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.

K+ Channel-SEC11 Binding Exchange Regulates SNARE Assembly for Secretory Traffic

“…S5), much as has been reported in the past (Hoshi, 1995;Gajdanowicz et al, 2009;Lefoulon et al, 2018). As before (Grefen et al, 2015;Zhang et al, 2015Zhang et al, , 2017Lefoulon et al, 2018), co-expression with SYP121 and with VAMP721 ( Fig. 5, A and B) showed opposing effects on KAT1 gating: SYP121 co-expression .…”

“…KAT1 assembles as a homotetramer, but its close homolog, the so-called "silent" KC1 channel monomer, normally functions in vivo as a heterotetramer with the AKT1 K 1 channel subunit (Duby et al, 2008;Honsbein et al, 2009). Both channel assemblies show gating that is significantly altered, with opposing effects, by SYP121 and VAMP721 (Honsbein et al, 2009;Grefen et al, 2010Grefen et al, , 2015Zhang et al, 2015Zhang et al, , 2017Lefoulon et al, 2018). However, it is not known whether these characteristics are retained in combination with the cognate Qbc-SNARE SNAP33.…”

“…To address this question, we assayed channel gating as a proxy to assess the effects of SNARE protein combinations. KAT1 was expressed alone and in combinations with SYP121, VAMP721, and SNAP33 in Xenopus laevis oocytes for analysis by two-electrode voltage clamp (Honsbein et al, 2009;Lefoulon et al, 2018). Using KAT1 avoided the technical complications of coexpressing KC1 with AKT1, CIPK23, and CBL1, which are necessary for KC1-AKT1 channel activity (Xu et al, 2006;Honsbein et al, 2009).…”

“…Most important, through the F 9 xRF motif near its N terminus, SYP121 binds directly with a subset of K 1 channels, including KAT1 and KC1, already present at the plasma membrane to promote channel gating (Honsbein et al, 2009(Honsbein et al, , 2011Grefen et al, 2010). The K 1 channels incorporate a unique RYxxWE motif at the cytosolic surface of their voltage sensor domain (VSD), and binding of the SNARE through this motif effectively commandeers the voltage-dependent conformations of the VSD to enhance secretion in parallel with voltagedriven K 1 uptake (Grefen et al, 2015;Lefoulon et al, 2018). Significantly, both binding motifs are unique to land plants and are highly conserved within a subset of plasma membrane K 1 channels and Qa-SNAREs, suggesting that their functional coordination was an important evolutionary step in the colonization of the dry land environment (Honsbein et al, 2011;Grefen et al, 2015;Karnik et al, 2017).…”

The roles of endomembrane trafficking in plant abiotic stress responses

“…Subsequent studies have demonstrated a direct interaction between SYP121 and another K + channel KAT1, and have shown that the addition of SYP121 could enhance the whole cell current when coexpressed with KAT1 in Xenopus oocytes . Studies with temporal kinetics of the channel gating showed that expression of SYP121 significantly alters the lifetime of KAT1 in both open and close states (Figure 1D) (Lefoulon et al 2018). These findings confirmed the unique function of SYP121 in the direct regulation of K + channel activities.…”