Gary Yellen

Mutations affecting internal TEA blockade identify the probable pore-forming region of a K+ channel

The active site of voltage-activated potassium channels is a transmembrane aqueous pore that permits ions to permeate the cell membrane in a rapid yet highly selective manner. A useful probe for the pore of potassium-selective channels is the organic ion tetraethylammonium (TEA), which binds with millimolar affinity to the intracellular opening of the pore and blocks potassium current. In the potassium channel encoded by the Drosophila Shaker gene, an amino acid residue that specifically affects the affinity for intracellular TEA has now been identified by site-directed mutagenesis. This residue is in the middle of a conserved stretch of 18 amino acids that separates two locations that are both near the external opening of the pore. These findings suggest that this conserved region is intimately involved in the formation of the ion conduction pore of voltage-activated potassium channels. Further, a stretch of only eight amino acid residues must traverse 80 percent of the transmembrane electric potential difference.

The voltage-gated potassium channels and their relatives

The voltage-gated potassium channels are the prototypical members of a family of membrane signalling proteins. These protein-based machines have pores that pass millions of ions per second across the membrane with astonishing selectivity, and their gates snap open and shut in milliseconds as they sense changes in voltage or ligand concentration. The architectural modules and functional components of these sophisticated signalling molecules are becoming clear, but some important links remain to be elucidated.

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.

Dynamic Rearrangement of the Outer Mouth of a K+ Channel during Gating

With prolonged stimulation, voltage-activated K+ channels close by a gating process called inactivation. This inactivation gating can occur by two distinct molecular mechanisms: N-type, in which a tethered particle blocks the intracellular mouth of the pore, and C-type, which involves a closure of the external mouth. The functional motion involved in C-type inactivation was studied by introducing cysteine residues at the outer mouth of Shaker K+ channels through mutagenesis, and by measuring state-dependent changes in accessibility to chemical modification. Modification of three adjacent residues in the outer mouth was 130-10,000-fold faster in the C-type inactivated state than in the closed state. At one position, state-dependent bridging or crosslinking between subunits was also possible. These results give a consistent picture in which C-type inactivation promotes a local rearrangement and constriction of the channel at the outer mouth.

THE MOVING PARTS OF VOLTAGE-GATED ION CHANNELS

.    . Early evidence for an activation gate at the intracellular mouth  .. Open channel blockade  .. The ‘foot-in-the-door ’ effect  .. Trapping of blockers behind closed activation gates  . Site-directed mutagenesis and the difficulty of inferring structural roles from functional effects  . State-dependent cysteine modification as a reporter of position and motion  . Localization of activation gating  .. The trapping cavity  .. The activation gate  .. Is there more than one site of activation gating? 

Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels.

Voltage-activated K+ channels are a family of closely related membrane proteins that differ in their gating behavior, conductance, and pharmacology. A prominent and physiologically important difference among K+ channels is their rate of inactivation. Inactivation rates range from milliseconds to seconds, and K+ channels with different inactivation properties have very different effects on signal integration and repetitive firing properties of neurons. The cloned Shaker B (H4) potassium channel is an example of a K+ channel that inactivates in a few milliseconds. Recent experiments have shown that removal of an N-terminal region of the Shaker protein by site-directed deletion practically abolishes this fast inactivation, but the modified channel does still inactivate during a prolonged depolarization lasting many seconds. Here we report that this remnant inactivation must occur by a distinct mechanism from the rapid inactivation of the wild-type Shaker channel. Like the inactivation of another K+ channel [Grissmer, S. & Calahan, M. (1989) Biophys. J. 55, 203-206], this slow inactivation is retarded by the application of a channel blocker, tetraethylammonium, to the extracellular side of the channel. By contrast, the fast inactivation of the wild-type Shaker channel is sensitive only to intracellular application of tetraethylammonium. Intracellular tetraethylammonium slows down the fast inactivation process, as though it competes with the binding of the inactivation particle.

Modulation of K+ current by frequency and external [K+]: A tale of two inactivation mechanisms

Voltage-activated K+ currents and their inactivation properties are important for controlling frequency-dependent signaling in neurons and other excitable cells. Two distinct molecular mechanisms for K+ channel inactivation have been described: N-type, which involves rapid occlusion of the open channel by an intracellular tethered blocker, and C-type, which involves a slower change at the extracellular mouth of the pore. We find that frequency-dependent cumulative inactivation of Shaker channels is very sensitive to changes of extracellular [K+] in the physiological range, with much more inactivation at low [K+]out, and that it results from the interaction of N- and C-type inactivation. N-type inactivation enhances C-type inactivation by two mechanisms. First, it inhibits outward K+ flux, which normally fills an external ion site and thus prevents C-type inactivation. Second, it keeps the channels activation gate open even after repolarization, allowing C-type inactivation to occur for a prolonged period.

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.

Single Ca2+-activated nonselective cation channels in neuroblastoma.

Recent work suggests an important role for intracellular agents in controlling ion channels in the membranes of nerve cells and other excitable tissues. Calcium ions1,2, cyclic nucleotides3 and protein kinases4,5 can all act on the inner surface of the membrane to influence ion channel activity. Earlier studies of these effects on intact cells could not, however, effectively control or measure conditions on the inner membrane surface. The technique of recording from detached membrane patches6,7permits free access to the intracellular face of ion channels and makes it possible to study them in isolation from the many components of the cytoplasm. With this technique, I have studied the response of membrane patches from neuroblastoma cells to intracellular Ca2+ ions, and have found a class of nonselective cation channels activated by micromolar concentrations of Ca2+ on the intracellular face of the membrane. These channels are almost equally permeable to Na+, K+, Li+ and Cs+ ions, but are practically impermeable to Ca2+ ions. Similar channels were first found recently in cultured heart cells8, where they probably account for the previously reported ‘transient inward’ current9. Their discovery in neuronal cells as well as heart cells suggests that this hitherto scarcely recognized channel species may be more widely distributed than previously supposed.

A genetically encoded fluorescent reporter of ATP:ADP ratio

We constructed a fluorescent sensor of adenylate nucleotides by combining a circularly permuted variant of GFP with a bacterial regulatory protein, GlnK1, from Methanococcus jannaschii. The sensors affinity for Mg-ATP was <100 nM, as seen for other members of the bacterial PII regulator family, a surprisingly high affinity given that normal intracellular ATP concentration is in the millimolar range. ADP bound the same site of the sensor as Mg-ATP, competing with it, but produced a smaller change in fluorescence. At physiological ATP and ADP concentrations, the binding site is saturated, but competition between the two substrates causes the sensor to behave as a nearly ideal reporter of the ATP:ADP concentration ratio. This principle for sensing the ratio of two analytes by competition at a high-affinity site probably underlies the normal functioning of PII regulatory proteins. The engineered sensor, Perceval, can be used to monitor the ATP:ADP ratio during live-cell imaging.