Gating control and K+ uptake by the KAT1 K+ channel leaveraged through membrane anchoring of the trafficking protein SYP121

Abstract Vesicle traffic is tightly coordinated with ion transport for plant cell expansion through physical interactions between subsets of vesicle‐trafficking (so‐called SNARE) proteins and plasma membrane Kv channels, including the archetypal inward‐rectifying K+ channel, KAT1 of Arabidopsis. Ion channels open and close rapidly over milliseconds, whereas vesicle fusion events require many seconds. Binding has been mapped to conserved motifs of both the Kv channels and the SNAREs, but knowledge of the temporal kinetics of their interactions, especially as it might relate to channel gating and its coordination with vesicle fusion remains unclear. Here, we report that the SNARE SYP121 promotes KAT1 gating through a persistent interaction that alters the stability of the channel, both in its open and closed states. We show, too, that SYP121 action on the channel open state requires SNARE anchoring in the plasma membrane. Our findings indicate that SNARE binding confers a conformational bias that encompasses the microscopic kinetics of channel gating, with leverage applied through the SNARE anchor in favour of the open channel.

the channel pore (Dreyer & Blatt, 2009;Labro, Lacroix, Villalba-Galea, Snyders, & Bezanilla, 2012;Lai, Grabe, Jan, & Jan, 2005;Lefoulon et al., 2014). VSD conformation thus serves the dual function of sensor and regulator for voltage control of K + flux, connecting channel activity to that of other transporters in the membrane, especially to the H + -ATPase and its role in membrane energization.
KAT1 and KC1, a close channel homolog, also bind directly and selectively with the plasma membrane protein SYP121 through a conserved RYxxWE motif located at the cytosolic surface of the VSD (Grefen et al., 2015;Honsbein, Blatt, & Grefen, 2011). SYP121 is one of two so-called Qa-SNAREs (Soluble NSF Attachment protein REceptor) proteins that facilitate accelerated vesicle traffic at the plasma membrane of Arabidopsis (Karnik et al., 2017). In general, SNAREs from target and vesicle membranes drive vesicle fusion and membrane intercalation by assembling in ternary complexes of cognate partner Qa-, Qb-, Qc-and R-SNAREs, designations defined by the respective residues at the core of the SNARE complex (Lipka, Kwon, & Panstruga, 2007). SYP121 binding with the K + channels is independent of channel traffic, however. Instead, SYP121 binds with the channels that are already located at the plasma membrane, and the interaction regulates SYP121-mediated vesicle traffic, enhancing vesicle fusion to facilitate secretion and membrane expansion (Grefen et al., 2015). SYP121 binding also moderates the activities of K + channels at the plasma membrane to promote K + uptake, thereby coordinating the rates of K + uptake with secretory traffic for growth (Honsbein et al., 2009;Karnik et al., 2017). Indeed, vesicle traffic and solute transport must be tightly coordinated to maintain solute content and cell surface area during plant cell expansion. Such coupling between SNAREs and channel VSDs may be common in plants. The binding motifs on both proteins are closely conserved within subsets of plasma membrane SNAREs and K + channels in vascular plants, implying their co-evolution as the number of SNARE genes expanded when plants colonized land (Karnik et al., 2017;Sanderfoot, 2007).
Clearly, the molecular mechanics of the SNARE-channel interaction is particularly important. Not only can this knowledge inform on how channel activity is integrated with the SNARE complex assembly during vesicle traffic, but it is also relevant to understanding the process of channel gating and K + uptake. In general, channel activity arises from millisecond transitions between open and closed conformations of the channel pore. What is not obvious is how these events might be coordinated temporally with the very much slower process of SNARE-mediated vesicle fusion (Jahn & Scheller, 2006). To address this question, we have made use of heterologous expression in Xenopus oocytes which ensures an environment uncomplicated by other, native plant proteins in which to study the SNARE-channel interactions in isolation. Here, we report that SYP121 binding to KAT1 alters the stability of the channel "open" and "closed" states to promote channel activity. Additionally, we show that the bias introduced by binding depends on membrane anchoring of SYP121. Thus, the SNARE alters KAT1 activity through long-lived alterations to the conformational stability of its VSD.
These findings suggest, too, that the bound channel may be integrated stably within the SNARE complex assembly during vesicle fusion.
