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Two-pore domain K+ (K2P) channel activity was previously thought to be controlled primarily via a selectivity filter (SF) gate. However, recent crystal structures of TASK-1 and TASK-2 revealed a lower gate at the cytoplasmic pore entrance. Here, we report functional evidence of such a lower gate in the K2P channel K2P17.1 (TALK-2, TASK-4). We identified compounds (drugs and lipids) and mutations that opened the lower gate allowing the fast modification of pore cysteine residues. Surprisingly, stimuli that directly target the SF gate (i.e., pHe., Rb+ permeation, membrane depolarization) also opened the cytoplasmic gate. Reciprocally, opening of the lower gate reduced the electric work to open the SF via voltage driven ion binding. Therefore, it appears that the SF is so rigidly locked into the TALK-2 protein structure that changes in ion occupancy can pry open a distant lower gate and, vice versa, opening of the lower gate concurrently promote SF gate opening. This concept might extent to other K+ channels that contain two gates (e.g., voltage-gated K+ channels) for which such a positive gate coupling has been suggested, but so far not directly demonstrated. Here, the authors identify a cytoplasmic gate in the K2P channel TALK-2 that is strongly positively-coupled to a second gate made up by the selectivity filter. Thereby, stimuli that converge at either gate open both gates, allowing effective polymodal regulation. [ABSTRACT FROM AUTHOR]

Polyamines cause inward rectification of (Kir) [K.sup.+] channels, but the mechanism is controversial. We employed scanning mutagenesis of Kir6.2, and a structural series of blocking diamines, to combinatorially examine the role of both channel and blocker charges. We find that introduced glutamates at any pore-facing residue in the inner cavity, up to and including the entrance to the selectivity filter, can confer strong rectification. As these negative charges are moved higher (toward the selectivity filter), or lower (toward the cytoplasm), they. preferentially enhance the potency of block by shorter, or longer, diamines, respectively. MTSE[A.sup.+] modification of engineered cysteines in the inner cavity reduces rectification, but modification below the inner cavity slows spermine entry and exit, without changing steady-state rectification. The data provide a coherent explanation of classical strong rectification as the result of polyamine block in the inner cavity and selectivity filter. KEY WORDS: inward rectifier * spermine * diamine * rectification * selectivity filter

Voltage-gated potassium (Kv) channels control action potential repolarization, interspike membrane potential and action potential frequency in excitable cells. It is thought that the combinatorial association between distinct α and β subunits determines whether Kv channels function as non-inactivating delayed rectifiers or as rapidly inactivating A-type channels. We show that membrane lipids can convert A-type channels into delayed rectifiers and vice versa. Phosphoinositides remove N-type inactivation from A-type channels by immobilizing the inactivation domains. Conversely, arachidonic acid and its amide anandamide endow delayed rectifiers with rapid voltage-dependent inactivation. The bidirectional control of Kv channel gating by ENds may provide a mechanism for the dynamic regulation of electrical signaling in the nervous system., The action potential (AP) is the fundamental unit of information in the brain (1). Its shape is of critical importance in many forms of neuronal signaling (2-5). Voltage-gated potassium (Kv) [...]

Many blockers of [Na.sup.+] and [K.sup.+] channels act by blocking the pore from the intracellular side. For Shaker [K.sup.+] channels, such intracellular blockers vary in their functional effect on slow (C-type) inactivation: Some blockers interfere with C-type inactivation, whereas others do not. These functional differences can be explained by supposing that there are two overlapping 'subsites' for blocker binding, only one of which inhibits C-type inactivation through an allosteric effect. We find that the ability to bind to these subsites depends on specific structural characteristics of the blockers, and correlates with the effect of mutations in two distinct regions of the channel protein. These interactions are important because they affect the ability of blockers to produce use-dependent inhibition.

A study was conducted on the inward rectification in HERG cardiac potassium channels expressed in mammalian cells. The inward rectification was found to be caused by a rapid voltage-dependent inactivation process that decreases conductance at positive voltages. The observed gating mechanism indicates the influence of the HERG channel in the normal physiological suppression of arrhythmias, particularly in suppressing premature afterbeats.

Recent work introduced two distinct classes of lipids as potent regulators of Kir channels: long-chain acyl-CoA (LC-CoA) esters (e.g., oleoyl-CoA) and phosphatidylinositol phosphates (e.g., PIP2). PIP2 is known to activate all members of the Kir family whereas LC-CoA esters appear to be a specific activators of KATP channels. Here we studied the effect of LC-CoA esters on different Kir channels heterologously expressed in Xenopus laevis oocytes. In agreement with previous reports, none of the Kir channels tested (Kir1.1, Kir2.1, Kir3.4, Kir4.1, and Kir7.1) were activated by oleoyl-CoA. Quite the contrary, we found LC-CoA esters to be potent inhibitors of all Kir channels (except Kir6.2). PIP2 reversed the inhibitory effect of oleoyl-CoA, suggesting a displacement of PIP2 by oleoly-CoA from a common binding site as the mechanism behind the oleoyl-CoA inhibition. This notion is supported by mutant channels with reduced PIP2 affinity and biochemical studies assaying the binding of PIP2 and oleoyl-CoA on N[H.sub.2]- and COOH-terminal fragments of Kir2.1 and Kir6.2. In addition, we provide evidence that PIP2 and LC-CoA regulate the activity of Kir channels via strucmral changes of in the selectivity filter of the channel's pore.