Molecular driving forces determining potassium channel slow inactivation

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassiumselective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.solution NMR | ion channels | membrane protein | protein dynamics

Section: Discussionsupporting

confidence: 78%

mentioning

confidence: 99%

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

Brettmann

Urusova

Tonelli

et al. 2015

“…This discrepancy between what the crystal structure and the functional data show is explained by the results of Cordero-Morales et al (23) who found that inactivation in KcsA is the result of a conformational change in the selectivity filter region which blocks ion conduction even though the bundle-crossing gate is open. Several features of slow inactivation found in KcsA have counterparts in eukaryotic voltage-gated channels, making the Cordero-Morales explanation a viable one for the molecular basis of slow inactivation in voltage-gated channels (24). Therefore, the most likely functional state that correlates to the crystal structure of both Kv1.2 and the chimera of Kv1.2 with Kv2.1 is the open-inactivated state that has the sensor in the relaxed state, the bundle crossing is in the open state, but the pore is nonconducting because the selectivity filter is in the inactivated state.…”

Section: Discussionmentioning

confidence: 99%

S4-based voltage sensors have three major conformations

Villalba-Galea

Sandtner

Starace

et al. 2008

197

370

Voltage sensors containing the charged S4 membrane segment display a gating charge vs. voltage (Q-V) curve that depends on the initial voltage. The voltage-dependent phosphatase (Ci-VSP), which does not have a conducting pore, shows the same phenomenon and the Q-V recorded with a depolarized initial voltage is more stable by at least 3RT. The leftward shift of the Q-V curve under prolonged depolarization was studied in the Ci-VSP by using electrophysiological and site-directed fluorescence measurements. The fluorescence shows two components: one that traces the time course of the charge movement between the resting and active states and a slower component that traces the transition between the active state and a more stable state we call the relaxed state. Temperature dependence shows a large negative enthalpic change when going from the active to the relaxed state that is almost compensated by a large negative entropic change. The Q-V curve midpoint measured for pulses that move the sensor between the resting and active states, but not long enough to evolve into the relaxed states, show a periodicity of 120°, indicating a 3 10 secondary structure of the S4 segment when determined under histidine scanning. We hypothesize that the S4 segment moves as a 3 10 helix between the resting and active states and that it converts to an ␣-helix when evolving into the relaxed state, which is most likely to be the state captured in the crystal structures.A number of membrane proteins respond to changes in the membrane electric field via an intrinsic voltage sensor in the protein structure (1). The classical examples, first described by Hodgkin and Huxley (2), are the voltage-dependent sodium and potassium conductances, which are crucial players in the generation and propagation of the nerve impulse. In the voltagegated ion channels that generate these conductances, the movement of the voltage sensor generates a transient current that has been traditionally called gating current, because in the original recordings it was correlated with the opening of the conduction pathway in Na channels (3, 4). Gating currents are transient because the sensing or gating charges are tethered to the protein and so their movement is restricted within the membrane electric field. Most of the moving charges that produce gating currents have been identified as the four most extracellular basic residues of the fourth transmembrane segment (S4) in voltage-gated channels (5, 6).At extremely negative and positive membrane potentials the gating charges are driven to extreme positions so that a plot of the transported charge as a function of the membrane potential (the Q-V curve) has a sigmoid shape that saturates at extreme potentials. The voltage dependence of this charge movement is an expression of the amount of charge involved and the number of states that the sensing charge populates when moving between the extreme positions. The steepness of the Q-V curve in going from one extreme position to the other increases by increasing the moving charge o...

“…In KcsA, the selectivity filter is stabilized by a hydrogen-bond network between E71 and D80, W67 and D80, and a water molecule bound to the backbone nitrogens of G79 and L81 (6). It has been proposed recently that the conformational change in the inner helix during gating propagates to the selectivity filter to trigger the slow inactivation by altering the interaction between E71 and D80 (18). In WSK3, the tertiary contact corresponding to E71 and D80 (E50 and D59) is replaced by a quaternary 3-residue contact among E50 and D59 of one subunit and R68 of the adjacent subunit.…”

Section: Discussionmentioning

confidence: 99%

“…A noteworthy difference between WSK3 and detergentsolubilized KcsA is that in KcsA, the selectivity filter appears as rigid as the TM helices (15), whereas in WSK3, the selectivity filter exhibits relatively higher flexibility. A recent structural analysis of the KcsA slow-inactivation mutants (18) seems to suggest that the selectivity filter is ''intrinsically unstable'' and thus prone to structural rearrangement leading to an inactivated open state. Molecular-dynamics simulations seem to support this conclusion (19).…”

Section: Wsk3 Forms Helical Tetramers In Watermentioning

confidence: 99%

NMR studies of a channel protein without membranes: Structure and dynamics of water-solubilized KcsA

Ma¹,

Tillman²,

Tang³

et al. 2008

Structural studies of polytopic membrane proteins are often hampered by the vagaries of these proteins in membrane mimetic environments and by the difficulties in handling them with conventional techniques. Designing and creating water-soluble analogues with preserved native structures offer an attractive alternative. We report here solution NMR studies of WSK3, a water-soluble analogue of the potassium channel KcsA. The WSK3 NMR structure (PDB ID code 2K1E) resembles the KcsA crystal structures, validating the approach. By more stringent comparison criteria, however, the introduction of several charged residues aimed at improving water solubility seems to have led to the possible formations of a few salt bridges and hydrogen bonds not present in the native structure, resulting in slight differences in the structure of WSK3 relative to KcsA. NMR dynamics measurements show that WSK3 is highly flexible in the absence of a lipid environment. Reduced spectral density mapping and model-free analyses reveal dynamic characteristics consistent with an isotropically tumbling tetramer experiencing slow (nanosecond) motions with unusually low local ordering. An altered hydrogen-bond network near the selectivity filter and the pore helix, and the intrinsically dynamic nature of the selectivity filter, support the notion that this region is crucial for slow inactivation. Our results have implications not only for the design of water-soluble analogues of membrane proteins but also for our understanding of the basic determinants of intrinsic protein structure and dynamics. membrane protein ͉ protein design ͉ potassium channels ͉ slow inactivation L ess than 1% of known protein structures belong to transmembrane (TM) proteins. The scarcity of structural data is due to the difficulties associated with handling membrane proteins for structure determination. With few exceptions, membrane proteins are often present at low levels in natural tissues, and the available cellular machinery for membrane insertion often limits the capacity of high-level expression systems. Outside their native environment, membrane proteins are unpredictable in behavior, with proper folding and stability depending on the choice of the membrane mimetic environment in addition to the common variables affecting soluble proteins. Although some membrane proteins can be correctly refolded after misfolding or aggregation during expression and purification, others are affected by these processes irreversibly. Currently, no reliable method is available to predict which membrane mimetic will stabilize a given protein in its native conformation without aggregation, and often an arduous process of trial and error using expensive reagents must be carried out to find appropriate conditions.An intriguing alternative to inserting TM proteins into a membrane mimetic environment for structure determination is to alter the proteins such that a membrane is no longer required. Designing water-soluble analogues of membrane proteins challenges the basic understanding of what st...