For patch clamp studies, the vitelline membrane was first removed with forceps after exposing oocytes to a hypertonic solution of the standard buffer plus 245 mM sucrose. SYP121 ΔC was expressed and purified from Escherichia coli BL21 DE3 cells (Karnik et al., 2013) prior to transfer to the final recording buffer.
Borosilicate pipettes with input resistances of 7-10 MΩ were pulled with a PP-83 puller (Narishige, Tokyo). Patch and macropatch recordings were recorded with an Axopatch 200B amplifier driven by Heka software (Heka Elektronik, Lambrecht). Currents were recorded in symmetrical 135 mM K + -gluconate, with 4 mM MgCl 2 , 3 mM EGTA, 0.5 mM CaCl 2 , and 7 mM HEPES-NaOH, pH 7.2, at the outer face of the membrane and with 2 mM CaCl 2 , 6 mM MgCl 2 , and 10 mM HEPES-NaOH, pH 7.2, to give 70 nM free [Ca 2+ ] at the inner face of the membrane (Schoenmakers, Visser, Flik, & Theuvenet, 1992). SYP121 ΔC was added to the bath at 1 μM unless otherwise noted. Single-channel currents were filtered at 1 or 3 kHz with an 8-pole Bessel filter and recorded at 40 kHz. Data segments were collected offline with Acquire (Bruxton, Seattle, USA) and filtered digitally for recurrent noise with N-PRO (Y-Science, Glasgow UK). Single-channel events were reconstructed using the Hidden Markov Model approach with Bayesian restoration (Fredkin & Rice, 1992) of PANDORA! (YScience) and lifetime distributions determined using TAC and TACfit (Bruxton).

| Statistics
Data are reported as means ±SE of n independent experiments, with post-analysis carried out by ANOVA or Student's t test. Joint non-linear, least squares fittings were carried out using the Marquardt-Levenberg algorthm of SigmaPlot v.11 (SPSS, Poole UK).

| SYP121 alters the voltage sensitivity of KAT1
We expressed KAT1 alone and together with SYP121 in Xenopus oocytes to compare the channel activities in isolation from the plant.
Initially, channel current was recorded by two-electrode voltage clamp for analysis of the ensemble current. Steps to −100 mV and more negative voltages yielded inward current relaxations that approached a new steady state within 500 ms (Figure 1a), and steady-state current-voltage (IV) analysis showed a clear inward rectification, much as has been described previously (Lefoulon et al., 2014). By contrast, oocytes injected with water or with SYP121 only showed no appreciable current under these same conditions (less than 0.3 nA inward current at −140 mV, not shown; see , Grefen, Karnik, et al. (2015), and Honsbein et al., 2009). Co-expression with SYP121 enhanced the KAT1 current amplitude and displaced the IV curve to more positive voltages. Binding of SYP121 is disrupted by mutating the R 58 YxxWE motif of KAT1 (Grefen et al., 2015). To confirm that the effects of SYP121 expression depended its interaction with KAT1, we co-expressed SYP121 with the KAT1 W62A mutant.
The amplitude of the current, both with and without SYP121, was reduced in this mutant, consistent with its lower expression ( Figure 1b). However, the KAT1 W62A current was unaffected by coexpression with SYP121, confirming the requirement for SYP121 binding to promote channel gating.
To estimate the effects of SYP121 on KAT1 gating, we fitted steady-state IV curves to a Boltzmann function (Figure 1, Equation (3)) to extract the macroscopic conductance, g max , and the gating characteristics defined by the apparent gating charge, δ, and the voltage yielding half-maximal conductance, V 1/2 . g max is affected by the number of channels at the membrane that are active; δ and V 1/2 reflect the intrinsic voltage sensitivity and free energy of channel gating, and so give insight into how the channels are regulated by voltage. SYP121 expression led to almost a twofold increase in g max and a +10 mV shift in V 1/2 , without an apparent change in δ. We validated these findings by recording tail current amplitudes at +20 mV following channel activation at each voltage ( Figure S1). Scaling the relative amplitudes against those determined at the previously estimated values for V 1/2 gave voltage dependencies that were indistinguishable from relative conductances derived from the Boltzmann fittings ( Figure 1b). Thus, like KC1 (Honsbein et al., 2009), co-expression of SYP121 with KAT1 has an appreciable effect on the voltage sensitivity of the channel gate as well as its macroscopic conductance.
