T.

Paldi: NaChBac independent-gating model

A NaChBac Independent-Gating Model Elucidates Exchanges between Closed- and Open-State Inactivation
Tzur Paldi

Abstract Voltage gated ion channels open their gates in response to membrane depolarization, allowing selective passage of ions into or out of the cell. During prolonged depolarizations, the activated channels may lose their conductivity in a complex process called inactivation. Here, the structural mechanisms of channel activation and inactivation were modelled using data from the bacterial sodium channel (NaChBac). It is shown that both activation and inactivation fit an independent model, in which the four subunits of the channel participate in gating in a non-cooperative manner. The model also reveals the relationship between closed-state inactivation and open-state inactivation, and draws key elements that determine the inactivation type in different ion channels. Keywords C-type inactivation ion channel modeling voltagegated ion channels voltage sensing domain

Introduction Voltage-gated ion channels (VGICs) are ubiquitous membrane proteins that are pivotal in cell excitability [1], and to signal transduction of non-excitable cells [2]. Channels belonging to the VGIC superfamily are tetrameric or pseudo-tetrameric proteins that sense changes in the membrane potential using a voltage sensing module (VSM), and respond by pore opening that enables selective ion flux. In many cases, VGICs undergo a timedependent loss of conductivity during sustained depolarization, due to specific channel conformational changes, called inactivation, that restrict ion access through the channels pore. In voltage gated K+ channels (Kvs) two main mechanisms of inactivation have been characterized. The first, known as N-type inactivation, entails a relatively fast ball and chain mechanism, in which a cytoplasmically located N-terminal portion of the channel peptide diffuses into the mouth of the inner vestibule
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Author: Tzur Paldi Email: tzur.paldi@outlook.com 2013 Tzur Paldi, All Rights Reserved
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through the open activation gate (lower gate), thereby blocking conduction [3-5]. The second inactivation mechanism is the slow inactivation (termed P/C-type or as referred here as C-type inactivation), which originates from transitions at the selectivity filter at the outer pore region (upper gate) [6, 7], and often develops with much slower kinetics that is highly modulated by permeant ions and blockers [8-14]. Inactivated channels cannot reopen before they undergo recovery at hyperpolarizing potentials (negative inside), where the duration of the recovery from C-type inactivation is usually much longer, compared to recovery from N-type inactivation. While the structural mechanism of N-type inactivation, and its association with channel opening is well known [3-5, 15], Ctype inactivation is not so well defined. Upon changes in membrane potential, VGICs may inactivate by closure of their upper gate via two pathways: an open state (open-state inactivation, OSI), at strongly depolarized membrane potentials, or via a pre-open closed-states (closed-state inactivation, CSI), at hyperpolarized or modestly depolarized membrane potentials [16]. Most VGICs use both of these pathways, but channels show preferential usage of either the OSI or the CSI pathway. Currently, the molecular mechanism underlies these two pathways is not fully understood. Hodgkin and Huxley assumed that potassium ions could only cross the membrane when four similar particles occupy a certain region of the membrane [17]. Their statement regarding potassium ions permeability constitutes a primary gating model of interdependency among homotetrameric VGIC subunits (the HH model). Their model is based on the independent operation of four gating particles (equivalent to four VSM subunits in a homotetrameric channel), each undergoing a single transition from a closed state to an active state; the channel only conducts when all four are in the active state. Addition of one state to the original HH model resulted with a three-state, four-subunit model, which better describes many aspects of the tetrameric K + channel behavior [18], and is presented as follows: (C1 C2 C3)4 O where each of the four subunits proceeds independently through hree closed states (C1 to C3) toward the open state (O). While non-cooperative (independent) model for channel activation is backed experimentally [19], C-type inactivation, which is

T. Paldi: NaChBac independent-gating model

coupled to channel activation [20, 21], is thought to be involved with cooperative transition of the four channel subunits [19, 22, 23]. The bacterial voltage-gated Na+ channel from Bacillus halodurans (NaChBac) is a tetrameric channel, which adopts only a C-type mechanism of inactivation [24-26], therefore it is beneficial system for studying C-type inactivation. Here, a noncooperative C-type inactivation mechanism that is based on fitting of the gating properties of NaChBac to an independentgating model is suggested. The model, which correlates VSM activation with gating of both the upper and the lower channels gates, indicates that both NaChBac activation and inactivation require independent transitions of the four channel's subunits. Here, the implications of this independent-gating model on the mechanism that partitions C-type inactivation between the OSI and the CSI pathways are discussed, and are tested qualitatively against previous work done on this subject.

