NMR structure of the "ball-and-chain" domain of KCNMB2, the beta 2-subunit of large conductance Ca2+- and voltage-activated potassium channels.

The auxiliary beta-subunit KCNMB2 (beta(2)) endows the non-inactivating large conductance Ca(2+)- and voltage-dependent potassium (BK) channel with fast inactivation. This process is mediated by the N terminus of KCNMB2 and closely resembles the "ball-and-chain"-type inactivation observed in voltage-gated potassium channels. Here we investigated the solution structure and function of the KCNMB2 N terminus (amino acids 1-45, BKbeta(2)N) using NMR spectroscopy and patch clamp recordings. BKbeta(2)N completely inactivated BK channels when applied to the cytoplasmic side; its interaction with the BK alpha-subunit is characterized by a particularly slow dissociation rate and an affinity in the upper nanomolar range. The BKbeta(2)N structure comprises two domains connected by a flexible linker: the pore-blocking "ball domain" (formed by residues 1-17) and the "chain domain" (between residues 20-45) linking it to the membrane segment of KCNMB2. The ball domain is made up of a flexible N terminus anchored at a well ordered loop-helix motif. The chain domain consists of a 4-turn helix with an unfolded linker at its C terminus. These structural properties explain the functional characteristics of BKbeta(2)N-mediated inactivation.

K(+)-dependent gating of K(ir)1.1 channels is linked to pH gating through a conformational change in the pore.

1. We have used giant patch-clamp recording to investigate the interaction between pH gating and K(+)-dependent gating in rat K(ir)1.1 (ROMK) channels heterologously expressed in Xenopus oocytes. 2. Gating by intracellular protons (pH gating) and extracellular K(+) ions (K(+)-dependent gating) is a hallmark of K(ir)1.1 channels that mediate K(+) secretion and control NaCl reabsorption in the kidney. pH gating is driven by protonation of an intracellular lysine residue (K80 in K(ir)1.1). K(+)-dependent gating occurs upon withdrawal of K(+) ions from the extracellular side of the channel. Both gating mechanisms are thought to interact allosterically. 3. K(+)-dependent gating was shown to be strictly coupled to pH gating; it only occurred when channels were in the pH-inactivated closed state, but not in the open state. Moreover, K(+)-dependent gating was absent in the non-pH-gated mutant K(ir)1.1(K80 M). 4. Channels inactivated by K(+)-dependent gating were reactivated upon addition of permeant ions to the extracellular side of the membrane, while impermeant ions failed to induce channel reactivation. Moreover, mutagenesis identified two residues in the P-helix (L136 and V140 in K(ir)1.1) that are crucial for K(+)-dependent gating. Replacement of these residues with the ones present in the non-K(+)-gated K(ir)2.1 abolished K(+)-dependent gating of K(ir)1.1 channels without affecting pH gating. 5. The results indicate that pH gating and K(+)-dependent gating are coupled to each other via structural rearrangements in the inner pore involving the P-helix.

Intracellular anions as the voltage sensor of prestin, the outer hair cell motor protein.

Outer hair cells (OHCs) of the mammalian cochlea actively change their cell length in response to changes in membrane potential. This electromotility, thought to be the basis of cochlear amplification, is mediated by a voltage-sensitive motor molecule recently identified as the membrane protein prestin. Here, we show that voltage sensitivity is conferred to prestin by the intracellular anions chloride and bicarbonate. Removal of these anions abolished fast voltage-dependent motility, as well as the characteristic nonlinear charge movement ("gating currents") driving the underlying structural rearrangements of the protein. The results support a model in which anions act as extrinsic voltage sensors, which bind to the prestin molecule and thus trigger the conformational changes required for motility of OHCs.

Memantine inhibits efferent cholinergic transmission in the cochlea by blocking nicotinic acetylcholine receptors of outer hair cells.