We used patch clamp methods to resolve single-channel currents of KAT1 expressed in the oocytes and thereby identify the FIGURE 1 SYP121 enhances whole-cell KAT1 current. (a) Steadystate current-voltage curves (means ±SE, n > 5 for each data set) and representative current traces recorded on expressing KAT1 (circles) and the non-interacting KAT1 W62A mutant (triangles) without (filled symbols) and with SYP121 (open symbols) using a 1:4 molar ratio of cRNAs (Grefen et al., 2015;Honsbein et al., 2009). Voltage was stepped from a holding voltage of −20 mV to voltages from +30 mV to −180 mV. Solid curves are the results of joint, least squares fitting to the Boltzmann function where g max is the conductance maximum, E K is the equilibrium voltage for K + , V 1/2 is the voltage at mid-point for maximal conductance; δ is the apparent gating charge or voltage sensitivity coefficient. F, R, and T have their usual meaning. Mean g max increased from 0.061 ± 0.009 to 0.096 ± 0.006 mS, and V 1/2 shifted from −132 ± 1 mV to −119 ± 1 mV with SYP121 (P < 0.001). KAT1 W62A current shown scaled (multiplied) by 4 for comparison; its parameters were unaffected by SYP121 (g max , 0.013 ± 0.002 mS; V 1/2 -136 ± 2 mV). Insets: Representative current traces crossreferenced by symbol. Scale: 5 μA (vertical), 1 s (horizontal). (b) Relative conductance-voltage curves for KAT1 ± SYP121 from (a).
Symbols are corresponding tail current amplitudes scaled with the midpoint to give 0.5. Solid line is the fitting of Boltzmann function derived as g/g max Equation (3). Dashed line marks 0.5 g max . Immunoblot analysis of myc-tagged KAT1 and SYP121 from representative oocytes collected after recordings. Ponceau is included as a loading control microscopic properties of channel gating affected by SYP121. KAT1 channels were identified initially on the basis of their inward rectification and single-channel conductance, around 6-9 pS (see Jezek & Blatt, 2017 and references therein), which is an order of magnitude smaller than the dominant Ca 2+ -activated Cl − channels that contribute to the so-called leak current of the oocytes (Sigel, 1990;Weber, Liebold, Reifarth, & Clauss, 1995). Single KAT1 channels showed amplitudes-and hence, a single-channel conductance-that were consistent with the major lifetime distributions previously reported for KAT1 (Zei & Aldrich, 1998

| SYP121 ΔC mediates a subset of KAT1 gating transitions
The truncated SNARE SYP121 ΔC lacks the C-terminal transmembrane anchor but retains the essential, channel-interacting domain and binds with the channel N-terminus in vitro (Grefen et al., 2015;Grefen, Chen, et al., 2010;Honsbein et al., 2011). We used SYP121 ΔC as a tool to dissect the impact on KAT1 gating of SYP121 anchoring and to explore the temporal characteristics of their interaction. Co-expression in whole oocytes showed that SYP121 ΔC altered KAT1 gating but, unlike the full-length SNARE, the effect was to displace the V 1/2 to more negative voltages with little effect on ensemble conductance g max (Figures 3a and S2). We observed a near-maximal effect of SYP121 ΔC with roughly 0.4 ng SYP121 ΔC per oocyte, or 0.14 μM protein on a cell volume basis ( Figure S3), and similar results were obtained with KAT1 in inside-out macropatches on applying 1 μM SYP121 ΔC to the cytosolic face of the membrane ( Figure S4).
Because the truncated SNARE is soluble, it was possible to add SYP121 ΔC directly to the membrane surface during experiments. We  Table 1).
We used these characteristics of KAT1 with τ C3 as a proxy SYP121 ΔC binding and debinding. We reasoned that if the SNAREchannel interaction was long-lived, as suggested by the distributed effects of the full-length SYP121 and the common actions of SYP121 and SYP121 ΔC on τ C3 (Figure 2 and Table 1), then KAT1 gating should not recover on SYP121 ΔC washout. Indeed, following SYP121 ΔC additions τ C3 failed to return to its pretreatment value, even a prolonged washout of 5 min (Figure 4a).
