Inactivation in the potassium channel KcsA

Figures

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  Fig. 1. Overview of the structure of homo tetrameric KcsA and the reported functions. The protein has been dissected into the outer month (Raghuraman et al., 2014), the selectivity filter (Zhou et al., 2001), the pore helix (Bhate and McDermott, 2012), the hinge, the N-terminal helices (Iwamoto and Oiki, n.d.), the pH gate (Thompson et al., 2008) and the C-terminal helices (Uysal et al., 2009).

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  Fig. 2. Sequence alignment of KcsA and other potassium channels. The stretch of residues highlighted in yellow is the most conserved region, and is termed the selectivity filter because of its involvement in binding and conduction of K+ ions. Residues highlighted in pink have been shown to be critical for the inactivation in KcsA.

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  Fig. 3. Inactivation of potassium channels. (a) The macroscopic current of KcsA in the patch-clamp measurement. KcsA is a pH-gated potassium channel. After activation by a pH drop from pH 8.0 to 3.0, the channel inactivates spontaneously and the current decreases gradually (b) The macroscopic current of Shaker measured by the patch-clamp technique. Shaker is a voltage-gated potassium channel, activated by switching the voltage from −100 to 50 mV. Shaker has two inactivation processes, the fast-decay current for the N-type inactivation and the slow-decay current for the C-type inactivation after the residues 6–46 were deleted. Adapted from reference (Sudha Hoshi et al., 1991). (c) Activation-coupled inactivation mechanism shown in KcsA. At pH 8.0, the channel is in the deactivated state; the activation gate is closed and the selectivity filter is in the conductive conformation. After the pH is dropped to 3.0, the channel converts to the activated state and both the activation gate and the selectivity filter are in the open conformation; potassium ions flow through the channel. The opening of the pH gate is also slowly coupled to the selectivity filter and biases the collapsed…

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  Fig. 4. NMR chemical shifts indicate conformational changes and a potassium affinity change at the selectivity filter. (a) Similar chemical shift changes imply similar structural transitions for the selectivity filter of KcsA, regardless of pH, as indicated by the 13C–13C marker peaks of residues at the selectivity filter (e.g., T74, T75, V76). Presumably these structural changes correspond to conformational change between the conductive and collapsed selective filter seen in crystal structures. (b) Population of the K+-apo (collapsed) and K+-bound (conductive) states in the structural transitions modulated by ambient [K+]. (c) Titration experiments show that the potassium affinity changes by more than three orders of magnitude from 4 ± 1 μM for pH 7.5 and 14 ± 1 mM at pH 3.5. This indicates that the pH gate and the selectivity filter is strongly coupled at WT-KcsA. While similar measurements on the mutant F103A, in which C-type inactivation is greatly suppressed, a much smaller affinity change is measured, indicating a reduced coupling network. (d) Single channel recording experiments show that F103A can rescue the enhanced C-type inactivation caused by E71H, thus F103A reduces…

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  Fig. 5. Buried water molecule(s) are critical to stabilize the collapsed conformation during inactivation, as shown by computational and SSNMR method. (a) Potential force calculation shows that when the water molecules are buried behind the selectivity filter, there is a high energy barrier to move K+ ion into the S2 site and convert the selectivity filter into a conductive conformation. However, when these waters are removed, the energy barrier for the transition is reduced. (b) SSNMR study confirms that there are buried water molecules behind the selectivity filter in the collapsed state, which are not present in the conductive conformation. Taken from reference (Ostmeyer et al., 2013, Weingarth et al., 2014).

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  Fig. 6. (a) NMR chemical shifts monitor a global structural change during the K+-apo (collapsed) and K+-bound (conductive) transition in response to ambient [K+] change. (b) NMR chemical shifts report \ on change of protonation state in the pH sensor in response to pH change. The pH sensor also gets protonated at pH 7.5, 0.2 μM [K+], indicating a shifted pKa caused by the coupling to the selectivity filter. Low ambient [K+] collapses the selectivity filter, which favors an open pH gate. (c) Residues of known functional importance display large chemical shift perturbations between conformational changes. This correlation demonstrates the power of NMR to study the channel conformal change and allostery. Taken from reference (Wylie et al., 2014).

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  Fig. 7. Crystal structures show that the extent of opening at the pH gate is coupled to the conformation and ion binding at the selectivity filter. Adapted from reference (Cuello et al., 2010a).

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  Fig. 8. Another scheme proposed to explain the inactivation process. (a) Various sites at the selectivity filter have been shown to be important for inactivation or recovery from inactivation. (b) In this hypothesis, ion binding at the S2 site leads to the deformation at the S3 site, blocking the ion flow and causing inactivation. Adapted from reference (Matulef et al., 2016).

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  Fig. 9. The ion dilation hypothesis proposed by Armstrong et al. to explain the inactivation process in Shaker channel. The C-type inactivation starts with a local deformation at the outer mouth of the selectivity filter, which blocks the ion flow. After the potassium ions are depleted from the selectivity filter, the channel dilates and allows partially dehydrated sodium ions to flow through the channel. The channel eventually enters a defunct state, in which the selectivity filter is in a collapsed conformation. Adapted from reference (Hoshi and Armstrong, 2013).