Materials and methods Recordings of macroscopic Na+ currents mediated by NaChBactransfected CHO cells were performed, using the whole-cell conguration of the patch-clamp technique, as previously described [27].

Results and discussion NaChBac steady-state activation and inactivation, fits an independent gating model The VSM is a four membrane-spanning domain, in which its fourth segment (S4) is typically embedded with up to eight positively charged residues at every third position. The VSM of ion channels is structurally [28-31] and functionally [32] independent of the channels pore domain. Nevertheless, delineating the nature of the gating process in the tetrameric channel is not straightforward since the interactions between the VSM and the pore domains are still ambiguous. Toward this goal, a model that correlates VSM activation to channel gating properties was developed and tested against experimental data. The interaction of S4 with the membrane electric field renders conformational changes in the VSM, which are reflected by a capacitive current (charge transfer) that precedes the ion current (the so called gating current) [33]. The probability of this charge transfer in a single VSM upon membrane depolarization should be the same as that of an independent activation of all the four VSMs in a tetrameric channel. Thus, as stated by the HH model:

(1) po = pQ4 Where po is the channel open probability and pQ is the probability of charge transfer in a single VSM upon membrane depolarization. Gating and ion current data from the work of Kuzmenkin et al. [25] was used to assess this relation in NaChBac. The fourth power of the Boltzmann fit of the normalized Q-V curve (relates to pQ), according to Eq. 1, gives an excellent fit with the normalized G-V curve of NaChBac (relates to po; Fig. 1 A). This steady-state activation data is consistent with the independent HH model for channel activation that requires activation of all four VSMs for channel opening. C-type inactivation is correlated with VSM activation [21, 34]. Although it requires activation of all four subunits [19], channel opening is not requisite for the occurrence of C-type inactivation since channels may inactivate via the CSI pathway (see review in ref. 16). The ability of NaChBac to inactivate by independent activation of the four VSMs is examine as follows: Since NaChBac inactivates also via the CSI pathway, it is hypothesized here that only those channels in which all four VSM remained deactivated (i.e., in their resting state) during membrane depolarization, are available for opening by a subsequent depolarizing pulse according to: (2) pavail* = (1-pQ)4 where pavail* is the theoretical probability of the channel to be found in the available state, and 1-pQ represents the probability of a single VSM to remain non-activated (i.e, at rest position). Thus, any channel state combination (at the single-channel level) that includes one or more activated subunits would reduce the channel availability. Insertion of the theoretical pQ (pQ*; Fig.1 B, Q-V*), calculated from the normalized G-V curve according to Eq. (1), into Eq. (2) gives: (3) pavail* = (1-po)4 Since NaChBac half-life time for inactivation decreases with larger depolarizations (Fig 1 C), channel availability was tested using two prepulse (pp) durations of 4 and 15 seconds in the voltage-clamp protocol (Fig. 1 D, bottom). The theoretical availability curve describing pavail* as a function of V (Iavail-V*), using the experimental G-V curve of NaChBac according to Eq. (3), fits between the two experimental availability curves (Fig. 1 B; Iavail-Vpp=4s: V0.5= -79 mV 0.7, n= 9; Iavail-Vpp=15s: V0.5= -87 mV 1.2, n= 7; and Iavail-V*: V0.5= -83 mV). These results fit channel inactivation with independent activation of its VSMs. According to Eq. (1) and Eq. (2) the G-V and the availability curves intersect when pQ = 0.5. Indeed, the steady-state availability curves, when the prepulse was 4 s or 15 s, intersect with the G-V curve at or a little below to this point, respectively (Fig. 1 B).

