Cadmium–cysteine coordination in the BK inner pore region and its structural and functional implications

Abstract

To probe structure and gating-associated conformational changes in BK-type potassium (BK) channels, we examined consequences of Cd 2+ coordination with cysteines introduced at two positions in the BK inner pore. At V319C, the equivalent of valine in the conserved Kv proline-valine-proline (PVP) motif, Cd 2+ forms intrasubunit coordination with a native glutamate E321, which would place the side chains of V319C and E321 much closer together than observed in voltage-dependent K + (Kv) channel structures, requiring that the proline between V319C and E321 introduces a kink in the BK S6 inner helix sharper than that observed in Kv channel structures. At inner pore position A316C, Cd 2+ binds with modest state dependence, suggesting the absence of an ion permeation gate at the cytosolic side of BK channel. These results highlight fundamental structural differences between BK and Kv channels in their inner pore region, which likely underlie differences in voltagedependent gating between these channels.H ow transmembrane potential influences the opening and closing of ion channels, a process known as gating, is central to understanding how cellular excitability is regulated (1). For voltage-dependent K + (Kv) channels, functional (2-4) and crystallographic (5, 6) studies have led to a compelling model of gating. In this model there are two key elements, both of which arise from properties of the cytosolic end of Kv channels: a cytosolic ion permeation gate formed by an interlaced arrangement of S6 inner helices termed the bundle crossing (2, 3), and a kink produced by the conserved proline-valine-proline (PVP) motif (Fig. 1A, boxed residues) to allow the C terminus of Kv S6 to form extensive contact with the S4-S5 linker (5, 6). Thus, the outward movement of the voltage sensors (VSDs) induced by transmembrane depolarization is thought to be transmitted to S6 through the S4-S5 linker to open the cytosolic ion permeation gate (6-8), and this enables access of cytosolic K + ions to the Kv inner pore region.Although this model is generally accepted for Kv channel gating, it is not clear to what extent it applies to other K + channels. For example, the large conductance, Ca 2+ -activated K + (BK or Slo1) channel shares with Kv channels a similar set of four VSDs attached to a central pore and gate domain (PGD), such that both channels are voltage dependent (9). However, unlike that of Kv channels, the inner pore of a closed BK channels is accessible to large molecules such as quaternary ammonium (QA) blockers (10, 11) and methanethiosulfonate ethyltrimethylammonium (MTSET) (12), indicating that the cytosolic end of a closed BK channel cannot completely occlude K + flow; this indicates that a gate extracellular to that proposed for Kv channels is required to securely prevent K + flow in a closed BK channel. As a corollary, the underlying structural and conformational details required to couple VSD activation to channel opening may differ between BK and Kv channels.To provide new insight into differences between BK and Kv in t...

Calcium ions open a selectivity filter gate during activation of the MthK potassium channel

“…The gating mechanisms in eukaryotic Ca 2+ - and voltage-gated BK channels have been explored using blockers 19 21 44 45 46 47 48 49 and thiol-modifying probes 20 22 to assess conformational changes within the pore. Taken together, most of these studies are suggestive of a gating mechanism ‘deeper' inside the pore than for K v channels 50 , though a molecular-gating model has yet to be firmly established 51 .…”

“…In the case of voltage-gated channels, numerous functional studies have shown that activation gating involves the opening of an intracellular gate 14 17 18 . Alternatively, several types of ligand-gated channels, including BK channels 19 20 21 22 , have been proposed to employ a different activation gating mechanism that involves changes at the selectivity filter 23 24 25 26 27 28 . However, the studies in support of selectivity filter gating in BK channels are not definitive due to the complexity of BK channel gating (voltage- and Ca 2+ -dependent gating) combined with the use of voltage-dependent blockers, leading to uncertainty in identification of the gate location.…”

Chemical Protein Modification through Cysteine

“…Previously, metal‐mediated reactions activating otherwise inert endogenous amino acids had been developed, resulting in the addition of reactions to the protein modification “toolbox” . Sulfur atoms within cysteine residues are capable of binding to a wide range of metal ions, including iron, zinc and cadmium (namely soft metal centres) . Protein immobilisation onto gold plates through cysteine residues for surface plasmon resonance (SPR) is well established .…”

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“…As development proceeds, P granules congregate and localize to the posterior size of the embryo, which helps to establish the asymmetric division and subsequent generation of germ cells (Schneider and Bowerman, 2003). Other membrane-less compartments resulting from protein/RNA interactions include nucleolus (Berry et al, 2015), the phase formed at the tail of RNA polymerase II during transcription (Kwon et al, 2013), stress granules (Wippich et al, 2013). …”