The participation of amino-terminal domains in human ether-a-go-go (eag)-related gene (HERG) K(+) channel gating was studied using deleted channel variants expressed in Xenopus oocytes. Selective deletion of the HERG-specific sequence (HERG Delta138-373) located between the conserved initial amino terminus (the eag or PAS domain) and the first transmembrane helix accelerates channel activation and shifts its voltage dependence to hyperpolarized values. However, deactivation time constants from fully activated states and channel inactivation remain almost unaltered after the deletion. The deletion effects are equally manifested in channel variants lacking inactivation. The characteristics of constructs lacking only about half of the HERG-specific domain (Delta223-373) or a short stretch of 19 residues (Delta355-373) suggest that the role of this domain is not related exclusively to its length, but also to the presence of specific sequences near the channel core. Deletion-induced effects are partially reversed by the additional elimination of the eag domain. Thus the particular combination of HERG-specific and eag domains determines two important HERG features: the slow activation essential for neuronal spike-frequency adaptation and maintenance of the cardiac action potential plateau, and the slow deactivation contributing to HERG inward rectification.

1. Modulation of the human ether-à-go-go-related gene (HERG) K+ channel was studied in two-electrode voltage-clamped Xenopus oocytes co-expressing the channel protein and the thyrotropin-releasing hormone (TRH) receptor. 2. Addition of TRH caused clear modifications of HERG channel gating kinetics. These variations consisted of an acceleration of deactivation, as shown by a faster decay of hyperpolarization-induced tail currents, and a slower time course of activation, measured using an envelope of tails protocol. The voltage dependence for activation was also shifted by nearly 20 mV in the depolarizing direction. Neither the inactivation nor the inactivation recovery rates were altered by TRH. 3. The alterations in activation gating parameters induced by TRH were demonstrated in a direct way by looking at the increased outward K+ currents elicited in extracellular solutions in which K+ was replaced by Cs+. 4. The effects of TRH were mimicked by direct pharmacological activation of protein kinase C (PKC) with beta-phorbol 12-myristate, 13-acetate (PMA). The TRH-induced effects were antagonized by GF109203X, a highly specific inhibitor of PKC that also abolished the PMA-dependent regulation of the channels. 5. It is concluded that a PKC-dependent pathway links G protein-coupled receptors that activate phospholipase C to modulation of HERG channel gating. This provides a mechanism for the physiological regulation of cardiac function by phospholipase C-activating receptors, and for modulation of adenohypophysial neurosecretion in response to TRH.

Within the potassium ion channel family, calcium activated potassium (KCa) channels are unique in their ability to couple intracellular Ca2+ signals to membrane potential variations. KCa channels are diversely distributed throughout the central nervous system and play fundamental roles ranging from regulating neuronal excitability to controlling neurotransmitter release. The physiological versatility of KCa channels is enhanced by alternative splicing and co-assembly with auxiliary subunits, leading to fundamental differences in distribution, subunit composition and pharmacological profiles. Thus, understanding specific KCa channels’ mechanisms in neuronal function is challenging. Based on their single channel conductance, KCa channels are divided into three subtypes: small (SK, 4–14 pS), intermediate (IK, 32–39 pS) and big potassium (BK, 200–300 pS) channels. This review describes the biophysical characteristics of these KCa channels, as well as their physiological roles and pathological implications. In addition, we also discuss the current pharmacological strategies and challenges to target KCa channels for the treatment of various neurological and psychiatric disorders.

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 Ca 2+ 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 Ca 2+ 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.arge-conductance voltage-and calcium-dependent (BK) potassium channels are characterized by both their large singlechannel conductance and their synergistic activation by Ca 2+ and membrane depolarization (1). These channels are crucial regulators of physiological processes such as neurosecretion, neuronal firing, and smooth muscle tone (2). In humans, defects in BK channels can cause hypertension, cancer, and epilepsy (3)(4)(5).BK channels at the plasma membrane are homotetramers of α-subunits, which can assemble with various accessory subunits. The α-subunits, encoded by the potassium large-conductance calcium-activated channel, subfamily M, α member 1 (KCNMA1) gene, consist of seven membrane-spanning regions (S0-S6) and a large intracellular C-terminal domain ( Fig. 1A) (1). In common with other voltage-gated channels, the voltage sensor resides within the membrane (6), whereas Ca 2+ binds to binding sites located at a large C-terminal intracellular region where eight regulator of conductance for K + (RCK) domains form a "gating ring" (7-11). Kinetic modeling of the Ca 2+ -and voltage-dependent activation of BK channels suggests that fairly complex mechanisms are required to describe channel activity (12)(13)(14)(15)(16) (11,18,19).Recently, structures of gating rings isolated from eukaryotic channels have been solved (9, 10), and in the most recent X-ray structure obtained in the presence of Ca 2+ (11) the layer formed by the four RCK1 domains of the tetramer is seen to be expanded relative to the previous structures by more than 10 Å. Because this region of the gating ring is directly linked to the channel's pore-forming helices, this expansion could be the direct link between Ca 2+ binding and the opening of the pore in BK channels. To measure directly the conformational changes between subunits at the level of the gating ring, during the activation of functional BK channels, we performed patch-clamp fluorometry (20) on membrane patches containing fluorescently labeled BK channels. These channels were selected from a library of functional YFP-or CFP-fusion proteins (21). Simultaneous optical and electrical recording revealed large changes in fluorescence resonance energy transfer (FRET) accompanying Ca 2+ binding and channel activation. ResultsHeterotetramers...

Epithelial sodium channel (ENaC) is a member of the ENaC/degenerin family of amiloride-sensitive, non-voltage gated sodium ion channels. ENaC alpha, beta and gamma subunits are abundantly expressed in epithelial tissues, where they have been well characterized. An ENaC delta subunit has also been described in the human nervous system, although its histological distribution pattern remains unexplored. We have now isolated a novel ENaC delta isoform (delta2) from human brain and studied the expression pattern of both the known (delta1) and the new (delta2) isoforms in the human and monkey telencephalon. ENaC delta2 is produced by a combination of alternative transcription start sites, a frame shift in exon 3 and alternative splicing of exon 4. It forms functional amiloride-sensitive sodium channels when co-expressed with ENaC beta and gamma accessory subunits. Comparison with the classical ENaC channel (alphabetagamma) indicates that the interaction between delta2, beta and gamma is functionally inefficient. Both ENaC delta isoforms are widely expressed in pyramidal cells of the human and monkey cerebral cortex and in different neuronal populations of telencephalic subcortical nuclei, but double-labelling experiments demonstrated a low level of co-localization between isoforms (5-8%), suggesting specific functional roles for each of them.