T. Paldi: NaChBac independent-gating model

The fraction of channels that inactivate via the OSI pathway (pOSI) is represented by po, therefore, the availability curve of NaChBac at steady state could be generally described as follows: pavail = 1-(pCSI + po) were pCSI represents the fraction of channels that are inactivated through the closed state (Fig. 1 B, CSI dotted curve). From this equation it is clear that the availability curve of channels who do not exhibit any preferential to the CSI pathway, would be a mirror image of their G-V curve (1-po).

inactivation is responsible for both OSI and the CSI [37, 38], but the mechanism that underlies the CSI pathway is still unclear. An independent-gating model fits subunit accumulation of inactivation Like activation, NaChBac inactivation fits an independent gating model of the VSM operation by fitting with Eq. (2) (Fig. 1 B), suggesting that in steady state, activation of at least single VSM (i.e., all channel formations except of the resting state) would eventuate in NaChBac inactivation. To comply with the requirement of four activated subunits [19, 22, 39] it is suggested here that the observed accumulation of channel inactivation with time (Fig. 1 C) also reflects changes at the level of a singlechannel, as follows: Upon membrane depolarization from the resting state channels are distributed among 15 voltagedependent initial formations in which each activated VSM independently triggers the inactivation component of its own/related subunit (referred here as triggered subunit); only the channels, whose four activation gates are open at once, could inactivate through the OSI pathway, while the non-conducting, partially activated channels, inactivate through the CSI pathway (Fig. 2). The accumulation of triggered subunits is enabled only when the OFF-inactivation constant (Ioff) is smaller than the ONinactivation constant (Ion). Under these conditions, CSI is favored at hyperpolarizing to moderate depolarizing potentials because triggering of inactivation, which is related to pQ, is substantially larger than channel opening (po; Fig. 1 A, B), which require coordinated activation of the four VSMs. For example, at subthreshold voltages for NaChBac opening, at around -80 mV, NaChBac opening is nearly undetectable (po = 0.24 = 0.16%), but the probability for VSM activation and inactivation triggering is substantially large with pQ value of 20%. At higher depolarizing potentials the CSI probability declines, up to the point where pQ (Q-V curve) and po (G-V curve) approach each other, where the OSI is favored to the detriment of the CSI pathway. Large Ion/Ioff ratio in C-type inactivation may underlie the slow recovery that leads to cumulative inactivation [40], and development of C-type inactivation during recovery from N-type inactivation [8]. Permeant ions within the pore effectively hold the channel open by preventing the protein structure from relaxing to close the gate. Accordingly, non-conducting channels, in which their pore is blocked by N-type inactivation particle [8, 20, 41] or by blockers [42-45], exhibit accelerated C-type inactivation, probably by inhibiting the ion flux, which normally delays Ctype inactivation [8, 42]. This ion-mediated regulation of gating is seems to be an inherent mechanism of the cation channels superfamily as was suggested recently [46]. To meet these requirement, the model incorporates distinct kinetics of inactivation for inactivation via the non-conducting (CSI, fast)

Figure 1 Charge movement, ionic conductance and steady-state inactivation relationship. (A) Q-V and G-V curves of NaChBac (Data from Kuzmenkin et al., 2004. See ref. 25). The exponential function of the normalized Q-V curve ([Q-V]4; dashed curve) fits the normalized ionic G-V curve. (B) Steady-state activation and availability curves of NaChBac, and the theoretical Q-V (Q-V*; yellow) and availability (Iavail-V*; blue) curves, are presented. The voltage dependence of the close state inactivation curve (CSI-V; turquoise) is calculated by subtraction of Po from (1 - Pavail*). (C) Normalized NaChBac current dependency on prepulse duration at the indicated voltages. (D) Voltage protocols for generating the G-V curves (top) or the availability curves (bottom).

When it comes to channel inactivation, NaChBac shows preferential CSI at all relevant voltages. While C-type inactivation is evidently associated with the activation of the VSM [19, 20, 35, 36], this does not provide an obvious explanation for the mechanism that underlies the distinct OSI and CSI pathways. More puzzling is the fact that the C-type

T. Paldi: NaChBac independent-gating model

Figure 2 Independent model for NaChBac inactivation. The activation of a VSM from the resting state (four large open circles; left) upon membrane depolarization, triggers the inactivation component of its own/related subunit (filled circle). The slow return from the inactivation (Ioff < Ion) in each subunit enables accumulation of the inactivation components. Only channels activated in coordination, would be slowly inactivated through the OSI pathway (activation elements are not shown).

