intervals were used to assess the kinetics of gating charge return by observing the recovery of IgON in the second pulse. (A) In the presence of internal K+, only gating currents are observed and recovery of peak IgON in the second pulse follows the falling phase kinetics of IgOFF. (B) In a K+-free internal Na+ solution, IgON shows an initial delay in recovery that coincides with the rising phase of the ionic tail current. This is followed by a recovery phase in which IgON peaks increase with a time course that parallels the deactivation phase of the ionic tail current. For dashed curves see text.

References [+] :

Baukrowitz, Use-dependent blockers and exit rate of the last ion from the multi-ion pore of a K+ channel. 1996, Pubmed