Current through voltage-gated K+ channels underlies the action potential encoding the electrical signal in excitable cells. The four subunits of a voltage-gated K+ channel each have six transmembrane segments (S1–S6), whereas some other K+ channels, such as eukaryotic inward rectifier K+ channels and the prokaryotic KcsA channel, have only two transmembrane segments (M1 and M2). A voltage-gated K+ channel is formed by an ion-pore module (S5–S6, equivalent to M1–M2) and the surrounding voltage-sensing modules. The S4 segments are the primary voltage sensors while the intracellular activation gate is located near the COOH-terminal end of S6, although the coupling mechanism between them remains unknown. In the present study, we found that two short, complementary sequences in voltage-gated K+ channels are essential for coupling the voltage sensors to the intracellular activation gate. One sequence is the so called S4–S5 linker distal to the voltage-sensing S4, while the other is around the COOH-terminal end of S6, a region containing the actual gate-forming residues.

Potassium channels, a group of specialized membrane proteins, enable K+ ions to flow selectively across cell membranes. Transmembrane K+ currents underlie electrical signalling in neurons and other excitable cells. The atomic structure of a bacterial K+ channel pore has been solved by means of X-ray crystallography. To the extent that the prokaryotic pore is representative of other K+ channels, this landmark achievement has profound implications for our general understanding of K+ channels. But serious doubts have been raised concerning whether the prokaryotic K+ channel pore does actually represent those of eukaryotes. Here we have addressed this fundamental issue by substituting the prokaryotic pore into eukaryotic voltage-gated and inward-rectifier K+ channels. The resulting chimaeras retain the respective functional hallmarks of the eukaryotic channels, which indicates that the ion conduction pore is indeed conserved among K+ channels.

A fundamental question regarding the gating mechanism of voltage-activated K + (Kv) channels is how five positively charged voltage-sensing residues in the fourth transmembrane (TM) segment 1, 2 are energetically stabilized, as they operate in a low-dielectric cell membrane. The simplest solution would be to pair them with negative charges 3 . However, too few negatively charged channel residues are positioned for such a role 4,5 . Recent studies suggest that some of the channel's positively charged residues are exposed to cell membrane phospholipids and interact with their head groups [5][6][7][8][9] . A key question nonetheless remains: Is the phospho-head of membrane lipids necessary for proper function of the voltage sensor itself? Here, we find that a given type of Kv channel may interact with several species of phospholipid, and that enzymatic removal of their negatively charged phospho-head creates an insuperable energy barrier for the positively charged voltage sensor to move through the initial gating step(s), thus immobilizing it, and also raises the energy barrier for the downstream step(s).Kv2.1 channels, expressed in Xenopus oocytes, interact with sphingomyelin 8 present mainly in the outer leaf of plasma membranes. To investigate the importance of phospho-head groups of membrane lipids in Kv channel gating we employed bacterial sphingomyelinases C and D (SMases C and D) 10,11 . Both enzymes specifically hydrolyze sphingomyelin but in different ways (Fig. 1a): SMase D removes only choline and leaves the lipid ceramide-1-phosphate behind in the membrane whereas SMase C removes phosphocholine, leaving ceramide behind 12,13 . A comparison of the effects of these two enzymes on the channels will thus help reveal the functional significance of the phosphodiester group in voltage gating.SMase D of Corynebacterium pseudotuberculosis 11 shifts the conductance-voltage (G-V) relation of Kv2.1 by about -30 mV 8 (Fig. 1d), allowing channels to be activated at a negative voltage where they otherwise remain largely deactivated (Fig. 1c). The effect is maximal within 2 minutes and persists for at least 24 hours ( Fig. 1c, d; cells cannot regenerate sphingomyelin from ceramide-1-phosphate). It does not require direct exposure of channels to SMase D as it also occurred in Kv2.1-expressing oocytes that had been treated with SMase D and then thoroughly washed prior to injection with Kv2.1 cRNA (Fig. 1e).* Please address correspondence to: Dr. Zhe Lu, University of Pennsylvania, Department of Physiology, D302A Richards Building, 3700 Hamilton Walk, Philadelphia, PA 19104, Tel: 215-573-7711, FAX: 215-573-1940, zhelu@mail.med.upenn Additional exposure of such oocytes to SMase D, as expected, caused no further shift. Thus, SMase D acts through its lipase activity, not by direct binding to the channel. Our previous study 8 supports an electrostatic mechanism whereby removal of the positively charged choline favors the activated state of the positively charged voltage sensors.Unlike SMase D, SMase C removes the nega...

Voltage-gated ion channels in excitable nerve, muscle, and endocrine cells generate electric signals in the form of action potentials. However, they are also present in non-excitable eukaryotic cells and prokaryotes, which raises the question of whether voltage-gated channels might be activated by means other than changing the voltage difference between the solutions separated by the plasma membrane. The search for so-called voltage-gated channel activators is motivated in part by the growing importance of such agents in clinical pharmacology. Here we report the apparent activation of voltage-gated K+ (Kv) channels by a sphingomyelinase.

C-type inactivation underlies important roles played by voltage-gated K+ (Kv) channels. Functional studies have provided strong evidence that a common underlying cause of this type of inactivation is an alteration near the extracellular end of the channel's ion selectivity filter. Unlike N-type inactivation, which is known to reflect occlusion of the channel's intracellular end, the structural mechanism of C-type inactivation remains controversial and may have many detailed variations. Here, we report that in voltage-gated Shaker K+ channels lacking N-type inactivation, a mutation enhancing inactivation disrupts the outermost K+ site in the selectivity filter. Furthermore, in a crystal structure of the Kv1.2-2.1 chimeric channel bearing the same mutation, the outermost K+ site, which is formed by eight carbonyl oxygen atoms, appears to be slightly too small to readily accommodate a K+ ion and in fact exhibits little ion density; this structural finding is consistent with the functional hallmark characteristic of C-type inactivation.