and the conducting (OSI, slow) pathways. This distinction is important to explain changes in the kinetics of C-type inactivation by a single non-cooperative gating model. A consolidated independent gating model for both channel activation and inactivation, harboring the aspects mentioned above, is presented in a form of a three-state four-subunit kinetic model in Scheme I, where C1 (Closed 1) and C2 (Closed 2) and AT/AR (Activated) represent the closed, intermediate closed and the activated states of the VSM, respectively. R, O and I represent rest, open and inactivated channel states, respectively. That is to say, that channel opening and triggering of inactivation require that all four subunits occupy an activated state. The suggested condition in which Ioff << Ion enables accumulation of inactivation elements in a single channel may also explain the left shift of the availability curve of NaChBac, with increased prepulse durations. AT and AR represent two structural isoforms of the activated VSM (tensed and relaxed), that were previously linked to C-type inactivation [47].

Scheme I A three-state four-subunit channel gating model.

Interconversions between the OSI and the CSI pathways by channel deactivating pulse The transition of a channel from being fully activated at high depolarizing potentials to a state that is partially activated at lower depolarizing potentials, and vice versa, reflects macroscopic interconversions between the OSI and the CSI pathways in many VGICs. However, it is quite possible that a single channel would shift from the OSI to the CSI pathway at certain circumstances. In several VGICs that do not show clear inactivation phase by using a simple activation pulse, such as in HERG and channels from the Kv7 family, it is possible to examine inactivation using a three-pulse protocol, in which a brief hyperpolarizing interpulse separates two activating pulses; the brief hyperpolarizing interpulse is essential to reveal the inactivation phase observed in the second activating pulse (Fig 3 A) [48-51]. The mechanism, by which a short deactivating interpulse exposes inactivation, is still unclear. An attempt to explain this accelerated inactivation as recovery of closed-state inactivated channels and enabling their inactivation via the open state [51] is inconsistent with the similar kinetics observed for recovery from CSI and OSI [52]. However, in Kvs exhibiting a clear slow inactivation phase after an activating pulse, accelerated inactivation is observed after a short deactivating interpulse [53] or during repetitive depolarizing pulses alternating with hyperpolarizing pulses (Fig. 3 B) [54, 55], implying that short deactivating pulse mediates a kinetic transition in the inactivation process. A more direct observation of this phenomenon was shown in a fluorometric measurement of labeled Shaker K+ channel reporting structural changes in the pore due to C-type inactivation. In their experiment, Loots and Isacoff [20] showed that shortening the time length of a deactivating hyperpolarizing interpulse between two test pulses, switches the kinetics associated with inactivation to a faster one. The authors explained this transition as an entry into a less stable, which they called P-type inactivated closed state, that is followed by a slower

T. Paldi: NaChBac independent-gating model

Figure 3 Repetitive three-pulse protocol reveals (A) inactivation of KCNQ1 channel. (Reprinted from J Physiol, Vol 545, Peretz, A., et al., pp. 751-66. Copyright 2002, with permission from Wiley) or (B) excessive cumulative inactivation in Kv3.1 (Reprinted from Biophys J, Vol 81, Klemic, K.G., et al., pp. 814-826. Copyright 2001, with permission from Elsevier)

Figure 4 Low pH-induced acceleration of C-type inactivation. The two pathways for C-type inactivation (upper gate closure) are dependent on the open probability of the activation gate (lower gate). (Bottom) Slow inactivation through the open state (OSI) prevails at large depolarization (>>V1) where all the VSMs are coordinately activated. (Top) A stochastic VSM activation leads to cumulative, fast inactivation through a closed state (CSI). Low pH (as well as cAMP depletion in HCN, or short hyperpolarizing pulses) diverts the channels to the CSI (fast) pathway. Permeant cations are presented as filled circle.

entry into the more stable closed C-type state which its stabilization is in part related to the extended position of S4. The transition from more stable C-type closed state to less stable P-type inactivation is interpreted here, and generally in agreement with an OSI to CSI transitions during the channel slow inactivation process. That is to say, P -type and C-type inactivation are related to the CSI and the OSI pathways, respectively, and are both related to closure of the same upper gate. The mechanism underlies OSI to CSI transition of a single channel as a result of a short hyperpolarizing prepulse is provided by the independent-gating model described above. As mentioned above, blocking the pore either by the N-type particle or by other exogenous blockers may accelerate C-type inactivation [8, 4245]. This phenomenon is explained by the foot-in-the-door mechanism, in which binding of permeant ions to sites in the pore stabilize its open state. In this sense, closure of the lower gate imitates pore blocking but on the same time it is usually linked to stabilizing the open state of the upper gate that enables channel recovery from inactivation. For this reason the effect of channel