Membrane voltage drives the channel between conformations that bury much of the VSD in the membrane (the "up" state) when  Figures 2, 3, and 4 and are indicated within each subtable. For Figure 4, the protocol is referenced in square brackets. Lower case letters indicate significant differences for any one time constant at P < 0.01. Upper case letters indicate significant differences for any one time constant at P < 0.001.
depolarized and that expose it to the cytosol (the "down" state) when hyperpolarized (Latorre et al., 2003;Lefoulon et al., 2014;Palovcak et al., 2014). To test whether depolarized voltages might effect SYP121 ΔC release, we repeated these experiments also following washout with the membrane clamped to 0 mV (Figure 4b). The results showed that, following SYP121 ΔC additions, τ C3 failed to return to its pretreatment value, suggesting that the Qa-SNARE remains bound to KAT1 even when the channel is closed.

| DISCUSSION
A major action of the Qa-SNARE protein SYP121 is on the activity of K + channels already situated in the plasma membrane and independent of K + channel traffic (Karnik et al., 2017). Previous work showed that the SYP121 interacts with KAT1 and the closely related KC1 channel subunits in vitro (Honsbein et al., 2009;Honsbein et al., 2011), and binding with KC1 in roots was essential for channel mediated K + uptake in vivo and growth (Honsbein et al., 2009). These and additional studies isolated the binding motifs to the N-terminal F 9 xRF motif of Insets: Representative current traces cross-referenced by symbol. Scale: 2 μA (vertical), 1 s (horizontal). Corresponding Immunoblot analysis shown in Figure S1 and relative conductance curves shown in Figure S3. (b) Representative single-channel currents recorded at −120 mV from one inside-out patch with KAT1 alone (above) and after adding SYP121 ΔC to the bath (below , and after its washout from the bath (black bars). Shown is the largest time constant τ C3 used as a proxy for SNARE-channel interactions (see Figure 3). Protocols above summarize the sequence of treatments and clamp voltages used. Mean dwell time constants ±SE (n = 4) are indicated below each protocol and in Table 1. Adding SYP121 ΔC at −120 mV led to an increase in τ C3 that did not recover on washout at −120 mV (left protocol) or 0 mV (centre protocol). SYP121 ΔC had no effect on τ C3 when added at 0 mV (right protocol) at which the channel is normally closed [Colour figure can be viewed at wileyonlinelibrary. com] SYP121 and the RYxxWE motif immediately preceding the first transmembrane α-helices of the K + channels (Grefen et al., 2015;Grefen, Chen, et al., 2010;Karnik et al., 2017). Although SNARE-channel binding promotes vesicle traffic as well as channel-mediated K + influx, how these two, physiologically distinct processes might be temporally coordinated has remained obscure. Indeed, the kinetics of the SNAREchannel interaction is critical to understanding how channel activity is integrated with the SNARE cycle for vesicle fusion.
Direct access to these kinetics is most readily achieved by monitoring single-channel gating, which can report on the temporal behav-

| SYP121 action on KAT1 gating is distributed between state transitions
Co-expression studies in oocytes and analysis of SYP121 action on heterotetrameric channels assembled of KC1 and AKT1 in the plant had shown that SYP121 selectively displaces the V 1/2 by as much as +40 mV and enhances the ensemble conductance (Grefen, Chen, et al., 2010;Honsbein et al., 2009). Although we note some quantitative differences, the overall effect of SYP121 on the macroscopic KAT1 current is qualitatively similar. Like the KC1 channel, SYP121 action also depended on the conserved RYxxWE motif at the cytosolic face of the VSD (Grefen et al., 2015;Karnik et al., 2017), and mutation of Trp 62 in KAT1 was sufficient to eliminate the effects of SYP121 on KAT1 gating (Figure 1a). Heterologous function of the KC1-AKT1 assembly requires CBL-dependent kinase expression in oocytes (Honsbein et al., 2009). By contrast, a KAT1 current is recovered in oocytes when the channel subunit is expressed on its own, thus greatly simplifying heterologous analysis.