Memantine is a blocker of Ca(2+)-permeable glutamate and nicotinic acetylcholine receptors (nAChR). We investigated the action of memantine on cholinergic synaptic transmission at cochlear outer hair cells (OHCs). At this inhibitory synapse, hyperpolarization of the postsynaptic cell results from opening of SK-type Ca(2+)-activated K(+) channels via a highly Ca(2+)-permeable nAChR containing the alpha 9 subunit. We show that inhibitory postsynaptic currents recorded from OHCs were reversibly blocked by memantine with an IC(50) value of 16 microM. RT-PCR revealed that a newly cloned nAChR subunit, alpha 10, is expressed in OHCs. In contrast to homomeric expression, coexpression of alpha 9 and alpha 10 subunits in Xenopus laevis oocytes resulted in robust acetylcholine-induced currents, indicating that the OHC nAChR may be an alpha 9/alpha 10 heteromer. Accordingly, nAChR currents evoked by application of the ligand to OHCs and currents through alpha 9/alpha 10 were blocked by memantine with a similar IC(50) value of about 1 microM. Memantine block of alpha 9/alpha 10 was moderately voltage dependent. The lower efficacy of memantine for inhibition of inhibitory postsynaptic currents (IPSCs) most probably results from a blocking rate that is slow with respect to the short open time of the receptor channels during an IPSC. Thus, synaptic transmission in OHCs is inhibited by memantine block of Ca(2+) influx through nAChRs. Importantly, prolonged receptor activation and consequently massive Ca(2+) influx, as might occur under pathological conditions, is blocked at low micromolar concentrations, whereas the fast IPSCs initiated by short receptor activation are only blocked at concentrations above 10 microM.

Reciprocal electromechanical properties of rat prestin: the motor molecule from rat outer hair cells.

Cochlear outer hair cells (OHCs) are responsible for the exquisite sensitivity, dynamic range, and frequency-resolving capacity of the mammalian hearing organ. These unique cells respond to an electrical stimulus with a cycle-by-cycle change in cell length that is mediated by molecular motors in the cells' basolateral membrane. Recent work identified prestin, a protein with similarity to pendrin-related anion transporters, as the OHC motor molecule. Here we show that heterologously expressed prestin from rat OHCs (rprestin) exhibits reciprocal electromechanical properties as known for the OHC motor protein. Upon electrical stimulation in the microchamber configuration, rprestin generates mechanical force with constant amplitude and phase up to a stimulus frequency of at least 20 kHz. Mechanical stimulation of rprestin in excised outside-out patches shifts the voltage dependence of the nonlinear capacitance characterizing the electrical properties of the molecule. The results indicate that rprestin is a molecular motor that displays reciprocal electromechanical properties over the entire frequency range relevant for mammalian hearing.

Developmental expression of the small-conductance Ca(2+)-activated potassium channel SK2 in the rat retina.

Small-conductance Ca(2+)-activated potassium (SK) channels are present in most central neurons, where they mediate the afterhyperpolarizations (AHPs) following action potentials. SK channels integrate changes in intracellular Ca(2+) concentration with membrane potential and thus play an important role in controlling firing pattern and excitability. Here, we characterize the expression pattern of the apamin-sensitive SK subunits, SK2 and SK3, in the developing and adult rat retina using in situ hybridization and immunohistochemistry. The SK2 subunit showed a distinct and developmentally regulated pattern of expression. It appeared during the first postnatal week and located to retinal ganglion cells and to subpopulations of neurons in the inner nuclear layer. These neurons were identified as horizontal cells and dopaminergic amacrine cells by specific markers. In contrast to SK2, the SK3 subunit was detected neither in the developing nor in the adult retina. These results show cell-specific expression of the SK2 subunit in the retina and suggest that this channel underlies the apamin-sensitive AHP currents described in retinal ganglion cells.

Control of electrical activity in central neurons by modulating the gating of small conductance Ca2+-activated K+ channels.

