Miguel Holmgren

Gated Access to the Pore of a Voltage-Dependent K+ Channel

Voltage-activated K+ channels are integral membrane proteins that open or close a K(+)-selective pore in response to changes in transmembrane voltage. Although the S4 region of these channels has been implicated as the voltage sensor, little is known about how opening and closing of the pore is accomplished. We explored the gating process by introducing cysteines at various positions thought to lie in or near the pore of the Shaker K+ channel, and by testing their ability to be chemically modified. We found a series of positions in the S6 transmembrane region that react rapidly with water-soluble thiol reagents in the open state but not the closed state. An open-channel blocker can protect several of these cysteines, showing that they lie in the ion-conducting pore. At two of these sites, Cd2+ ions bind to the cysteines without affecting the energetics of gating; at a third site, Cd2+ binding holds the channel open. The results suggest that these channels open and close by the movement of an intracellular gate, distinct from the selectivity filter, that regulates access to the pore.

Blocker protection in the pore of a voltage-gated K+ channel and its structural implications.

The structure of the bacterial potassium channel KcsA has provided a framework for understanding the related voltage-gated potassium channels (Kv channels) that are used for signalling in neurons. Opening and closing of these Kv channels (gating) occurs at the intracellular entrance to the pore, and this is also the site at which many open channel blockers affect Kv channels. To learn more about the sites of blocker binding and about the structure of the open Kv channel, we investigated here the ability of blockers to protect against chemical modification of cysteines introduced at sites in transmembrane segment S6, which contributes to the intracellular entrance. Within the intracellular half of S6 we found an abrupt cessation of protection for both large and small blockers that is inconsistent with the narrow ‘inner pore’ seen in the KcsA structure. These and other results are most readily explained by supposing that the structure of Kv channels differs from that of the non-voltage-gated bacterial channel by the introduction of a sharp bend in the inner (S6) helices. This bend would occur at a Pro-X-Pro sequence that is highly conserved in Kv channels, near the site of activation gating.

The Activation Gate of a Voltage-Gated K+ Channel Can Be Trapped in the Open State by an Intersubunit Metal Bridge

Voltage-activated K+ channels are integral membrane proteins containing a potassium-selective transmembrane pore gated by changes in the membrane potential. This activation gating (opening) occurs in milliseconds and involves a gate at the cytoplasmic side of the pore. We found that substituting cysteine at a particular position in the last transmembrane region (S6) of the homotetrameric Shaker K+ channel creates metal binding sites at which Cd2+ ions can bind with high affinity. The bound Cd2+ ions form a bridge between the introduced cysteine in one channel subunit and a native histidine in another subunit, and the bridge traps the gate in the open state. These results suggest that gating involves a rearrangement of the intersubunit contacts at the intracellular end of S6. The recently solved structure of a bacterial K+ channel shows that the S6 homologs cross in a bundle, leaving an aperture at the bundle crossing. In the context of this structure, the metal ions form a bridge between a cysteine above the bundle crossing and a histidine below the bundle crossing in a neighboring subunit. Our results suggest that gating occurs at the bundle crossing, possibly through a change in the conformation of the bundle itself.

On the use of thiol-modifying agents to determine channel topology

A powerful tool in the study of cloned ion channels is the combined use of site-directed mutagenesis and chemical modification. Site-directed mutagenesis is used to introduce new cysteine residues at specific positions in a channel protein, and chemical modification by thiol-specific reagents is then used to assess the exposure of the introduced cysteins. This method has been used to assess secondary structure, membrane topology and conformational changes. We report that one commonly used, charged reagent (MTSEA; aminoethyl methanethiosulfonate) can cross the membrane quite readily. We also find that other reagents that are quite membrane-impermeant can cross the membrane when patches are electrically leaky. Both of these undesired effects can be controlled by the use of a thiol scavenger. These findings argue for caution in the use of modifying reagents to determine the membrane topology of channels and other membrane proteins.

Control of human potassium channel inactivation by editing of a small mRNA hairpin

Genomic recoding by A→I RNA editing plays an important role in diversifying the proteins involved in electrical excitability. Here, we describe editing of an intronless potassium channel gene. A small region of human KV1.1 mRNA sequence directs efficient modification of one adenosine by human adenosine deaminase acting on RNA 2 (hADAR2). Mutational analysis shows that this region adopts a hairpin structure. Electrophysiological characterization reveals that the editing event (I/V) profoundly affects channel inactivation conferred by accessory β subunits. Drosophila melanogaster Shaker channels, mimicking this editing event through mutation, exhibit a similar effect. In addition, we demonstrate that mRNAs for the paralogous D. melanogaster Shab potassium channel are edited at the same position by fly ADAR—a clear example of convergent evolution driven by adenosine deamination. These results suggest an ancient and key regulatory role for this residue in KV channels.

Three distinct and sequential steps in the release of sodium ions by the Na+/K+-ATPase.

The Na+/K+ pump, a P-type ion-motive ATPase, exports three sodium ions and then imports two potassium ions in each transport cycle. Ions on one side of the membrane bind to sites within the protein and become temporarily occluded (trapped within the protein) before being released to the other side, but details of these occlusion and de-occlusion transitions remain obscure for all P-type ATPases. If it is deprived of potassium ions, the Na+/K+ pump is restricted to sodium translocation steps, at least one involving charge movement through the membranes electric field. Changes in membrane potential alter the rate of such electrogenic reactions and so shift the distribution of enzyme conformations. Here we use high-speed voltage jumps to initiate this redistribution and show that the resulting pre-steady-state charge movements relax in three identifiable phases, apparently reflecting de-occlusion and release of the three sodium ions. Reciprocal relationships among the sizes of these three charge components show that the three sodium ions are de-occluded and released to the extracellular solution one at a time, in a strict order.

