Intracellular gate opening in Shaker K+ channels defined by high-affinity metal bridges

161

218

It is now well established that the voltage-sensing S4 segment in voltage-dependent ion channels undergoes a conformational change in response to varying membrane potential. However, the magnitude of the movement of S4 relative to the membrane and the rest of the protein remains controversial. Here, by using histidine scanning mutagenesis in the Shaker K channel, we identified mutants I241H (S1 segment) and I287H (S2 segment) that generate inward currents at hyperpolarized potentials, suggesting that these residues are part of a hydrophobic plug that separates the water-accessible crevices. Additional experiments with substituted cysteine residues showed that, at hyperpolarized potentials, both I241C and I287C can spontaneously form disulphide and metal bridges with R362C, the position of the first charge-carrying residue in S4. These results constrain unambiguously the closedstate positions of the S4 segment with respect to the S1 and S2 segments, which are known to undergo little or no movement during gating. To satisfy these constraints, the S4 segment must undergo an axial rotation of Ϸ180°and a transmembrane (vertical) movement of Ϸ6.5 Å at the level of R362 in going from the open to the closed state of the channel, moving the gating charge across a focused electric field.gating current ͉ metal bridge ͉ omega current ͉ S-S bridge V oltage-gated channels are crucial players in excitability and cell homeostasis. Voltage-dependent sodium and potassium conductances are responsible for the generation and propagation of the nerve impulse (1). The salient property of these channels is the steep membrane potential dependence of their open probability. This voltage dependence is conferred by the voltage sensor that has been identified with their first four transmembrane segments (S1-S4). It is now clear that the four most extracellular basic residues of the S4 segment and the most intracellular acidic residue in the S2 segment in the Shaker K channel are the gating charge-carrying residues that move in the field in response to changes in membrane potential (2, 3). The movement and conformational change of segments S1-S4 within the voltage-sensing module in response to membrane depolarization is somehow coupled to the intracellular gate of the pore domain (S5-S6) to open and close the channel. A multitude of biophysical experiments have attempted to delineate the conformation of the voltage sensor and the position of its charges in the open and closed states. The structural restraints deduced from those experiments have subsequently been shown to be consistent with the overall conformation of the open state from the crystallographic structure of the Kv1.2 channel. However, the experiments providing structural constraints about the closed state conformation of the voltage sensor are more uncertain. Although it is generally accepted that the S1 and S2 helical segments do not move extensively upon gating (4-8), there is disagreement concerning the magnitude of movements associated with the S3 and S4 segments. For exampl...

mentioning

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Two atomic constraints unambiguously position the S4 segment relative to S1 and S2 segments in the closed state of Shaker K channel

Campos

Chanda

Roux

et al. 2007

161

218

“…In the Shaker channel, it has been suggested that opening of the gate is expected to be accomplished by swiveling the lower half of the S6 below the PXP motif (32,40), with only modest changes in dimension at the position of V474C within the Shaker PVP motif (23,32). Although gating shifts reported for V474 mutation may somewhat contradict the idea that V474 changes little during gating (34,35), it is the case that Shaker S6 positions with the strongest energetic effects on gating extend largely from V474 down to perhaps N482.…”

Section: Discussionmentioning

confidence: 99%

“…In Shaker channels, Cd 2+ irreversibly inhibits two S6 cysteine mutants (I470C and V474C, corresponding to F315C and V319C in BK, respectively, based on linear alignment) by forming strong coordination with three or four cysteines individually introduced at these pore lining sites (23). This coordination by Cd 2+ defines a diameter in Shaker of approximately 8 to 9 Å separating diagonal cysteines (32). In BK channels, however, we observed no irreversible inhibition produced by Cd 2+ treatment at any of the S6-substituted cysteine mutants.…”

Section: Mtset Modification At the Single Channel Level Defines A Lowmentioning

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...

“…Despite the position of this proline residue, these channels are activated by depolarization. Moreover, this proline residue is thought to provide an essential bend in the S6 helix that is important for K V channel gating (26). It will be interesting to determine whether substitution of other amino acid residues at this position in K V channels can produce hyperpolarizationactivated gating.…”

Section: Bending Of S6 Segments Determines Both Voltage Dependence Andmentioning

confidence: 99%

Reversed voltage-dependent gating of a bacterial sodium channel with proline substitutions in the S6 transmembrane segment

Zhao

Scheuer

Catterall

2004

Members of the voltage-gated-like ion channel superfamily have a conserved pore structure. Transmembrane helices that line the pore (M2 or S6) are thought to gate it at the cytoplasmic end by bending at a hinge glycine residue. Proline residues favor bending of ␣-helices, and substitution of proline for this glycine (G219) dramatically stabilizes the open state of a bacterial Na ؉ channel NaChBac. Here we have probed S6 pore-lining residues of NaChBac by proline mutagenesis. Five of 15 proline-substitution mutants yielded depolarization-activated Na ؉ channels, but only G219P channels have strongly negatively shifted voltage dependence of activation, demonstrating specificity for bending at G219 for depolarization-activated gating. Remarkably, three proline-substitution mutations on the same face of S6 as G219 yielded channels that activated upon hyperpolarization and inactivated very slowly. Studies of L226P showed that hyperpolarization to ؊147 mV gives half-maximal activation, 123 mV more negative than WT. Analysis of combination mutations and studies of block by the local anesthetic etidocaine favored the conclusion that hyperpolarizationactivated gating results from opening of the cytoplasmic gate formed by S6 helices. Substitution of multiple amino acids for L226 indicated that hyperpolarization-activated gating was correlated with a high propensity for bending, whereas depolarization-activated gating was favored by a low propensity for bending. Our results further define the dominant role of bending of S6 in determining not only the voltage dependence but also the polarity of voltage-dependent gating. Native hyperpolarization-activated gating of hyperpolarization-and cyclic nucleotide-gated (HCN) channels in animals and KAT channels in plants may involve bending at analogous S6 amino acid residues. excitability ͉ sodium channels ͉ proline mutagenesis ͉ pore-gating V oltage-gated Na ϩ channels are large transmembrane proteins that generate and propagate action potentials (1, 2). In eukaryotes, the pore-forming ␣-subunit is composed of four homologous domains containing six probable transmembrane ␣-helices (TM) (S1 to S6; reviewed in ref.3). The lining of the inner pore is formed by the S6 segments of each domain, and the narrow ion selectivity filter is formed by the P loop in the linker between the S5 and S6 segments (3). The S4 segments of each domain contain positively charged amino acids at every third position that serve as voltage sensors (3). Opening and closing of the pore are controlled by changes in membrane potential (3). The pore is closed at the resting membrane potential and opens in response to depolarization (3). Na ϩ channels are the founding members of a large family of 143 voltage-gated-like ion channels (4). Their similar pore structures suggest fundamental similarities in pore-gating mechanisms despite the marked differences in their activation by changes in membrane voltage and͞or by binding of intracellular messengers like cyclic nucleotides, lipids, and Ca 2ϩ .Ion channels discovere...