In most central neurons, action potentials are followed by an afterhyperpolarization (AHP) that controls firing pattern and excitability. The medium and slow components of the AHP have been ascribed to the activation of small conductance Ca(2+)-activated potassium (SK) channels. Cloned SK channels are heteromeric complexes of SK alpha-subunits and calmodulin. The channels are activated by Ca(2+) binding to calmodulin that induces conformational changes resulting in channel opening, and channel deactivation is the reverse process brought about by dissociation of Ca(2+) from calmodulin. Here we show that SK channel gating is effectively modulated by 1-ethyl-2-benzimidazolinone (EBIO). Application of EBIO to cloned SK channels shifts the Ca(2+) concentration-response relation into the lower nanomolar range and slows channel deactivation by almost 10-fold. In hippocampal CA1 neurons, EBIO increased both the medium and slow AHP, strongly reducing electrical activity. Moreover, EBIO suppressed the hyperexcitability induced by low Mg(2+) in cultured cortical neurons. These results underscore the importance of SK channels for shaping the electrical response patterns of central neurons and suggest that modulating SK channel gating is a potent mechanism for controlling excitability in the central nervous system.

Polyamines as gating molecules of inward-rectifier K+ channels.

Inward-rectifier potassium (Kir) channels comprise a superfamily of potassium (K+) channels with unique structural and functional properties. Expressed in virtually all types of cells they are responsible for setting the resting membrane potential, controlling the excitation threshold and secreting K+ ions. All Kir channels present an inwardly rectifying current-voltage relation, meaning that at any given driving force the inward flow of K+ ions exceeds the outward flow for the opposite driving force. This inward-rectification is due to a voltage-dependent block of the channel pore by intracellular polyamines and magnesium. The present molecular-biophysical understanding of inward-rectification and its physiological consequences is the topic of this review. In addition to polyamines, Kir channels are gated by intracellular protons, G-proteins, ATP and phospholipids depending on the respective Kir subfamily as detailed in the following review articles.

Gating of inward-rectifier K+ channels by intracellular pH.

Inward rectifier K+ channels of the Kir1.1 (ROMK) and Kir4.1 subtype are predominantly expressed in epithelial cells where they are responsible for K+ transport across the plasma membrane. Uniquely among the members of the Kir family, these channels are gated by intracellular pH in the physiological range. pH-gating involves structural rearrangements in cytoplasmic domains and the P-loop of the Kir protein. The energy for the gating transition is delivered by protonation of a lysine residue that is located prior to the first transmembrane segment and serves as a 'pH sensor'. The anomalous titration required for lysine operating in the neutral pH range results from its close interaction with two positively charged arginines from the distant N- and C-termini termed the R/K/R triad. Disturbance of this triad as results from a number of point mutations found in patients with hyperprostaglandin E syndrome (HPS) increases the pKa of the pH sensor and results in channels being permanently inactivated under physiological conditions. This article will focus on the mechanism of pH-gating, its implications for the tertiary structure of Kir proteins and on its significance for the pathogenesis of HPS.

KATP channels gated by intracellular nucleotides and phospholipids.

The KATP channel is a heterooctamer composed of two different subunits, four inwardly rectifying K+ channel subunits, either Kir6. 1 or Kir6.2, and four sulfonylurea receptors (SUR), which belong to the family of ABC transporters. This unusual molecular architecture is related to the complex gating behaviour of these channels. Intracellular ATP inhibits KATP channels by binding to the Kir6.x subunits, whereas Mg-ADP increases channel activity by a hydrolysis reaction at the SUR. This ATP/ADP dependence allows KATP channels to link metabolism to excitability, which is important for many physiological functions, such as insulin secretion and cell protection during periods of ischemic stress. Recent work has uncovered a new class of regulatory molecules for KATP channel gating. Membrane phospholipids such as phosphoinositol 4, 5-bisphosphate and phosphatidylinositiol 4-monophosphate were found to interact with KATP channels resulting in increased open probability and markedly reduced ATP sensitivity. The membrane concentration of these phospholipids is regulated by a set of enzymes comprising phospholipases, phospholipid phosphatases and phospholipid kinases providing a possible mechanism for control of cell excitability through signal transduction pathways that modulate activity of these enzymes. This review discusses the mechanisms and molecular determinants that underlie gating of KATP channel by nucleotides and phospholipids and their physiological implications.

K(ATP) channels: linker between phospholipid metabolism and excitability.