Gating at the selectivity filter in cyclic nucleotide-gated channels

By opening and closing the permeation pathway (gating) in response to cGMP binding, cyclic nucleotide-gated (CNG) channels serve key roles in the transduction of visual and olfactory signals. Compiling evidence suggests that the activation gate in CNG channels is not located at the intracellular end of pore, as it has been established for voltage-activated potassium (KV) channels. Here, we show that ion permeation in CNG channels is tightly regulated at the selectivity filter. By scanning the entire selectivity filter using small cysteine reagents, like cadmium and silver, we observed a state-dependent accessibility pattern consistent with gated access at the middle of the selectivity filter, likely at the corresponding position known to regulate structural changes in KcsA channels in response to low concentrations of permeant ions.

Ouabain binding site in a functioning Na+/K+ ATPase

Background: Ouabain binds at the permeation pathway of the Na+/K+ ATPase. Results: We have identified two binding sites for ouabain along the ion conductive pathway of the Na+/K+ pump that are mutually exclusive and differ in their affinities by about an order of magnitude. Conclusion: Ouabain reaches its high affinity binding site at the inner end of the permeation pathway by a sequential mechanism. Significance: This work unifies all available functional and structural data on the interactions of ouabain with the Na+/K+ pump. The Na+/K+ ATPase is an almost ubiquitous integral membrane protein within the animal kingdom. It is also the selective target for cardiotonic derivatives, widely prescribed inhibitors for patients with heart failure. Functional studies revealed that ouabain-sensitive residues distributed widely throughout the primary sequence of the protein. Recently, structural work has brought some consensus to the functional observations. Here, we use a spectroscopic approach to estimate distances between a fluorescent ouabain and a lanthanide binding tag (LBT), which was introduced at five different positions in the Na+/K+ ATPase sequence. These five normally functional LBT-Na+/K+ ATPase constructs were expressed in the cell membrane of Xenopus laevis oocytes, operating under physiological internal and external ion conditions. The spectroscopic data suggest two mutually exclusive distances between the LBT and the fluorescent ouabain. From the estimated distances and using homology models of the LBT-Na+/K+ ATPase constructs, approximate ouabain positions could be determined. Our results suggest that ouabain binds at two sites along the ion permeation pathway of the Na+/K+ ATPase. The external site (low apparent affinity) occupies the same region as previous structural findings. The high apparent affinity site is, however, slightly deeper toward the intracellular end of the protein. Interestingly, in both cases the lactone ring faces outward. We propose a sequential ouabain binding mechanism that is consistent with all functional and structural studies.

Access of Quaternary Ammonium Blockers to the Internal Pore of Cyclic Nucleotide-gated Channels: Implications for the Location of the Gate

Cyclic nucleotide-gated (CNG) channels play important roles in the transduction of visual and olfactory information by sensing changes in the intracellular concentration of cyclic nucleotides. We have investigated the interactions between intracellularly applied quaternary ammonium (QA) ions and the α subunit of rod cyclic nucleotide-gated channels. We have used a family of alkyl-triethylammonium derivatives in which the length of one chain is altered. These QA derivatives blocked the permeation pathway of CNG channels in a concentration- and voltage-dependent manner. For QA compounds with tails longer than six methylene groups, increasing the length of the chain resulted in higher apparent affinities of ∼1.2 RT per methylene group added, which is consistent with the presence of a hydrophobic pocket within the intracellular mouth of the channel that serves as part of the receptor binding site. At the single channel level, decyltriethyl ammonium (C10-TEA) ions did not change the unitary conductance but they did reduce the apparent mean open time, suggesting that the blocker binds to open channels. We provide four lines of evidence suggesting that QA ions can also bind to closed channels: (1) the extent of C10-TEA blockade at subsaturating [cGMP] was larger than at saturating agonist concentration, (2) under saturating concentrations of cGMP, cIMP, or cAMP, blockade levels were inversely correlated with the maximal probability of opening achieved by each agonist, (3) in the closed state, MTS reagents of comparable sizes to QA ions were able to modify V391C in the inner vestibule of the channel, and (4) in the closed state, C10-TEA was able to slow the Cd2+ inhibition observed in V391C channels. These results are in stark contrast to the well-established QA blockade mechanism in Kv channels, where these compounds can only access the inner vestibule in the open state because the gate that opens and closes the channel is located cytoplasmically with respect to the binding site of QA ions. Therefore, in the context of Kv channels, our observations suggest that the regions involved in opening and closing the permeation pathways in these two types of channels are different.

State-dependent FRET reports calcium- and voltage-dependent gating-ring motions in BK channels

Large-conductance voltage- and calcium-dependent potassium channels (BK, “Big K+”) are important controllers of cell excitability. In the BK channel, a large C-terminal intracellular region containing a “gating-ring” structure has been proposed to transduce Ca2+ binding into channel opening. Using patch-clamp fluorometry, we have investigated the calcium and voltage dependence of conformational changes of the gating-ring region of BK channels, while simultaneously monitoring channel conductance. Fluorescence resonance energy transfer (FRET) between fluorescent protein inserts indicates that Ca2+ binding produces structural changes of the gating ring that are much larger than those predicted by current X-ray crystal structures of isolated gating rings.