C-type inactivation is a time-dependent process observed in many K + channels whereby prolonged activation by an external stimulus leads to a reduction in ionic conduction. While C-type inactivation is thought to be a result of a constriction of the selectivity filter, the local dynamics of the process remain elusive. Here, we use molecular dynamics (MD) simulations of the KcsA channel to elucidate the nature of kinetically delayed activation/inactivation gating coupling. Microsecond-scale MD simulations based on the truncated form of the KcsA channel (C-terminal domain deleted) provide a first glimpse of the onset of C-type inactivation. We observe over multiple trajectories that the selectivity filter consistently undergoes a spontaneous and rapid (within 1–2 µs) transition to a constricted conformation when the intracellular activation gate is fully open, but remains in the conductive conformation when the activation gate is closed or partially open. Multidimensional umbrella sampling potential of mean force calculations and nonequilibrium voltage-driven simulations further confirm these observations. Electrophysiological measurements show that the truncated form of the KcsA channel inactivates faster and greater than full-length KcsA, which is consistent with truncated KcsA opening to a greater degree because of the absence of the C-terminal domain restraint. Together, these results imply that the observed kinetics underlying activation/inactivation gating reflect a rapid conductive-to-constricted transition of the selectivity filter that is allosterically controlled by the slow opening of the intracellular gate.
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Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel
C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K+ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K+ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-μs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D.
more » « less- NSF-PAR ID:
- 10284885
- Publisher / Repository:
- DOI PREFIX: 10.1085
- Date Published:
- Journal Name:
- Journal of General Physiology
- Volume:
- 153
- Issue:
- 9
- ISSN:
- 0022-1295
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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