blocking by the lower gate on C-type inactivation is somehow elusive. Nevertheless, brief deactivating interpulse that would block ion passage by momentary shutting the lower gate, but is still fast enough not to allow full recovery of triggered subunits, would initiate faster collapsing of the upper gate as described for the CSI pathway. Thus, the observed outcome of a short prepulse would be accelerated C-type inactivation by diverting more channels from the OSI to the CSI pathway (Fig. 4). Of note is that channels that enters to the CSI from the OSI pathway (opposed to entrance to CSI from the rest state) are not necessarily remained inactivated after the brief deactivating pulse, but rather undergo accelerated inactivation during the switch to the CSI pathway. Induction of OSI to CSI pathway conversion by channel modulators Ion channel modulators are substances that interact with ion channels and change their gating properties. Shin et al. [56] have shown that cAMP couples between the VSM and the pore

T. Paldi: NaChBac independent-gating model

activation gate (i.e., the lower gate) of HCN channels, and therefore depletion of cAMP enhances reclosure of the lower gate by decoupling it from the activated VSM. The authors concluded that reclosure of the lower gate in the absence of cAMP, enhances a closed state inactivation of spHCN and HCN channels, however, they explained the mechanism of inactivation as reduction in the open probability of the lower gate without referring to the likely systematic coupling with the upper gate in the inactivation process (discussed below). Extracellular protons are widespread modulators of the VGIS superfamily. Extracellular acidification reduce the peak current and enhance current decay in many VGICs [1], but the mechanism underling acid-induced current attenuation is not fully clear. In Kvs, it was revealed that acid-induced current inhibition results from accelerated C-type inactivation [20, 57], and Fedida and coworkers further suggested that the major mechanism of pH-induced peak current reduction is inactivation of channels via the CSI pathway [37]. Recent finding showing that protons react with S4 arginines in the VSM of NaChBac, suggests that protonation of these arginine enhances an inward, deactivating movement of S4 in activated channels [27]. Furthermore, it was shown that point mutation on S4 that stabilizes the open activated state (R1K) is not affecting CSI at low pH, while mutation stabilizing the closed deactivated state (R4K) markedly enhances CSI at low pH. These findings support the model in which reclosure of the lower gates by deactivated VSMs (i.e., by inward movement of S4) has a major role in diverting channels to the CSI pathway. Ion current attenuation or inactivation of HCN channels by cAMP depletion or by extracellular protons on VGICs generally, fit nicely with the OSI to CSI conversion model presented in Fig. 4. In both of these instances, the VSMs of the channels are activated and thus enable triggering of the subunits, and the modulator mediates flickering closure of the activation gate, which diverts the channels to the CSI pathway. Flickering closure of the lower gate is equivalent to closure of this gate by short deactivating prepulse, which diverts channels to the faster CSI pathway to the detriment of the OSI pathway.

coupled to the VSMs, with slow C-type inactivation occurring in the upper gate. There are at least two opposing components affecting C-type inactivation that are coupled to activation of the VSMs: an allosteric coupling between opening of the lower gate and destabilization of the selectivity filter [58], and stabilization of the selectivity filter by occupancy of permeant ions. Thus, differences in the kinetics associated with these gate-affecting factors may show different gating properties in various channels. For example, channels in which permeant ions stabilize their selectivity filter in a very potent manner, would prevent or delay C-type inactivation at high Po, and would probably inactivate mainly via the CSI pathway while exhibiting U-shaped steady state inactivation curve (U-type inactivation). The model here shows that different variations in the kinetic parameters of the upper and lower gates (but is not limited to these gates), may explain distinct channel behavior that is currently considered as different gating mechanisms. Thus CSI and OSI may be two pathways of C-type inactivation if we focus our analysis only on the upper and the lower gates alone. From an evolutionary perspective, this model describes an economical solution in which relatively small structural variations can provide diverse autoregulation pathways in primordial and also in more complex VGICs such as the voltage gated sodium channels [59].

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T. Paldi: NaChBac independent-gating model

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