ATP-sensitive potassium (K(ATP)) channels couple electrical activity to cellular metabolism via their inhibition by intracellular ATP. When examined in excised patches, ATP concentrations required for half-maximal inhibition (IC(50)) varied among tissues and were reported to be as low as 10 microM. This set up a puzzling question on how activation of K(ATP) channels can occur under physiological conditions, where the cytoplasmic concentration of ATP is much higher than that required for channel inhibition. A new twist was added to this puzzle when two recent reports showed that phospholipids such as phosphatidylinositol-4,5-bisphosphate (PIP(2)) and phosphatidyl-4-phosphate (PIP) are able to shift ATP-sensitivity of K(ATP) channels from the micro- into the millimolar range and thus provide a mechanism for physiological activation of the channels. This commentary describes how phospholipids control ATP inhibition of K(ATP) channels and how this mechanism is regulated effectively by receptor-mediated stimulation of phospholipase C.

Gating of Ca2+-activated K+ channels controls fast inhibitory synaptic transmission at auditory outer hair cells.

Fast inhibitory synaptic transmission in the central nervous system is mediated by ionotropic GABA or glycine receptors. Auditory outer hair cells present a unique inhibitory synapse that uses a Ca2+-permeable excitatory acetylcholine receptor to activate a hyperpolarizing potassium current mediated by small conductance calcium-activated potassium (SK) channels. It is shown here that unitary inhibitory postsynaptic currents at this synapse are mediated by SK2 channels and occur rapidly, with rise and decay time constants of approximately 6 ms and approximately 30 ms, respectively. This time course is determined by the Ca2+ gating of SK channels rather than by the changes in intracellular Ca2+. The results demonstrate fast coupling between an excitatory ionotropic neurotransmitter receptor and an inhibitory ion channel and imply rapid, localized changes in subsynaptic calcium levels.

pH gating of ROMK (K(ir)1.1) channels: control by an Arg-Lys-Arg triad disrupted in antenatal Bartter syndrome.

Inward-rectifier K(+) channels of the ROMK (K(ir)1.1) subtype are responsible for K(+) secretion and control of NaCl absorption in the kidney. A hallmark of these channels is their gating by intracellular pH in the neutral range. Here we show that a lysine residue close to TM1, identified previously as a structural element required for pH-induced gating, is protonated at neutral pH and that this protonation drives pH gating in ROMK and other K(ir) channels. Such anomalous titration of this lysine residue (Lys-80 in K(ir)1.1) is accomplished by the tertiary structure of the K(ir) protein: two arginines in the distant N and C termini of the same subunit (Arg-41 and Arg-311 in K(ir)1.1) are located in close spatial proximity to the lysine allowing for electrostatic interactions that shift its pK(a) into the neutral pH range. Structural disturbance of this triad as a result from a number of point mutations found in patients with antenatal Bartter syndrome shifts the pK(a) of the lysine residue off the neutral pH range and results in channels permanently inactivated under physiological conditions. Thus, the results provide molecular understanding for normal pH gating of K(ir) channels as well as for the channel defects found in patients with antenatal Bartter syndrome.

NMR structure and functional characteristics of the hydrophilic N terminus of the potassium channel beta-subunit Kvbeta1.1.

Rapid N-type inactivation of voltage-dependent potassium (Kv) channels controls membrane excitability and signal propagation in central neurons and is mediated by protein domains (inactivation gates) occluding the open channel pore from the cytoplasmic side. Inactivation domains (ID) are donated either by the pore-forming alpha-subunit or certain auxiliary beta-subunits. Upon coexpression, Kvbeta1.1 was found to endow non-inactivating members of the Kv1alpha family with fast inactivation via its unique N terminus. Here we investigated structure and functional properties of the Kvbeta1.1 N terminus (amino acids 1-62, betaN-(1-62)) using NMR spectroscopy and patch clamp recordings. betaN-(1-62) showed all hallmarks of N-type inactivation: it inactivated non-inactivating Kv1.1 channels when applied to the cytoplasmic side as a synthetic peptide, and its interaction with the alpha-subunit was competed with tetraethylammonium and displayed an affinity in the lower micromolar range. In aequous and physiological salt solution, betaN-(1-62) showed no well defined three-dimensional structure, it rather existed in a fast equilibrium of multiple weakly structured states. These structural and functional properties of betaN-(1-62) closely resemble those of the "unstructured" ID from Shaker B, but differ markedly from those of the compactly folded ID of the Kv3.4 alpha-subunit.

