Ogielska EM and Aldrich RW (1999), Functional consequences of a decreased potassiu...

XB-ART-13583

J Gen Physiol 1999 Feb 01;1132:347-58. doi: 10.1085/jgp.113.2.347.

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Functional consequences of a decreased potassium affinity in a potassium channel pore. Ion interactions and C-type inactivation.

Ogielska EM , Aldrich RW .

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Ions bound near the external mouth of the potassium channel pore impede the C-type inactivation conformational change (Lopez-Barneo, J., T. Hoshi, S. Heinemann, and R. Aldrich. 1993. Receptors Channels. 1:61- 71; Baukrowitz, T., and G. Yellen. 1995. Neuron. 15:951-960). In this study, we present evidence that the occupancy of the C-type inactivation modulatory site by permeant ions is not solely dependent on its intrinsic affinity, but is also a function of the relative affinities of the neighboring sites in the potassium channel pore. The A463C mutation in the S6 region of Shaker decreases the affinity of an internal ion binding site in the pore (Ogielska, E.M., and R.W. Aldrich, 1998). However, we have found that this mutation also decreases the C-type inactivation rate of the channel. Our studies indicate that the C-type inactivation effects observed with substitutions at position A463 most likely result from changes in the pore occupancy of the channel, rather than a change in the C-type inactivation conformational change. We have found that a decrease in the potassium affinity of the internal ion binding site in the pore results in lowered (electrostatic) interactions among ions in the pore and as a result prolongs the time an ion remains bound at the external C-type inactivation site. We also present evidence that the C-type inactivation constriction is quite local and does not involve a general collapse of the selectivity filter. Our data indicate that in A463C potassium can bind within the selectivity filter without interfering with the process of C-type inactivation.

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	Figure 2. Properties of C-type inactivation with potassium as the conducting ion. Experiments were done in inside-out patches. Current traces were elicited by voltage steps to +50 mV. Except for F, all experiments were performed in the presence of 140 mM internal potassium, and currents were elicited by 10-s voltage steps. Data from the wild-type Shaker channel are shown in A and B, while data from A463C are shown in C–F. (A) The wild-type Shaker channel inactivates with a time constant of 1.4 s in the presence of 2 mM K and 140 mM Na in the external solution. (B) Increasing the external potassium to 140 mM slows C-type inactivation in the wild-type Shaker channel (τ = 2.6 s). (C) C-type inactivation is slow in A463C with 2 mM K and 140 mM Na in the external solutions. Neither increasing the external potassium concentration to 140 mM (D), nor replacing it with external NMG+ (E) has any effect on the time course of C-type inactivation in A463C. However, with 5 mM internal and no external potassium (NMG+), the efflux through the pore is decreased and the A463C channel readily inactivates (F).
	Figure 3. Properties of C-type inactivation with sodium as the conducting ion. All traces were elicited with voltage steps to +100 mV and in the presence of 140 mM internal sodium. Experiments were done in inside-out patches. (A) The wild-type Shaker channel inactivates rapidly (<20 ms) in the absence of added potassium and in the presence of symmetrical 140 mM sodium. Currents were elicited by a 100-ms voltage step. (B) In contrast, no inactivation is observed in the A463C mutant under the same ionic conditions. Currents were elicited by a 400-ms voltage step. (C) A slow decline in current is observed with longer pulses (2,000 ms). (D) In the presence of external NMG+, the A463C mutant rapidly inactivates to a steady state level. Pulse duration was 400 ms.
	Figure 4. Both external sodium and internal potassium can slow inactivation in the A463C mutant. The control traces were elicited by a 400-ms voltage step to +100 mV in the presence of 140 mM internal sodium and external NMG+. The experiment in A was performed with an outside-out patch, while the experiment in B was with an inside-out patch. Inactivation can be slowed by 1 mM external sodium (A) and by 2.5 mM internal potassium (B). For easier comparison, the current amplitudes are scaled and the baselines subtracted in the bottom sections of both panels.
	Figure 5. Increased occupancy of the external site of A463C results from decreased repulsive interactions between ions in the pore. (A) A model of the effects of increasing ion occupancy at the internal site on C-type inactivation in symmetrical sodium solutions. Potassium ions are depicted as • and sodium ions as ○. (B) The control trace was elicited by a 1,000-ms voltage step to +100 mV in an inside-out patch in symmetrical 140 mM NaCl. The addition of 1 mM internal potassium results in 13 ± 0.03% block and an increase in the C-type inactivation rate.
	Figure 6. External potassium blocks sodium currents in A463C with a high affinity but does not slow C-type inactivation. Data shown in A were obtained from an outside-out patch in the presence of external NMG+ and internal Na+. Currents were elicited by voltage steps to +100 mV and externally applied potassium blocked the observed currents in a concentration-dependent manner (A). The data were quantified by measuring percent current remaining (I/Imax) at +100 mV versus the external potassium concentration (B, •). The error bars represent the SEM. A fit to the data with a single binding isotherm yields a Kd value of 100 μM. The empty symbols represent the identical experiment except that it was performed in the presence of symmetrical sodium. Currents were elicited by voltage steps to +100 mV or by voltage ramps from −100 to +200 mV (data not shown). The blocking data were identical regardless of the voltage protocol used and as a result it was pooled. The error bars represent the SEM. A fit to the data with a single binding isotherm yields a Kd value of 300 μM. (C) The control current and the current in the presence of 50 μM external potassium from A were scaled in amplitude and the baselines were subtracted. No change in the C-type inactivation rate is observed in the presence of 50 μM external potassium.
	Figure 7. State diagram model of external potassium block of sodium currents in A463C. Sodium ions are represented by ○, while potassium ions are shown as •. (Outlined scheme) In the presence of external NMG+, external potassium first binds to the C-type inactivation site but proceeds to a deeper, higher affinity site in the pore. The occupancy of the deeper site by potassium does not interfere with the onset of C-type inactivation. (Shaded scheme) In the presence of external Na+, external potassium must first displace the bound sodium ion before it can proceed to the deeper site. After the higher affinity site is reached, the external (C-type) site is refilled by a sodium ion.

References [+] :

Baukrowitz, Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms. 1995, Pubmed