The VSDs of Kv channels, including that of KAT1, are known to transit between two well-defined conformational states, an "up" state in which significant portions of the VSD polypeptide are buried within the lipid bilayer, and a "down" state in which much of the inner half of the VSD is exposed to the aqueous phase of the cytosol (Palovcak et al., 2014). The "up" state of KAT1 is associated with the closed channel, whereas the "down" state, which is favoured by membrane hyperpolarization, leads to channel opening (Latorre et al., 2003;Lefoulon et al., 2014). Thus, in principle, in addition to any effects on single-channel conductance, we may envisage two different mechanisms of action for SNARE binding to alter channel gating. In the first, binding is transient, restricted to periods when the VSD is in the "down" state and largely exposed to the cytosol; in this case, binding may be seen to stabilize this state and prolong the associated channel open lifetime. In the second, binding is long-lived, occurring in both "up" and "down" states of the VSD, and may be expected to affect channel open as well as closed lifetimes.
We found that SYP121 had no measurable effect on the singlechannel conductance of KAT1, and its co-expression yielded no evidence of new kinetic components. Instead, SYP121 action on gating was distributed between three distinct kinetic transitions among those identified for the wild-type channel (Zei & Aldrich, 1998) (Ales et al., 1999;Fasshauer, Sutton, Brunger, & Jahn, 1998 (Latorre et al., 2003;Lefoulon et al., 2014;Palovcak et al., 2014), we anticipated that SYP121 ΔC binding might be favoured when KAT1 was held at voltages driving the "down" (open channel) state of the VSD.
Indeed, the effects on gating of SYP121 ΔC , as assayed by the longest closed lifetime τ C3 , could be circumvented if the SNARE was present only when the voltage clamped to 0 mV to drive KAT1 closed (VSD in the "up" conformation). These findings are consistent with bimolecular fluorescence complementation studies indicating a stronger interaction with the ensemble of open channels incorporating KC1 (Grefen et al., 2015). Furthermore, once SYP121 ΔC -mediated gating was altered, subsequent washout both at −120 and at 0 mV, failed to return KAT1 lifetimes to their pretreatment values ( Figure 4 and Table 1), at least over the 10-to 20-min periods of these recordings.
The observations appear counterintuitive, as the VSD assumes an "up" state conformation that buries much of the VSD in the membrane when KAT1 is closed (Lai et al., 2005;Latorre et  bound, the SNARE remains associated with the channel for extended periods of time in both "down" and "up" states of the VSD ( Figure 5).
These studies also highlight a need for SYP121 anchoring in the membrane, as challenging KAT1 with SYP121 ΔC returned a subset only of the kinetic alterations associated with the full-length SNARE (Figures 3 and S4 and Table 1). SYP121 is normally anchored to the plasma membrane through a single, C-terminal transmembrane domain, well removed from the channel binding motif located at its cytosolic N-terminus (Grefen, Chen, et al., 2010;Honsbein et al., 2011

| SYP121 action on KAT1 highlights a functional specificity among Kv channels
We stress that SYP121 action on KAT1, its effects on KC1, is fundamentally different from SNARE binding with Kv channels in animals.
The few animal Kv channels that are affected by SNARE binding are associated primarily with the mammalian Syntaxin1A, binding the H3 domain of this SNARE (Leung, Kwan, Ng, Kang, & Gaisano, 2007) which is also required for SNARE complex assembly. Such binding is difficult to reconcile with neurotransmitter secretion. By contrast, both KC1 and KAT1 lack SNARE-binding sequences homologous to the mammalian Kv channels. Instead, channel binding in the plant depends critically on a linear sequence of residues close to the N-terminus of SYP121, and the effects on K + uptake by roots and guard cells are straightforward and substantial (Eisenach, Chen, Grefen, & Blatt, 2012;Grefen et al., 2015;Honsbein et al., 2009).
Our findings also highlight more subtle differences between the K + channels that interact with SYP121 in the plant and its consequences for K + flux between tissues. Whereas the channels assembled of KC1 and AKT1 provide a major pathway for K + uptake from the soil, KAT1 expression is primarily foliar, notably in stomata (Jezek & Blatt, 2017), and plays a greater part in K + distribution within the plant. The syp121 mutation virtually eliminates the K + channel current in the root epidermis (Honsbein et al., 2009), but its effect in guard cells is a partial reduction only in basal K + current, independent of its action in slowing KAT1 recycling to the plasma membrane (Eisenach et al., 2012). These characteristics are evident also in the more modest impact of the SNARE on KAT1 gating and displacement in its voltage-dependence (Figures 1 and 3). Thus, the comparison shows up unexpected differences in SYP121 actions between the  (Geelen et al., 2002;Grefen et al., 2015).