Domains responsible for constitutive and Ca(2+)-dependent interactions between calmodulin and small conductance Ca(2+)-activated potassium channels.

Small conductance Ca(2+)-activated potassium channels (SK channels) are coassembled complexes of pore-forming SK alpha subunits and calmodulin. We proposed a model for channel activation in which Ca2+ binding to calmodulin induces conformational rearrangements in calmodulin and the alpha subunits that result in channel gating. We now report fluorescence measurements that indicate conformational changes in the alpha subunit after calmodulin binding and Ca2+ binding to the alpha subunit-calmodulin complex. Two-hybrid experiments showed that the Ca(2+)-independent interaction of calmodulin with the alpha subunits requires only the C-terminal domain of calmodulin and is mediated by two noncontiguous subregions; the ability of the E-F hands to bind Ca2+ is not required. Although SK alpha subunits lack a consensus calmodulin-binding motif, mutagenesis experiments identified two positively charged residues required for Ca(2+)-independent interactions with calmodulin. Electrophysiological recordings of SK2 channels in membrane patches from oocytes coexpressing mutant calmodulins revealed that channel gating is mediated by Ca2+ binding to the first and second E-F hand motifs in the N-terminal domain of calmodulin. Taken together, the results support a calmodulin- and Ca(2+)-calmodulin-dependent conformational change in the channel alpha subunits, in which different domains of calmodulin are responsible for Ca(2+)-dependent and Ca(2+)-independent interactions. In addition, calmodulin is associated with each alpha subunit and must bind at least one Ca2+ ion for channel gating. Based on these results, a state model for Ca2+ gating was developed that simulates alterations in SK channel Ca2+ sensitivity and cooperativity associated with mutations in CaM.

Expression density and functional characteristics of the outer hair cell motor protein are regulated during postnatal development in rat.

1. The non-linear capacitance (Cnon-lin) of postnatal outer hair cells (OHCs) of the rat was measured by a patch-clamp lock-in technique. Cnon-lin is thought to result from a membrane protein that provides the molecular basis for the unique electromotility of OHCs by undergoing conformational changes in response to changes in membrane potential (Vm). Protein conformation is coupled to Vm by a charged voltage sensor, which imposes Cnon-lin on the OHC. Cnon-lin was investigated in order to characterize the surface expression and voltage dependence of this motor protein during postnatal development. 2. On the day of birth (P0), Cnon-lin was not detected in OHCs of the basal turn of the cochlea, whilst it was 89 fF in apical OHCs. Cnon-lin increased gradually during postnatal development and reached 2.3 pF (basal turn, P9) and 7.5 pF (apical turn, P14) at the oldest developmental stages covered by our measurements. The density of the protein in the plasma membrane, deduced from non-linear charge movement per membrane area, increased steeply between P6 and P11 and reached steady state (4200 e- microm-2) at about P12. 3. Voltage at peak capacitance (V) shifted with development from hyperpolarized potentials shortly after birth (-88.3 mV, P2) to the depolarized potential characteristic of mature OHCs (-40.8 mV, P14). This developmental difference in V was also observed in outside-out patches immediately after patch excision. During subsequent wash-out V shifted towards the depolarized value found in the adult state, suggesting a direct modulation of the molecular motor. 4. Thus, the density of the motor protein in the plasma membrane and also its voltage dependence change concomitantly in the postnatal period and reach adult characteristics right at the onset of hearing.

Inward rectification in KATP channels: a pH switch in the pore.

Inward-rectifier potassium channels (Kir channels) stabilize the resting membrane potential and set a threshold for excitation in many types of cell. This function arises from voltage-dependent rectification of these channels due to blockage by intracellular polyamines. In all Kir channels studied to date, the voltage-dependence of rectification is either strong or weak. Here we show that in cardiac as well as in cloned KATP channels (Kir6.2 + sulfonylurea receptor) polyamine-mediated rectification is not fixed but changes with intracellular pH in the physiological range: inward-rectification is prominent at basic pH, while at acidic pH rectification is very weak. The pH-dependence of polyamine block is specific for KATP as shown in experiments with other Kir channels. Systematic mutagenesis revealed a titratable C-terminal histidine residue (H216) in Kir6.2 to be the structural determinant, and electrostatic interaction between this residue and polyamines was shown to be the molecular mechanism underlying pH-dependent rectification. This pH-dependent block of KATP channels may represent a novel and direct link between excitation and intracellular pH.

