Gating at the selectivity filter in cyclic nucleotide-gated channels

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...

Section: Significancementioning

confidence: 60%

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

Zhou

Xia

Lingle

2015

“…It should also be noted that the closed-state modification rates at the three BK pore-lining sites are approximately two orders higher than that of the Shaker channels (23). These rates are even several fold higher than that of closed cyclic nucleotide-gated channels (38), the ion perme- ation gate of which has been determined to be in the selectivity filter region (38,39). In accord with previous results that small molecules such as quaternary ammonium blockers may pass in and out of the BK inner pore in both open and closed states (17,20), this observation suggests that any constriction at the cytosolic end of BK S6 does not prevent access of molecules to the inner pore and thus is unlikely to form an ion permeation gate.…”

Section: Discussionmentioning

confidence: 99%

Cysteine scanning and modification reveal major differences between BK channels and Kv channels in the inner pore region

Zhou

Xia

Lingle

2011

105

BK channels are regulated by two distinct physiological signals, transmembrane potential and intracellular Ca 2+ , each acting through independent modular sensor domains. However, despite a presumably central role in the coupling of sensor activation to channel gating, the pore-lining S6 transmembrane segment has not been systematically studied. Here, cysteine substitution and modification studies of the BK S6 point to substantial differences between BK and Kv channels in the structure and function of the S6-lined inner pore. Gating shifts caused by introduction of cysteines define a pattern and direction of free energy changes in BK S6 distinct from Shaker. Modification of BK S6 residues identifies pore-facing residues that occur at different linear positions along aligned BK and Kv S6 segments. Periodicity analysis suggests that one factor contributing to these differences may be a disruption of the BK S6 α-helix from the unique diglycine motif at the position of the Kv hinge glycine. State-dependent MTS accessibility reveals that, even in closed states, modification can occur. Furthermore, the inner pore of BK channels is much larger than that of K + channels with solved crystal structures. The results suggest caution in the use of Kv channel structures as templates for BK homology models, at least in the pore-gate domain.cysteine modification | K channels | Slo1 channels A s a unique member of the family of K + channels, large conductance, Ca 2+ -activated K + (BK or Slo1) channels are activated in a highly synergistic manner by two distinct physiological signals: depolarization and intracellular Ca 2+ . To accomplish this, each of the four α-subunits in a functional BK channel is constructed of three modular parts: a voltage-sensing domain composed of S1 to S4 transmembrane segments, a huge cytosolic domain sensing various intracellular ligands, such as Ca 2+ (1, 2), Mg 2+ (3), and heme (4, 5), and a pore-gate domain (PGD) formed by S5-pore loop-S6 to allow selective K + permeation under the regulation of transmembrane potential and [Ca 2+ ] i (6-8).The BK channel shares homology in its membrane-associated domain, including the voltage-sensing domain and PGD, with voltage-dependent K + (Kv) channels. Thus, the X-ray crystallographic structures of Kv channels (9, 10) have been widely used as an important template to guide structure-function studies of BK channels (11-13). However, the distant evolutionary relationship between BK and Kv channels (14) raises the possibility that Kv channel structure must be used with caution as a guide to BK channel structure. Sequence alignment (Fig. 1A) shows that BK channels and Kv channels differ at two critical positions in their pore-lining S6 transmembrane segments. First, there are two consecutive glycines (i.e., diglycine) at the conserved glycine hinge (15) in BK channels, but only one in the corresponding region of Kv channels. Second, whereas Kv channels share a conserved PXP motif near the cytosolic entrance to the inner pore, in BK channels there is only one prol...

“…Cyclic nucleotide-gated (CNG) channels underlie sensory transduction in the retina and olfactory epithelium and share a high degree of homology with K + channels (8)(9)(10). In contrast to K + channels, CNG channels' primary gate is located at the selectivity filter (11), suggesting that the same protein region controls ion permeation and gating. In CNG channels the ionic species present inside the pore influences channel gating; however, the nature of this coupling is not well understood (12)(13)(14)(15)(16).…”

mentioning

confidence: 99%

A structural, functional, and computational analysis suggests pore flexibility as the base for the poor selectivity of CNG channels

Napolitano

Bisha

March

et al. 2015

Cyclic nucleotide-gated (CNG) ion channels, despite a significant homology with the highly selective K + channels, do not discriminate among monovalent alkali cations and are permeable also to several organic cations. We combined electrophysiology, molecular dynamics (MD) simulations, and X-ray crystallography to demonstrate that the pore of CNG channels is highly flexible. When a CNG mimic is crystallized in the presence of a variety of monovalent cations, including Na + , Cs + , and dimethylammonium (DMA + ), the side chain of Glu66 in the selectivity filter shows multiple conformations and the diameter of the pore changes significantly. MD simulations indicate that Glu66 and the prolines in the outer vestibule undergo large fluctuations, which are modulated by the ionic species and the voltage. This flexibility underlies the coupling between gating and permeation and the poor ionic selectivity of CNG channels.CNG channels | pore flexibility | X-ray crystallography | MD simulations I n K + selective channels, the opening and closing of the ion channel pore (gating) and the translocation of ions through the pore (permeation) are considered independent processes with distinct structural basis (1). Gating is controlled by the bundle crossing at the intracellular side, whereas permeation reflects ion-ion and ion-pore interactions within the selectivity filter (1-5). Based on these experimental observations, the current paradigm assumes that the 3D structure of the selectivity filter is relatively rigid during ion translocation and the mechanisms of ionic permeation can be deduced in essence from its crystal structure. This paradigm has been successfully applied to several K + channels (4, 6, 7). Cyclic nucleotide-gated (CNG) channels underlie sensory transduction in the retina and olfactory epithelium and share a high degree of homology with K + channels (8-10). In contrast to K + channels, CNG channels' primary gate is located at the selectivity filter (11), suggesting that the same protein region controls ion permeation and gating. In CNG channels the ionic species present inside the pore influences channel gating; however, the nature of this coupling is not well understood (12-16). In the presence of large cations, such as Rb + and Cs + , channel conductance and gating are also controlled by membrane voltage, and current-voltage relationships activated by 1 mM cGMP depend on the radius of the permeating ion (17, 18) (Fig. S1).Structural information on CNG channels is limited to a lowresolution electron microscopy map (19), partial crystal structures of the intracellular cyclic nucleotide-binding domains (20)(21)(22), and the crystal structure of a chimeric channel in which the CNG selectivity-filter sequence is engineered into a bacterial NaK channel, creating a CNG mimic (NaK2CNG; Fig. 1 A and B) that shares several properties of CNG channels (23). This CNG mimic provides a suitable model for understanding the properties of the pore underlying the low ionic selectivity and the coupling between gating and perme...