Molecular and functional characterization of s-KCNQ1 potassium channel from rectal gland of Squalus acanthias.

Functional and pharmacological data point to the involvement of KCNQ1/IsK potassium channels in the basolateral potassium conductance of secretory epithelia. In this study, we report the cloning and electrophysiological characterization of the KCNQ1 protein from the salt secretory rectal gland of the spiny dogfish (Squalus acanthias). The S. acanthias KCNQ1 (s-KCNQ1) cDNA was cloned by polymerase chain reaction (PCR) intensive techniques and showed overall sequence similarities with the KCNQ1 potassium channel subunits of Man, mouse and Xenopus laevis of 64, 70 and 77%, respectively, at the translated amino acid level. Analysis of s-KCNQ1 expression on a Northern blot containing RNA from heart, rectal gland, kidney, brain, intestine, testis, liver and gills revealed distinct expression of 7.4-kb s-KCNQ1 transcripts only in rectal gland and heart. Voltage-clamp analysis of s-KCNQ1 expressed in Xenopus oocytes showed pronounced electrophysiological similarities to human and murine KCNQ1 isoforms, with a comparable sensitivity to inhibition by the chromanol 293B. Coexpression of s-KCNQ1 with human-IsK (h-IsK) induced currents with faster activation kinetics and stronger rectification than observed after coexpression of human KCNQ1 with h-IsK, with the voltage threshold of activation shifted to more negative potentials. The low activation threshold at approximately -60 mV in combination with the high expression in rectal gland cells make s-KCNQ1 a potential candidate responsible for the basolateral potassium conductance.

Control of K+ channel gating by protein phosphorylation: structural switches of the inactivation gate.

Fast N-type inactivation of voltage-dependent potassium (Kv) channels controls membrane excitability and signal propagation in central neurons and occurs by a 'ball-and-chain'-type mechanism. In this mechanism an N-terminal protein domain (inactivation gate) occludes the pore from the cytoplasmic side. In Kv3.4 channels, inactivation is not fixed but is dynamically regulated by protein phosphorylation. Phosphorylation of several identified serine residues on the inactivation gate leads to reduction or removal of fast inactivation. Here, we investigate the structure-function basis of this phospho-regulation with nuclear magnetic resonance (NMR) spectroscopy and patch-clamp recordings using synthetic inactivation domains (ID). The dephosphorylated ID exhibited compact structure and displayed high-affinity binding to its receptor. Phosphorylation of serine residues in the N- or C-terminal half of the ID resulted in a loss of overall structural stability. However, depending on the residue(s) phosphorylated, distinct structural elements remained stable. These structural changes correlate with the distinct changes in binding and unbinding kinetics underlying the reduced inactivation potency of phosphorylated IDs.

pH-dependent gating of ROMK (Kir1.1) channels involves conformational changes in both N and C termini.

ROMK channels (Kir1.1) are members of the superfamily of inward rectifier potassium channels (Kir) and represent the channels underlying K+ secretion in the kidney. As their native counterparts, Kir1.1 channels are gated by intracellular pH, with acidification leading to channel closure. Although a lysine residue (Lys80) close to the first hydrophobic segment M1 has been identified as the pH sensor, little is known about how opening and closing of the channel is accomplished. Here we investigate the gating process of Kir1.1 channels exploiting their state-dependent modification by water-soluble oxidants and sulfhydryl reagents. Mutagenesis of all intracellular cysteines either alone or in combination revealed two residues targeted by these reagents, one in the N terminus (Cys49) and one in the C terminus (Cys308) of the channel protein. Both sites reacted with the thiol reagents only in the closed state and not in the open state. These results indicate that pH-dependent gating of Kir1.1 channels involves movement of protein domains in both N and C termini of the Kir1.1 protein.