Up-regulation of GABAB Receptor Signaling by Constitutive Assembly with the K+ Channel Tetramerization Domain-containing Protein 12 (KCTD12)

GABA_B receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous system. Native GABA_B receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits GABA_B1a, GABA_B1b, and GABA_B2 form fully functional heteromeric GABA_B(1a,2) and GABA_B(1b,2) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic proteins that influence agonist potency and G-protein signaling of GABA_B(1a,2) and GABA_B(1b,2) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABA_B receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we show that GABA_B receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal neurons reduces the magnitude of the GABA_B receptor-mediated K⁺ current response. In summary, our experiments support that the up-regulation of functional GABA_B receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.     

Caveolin-1 Facilitates the Direct Coupling between Large Conductance Ca2+-activated K+ (BKCa) and Cav1.2 Ca2+ Channels and Their Clustering to Regulate Membrane Excitability in Vascular Myocytes

L-type voltage-dependent Ca²⁺ channels (LVDCC) and large conductance Ca²⁺-activated K⁺ channels (BK_Ca) are the major factors defining membrane excitability in vascular smooth muscle cells (VSMCs). The Ca²⁺ release from sarcoplasmic reticulum through ryanodine receptor significantly contributes to BK_Ca activation in VSMCs. In this study direct coupling between LVDCC (Cav1.2) and BK_Ca and the role of caveoline-1 on their interaction in mouse mesenteric artery SMCs were examined. The direct activation of BK_Ca by Ca²⁺ influx through coupling LVDCC was demonstrated by patch clamp recordings in freshly isolated VSMCs. Using total internal reflection fluorescence microscopy, it was found that a large part of yellow fluorescent protein-tagged BK_Ca co-localized with the cyan fluorescent protein-tagged Cav1.2 expressed in the plasma membrane of primary cultured mouse VSMCs and that the two molecules often exhibited FRET. It is notable that each BKα subunit of a tetramer in BK_Ca can directly interact with Cav1.2 and promotes Cav1.2 cluster in the molecular complex. Furthermore, caveolin-1 deficiency in knock-out (KO) mice significantly reduced not only the direct coupling between BK_Ca and Cav1.2 but also the functional coupling between BK_Ca and ryanodine receptor in VSMCs. The measurement of single cell shortening by 40 mm K⁺ revealed enhanced contractility in VSMCs from KO mice than wild type. Taken together, caveolin-1 facilitates the accumulation/clustering of BK_Ca-LVDCC complex in caveolae, which effectively regulates spatiotemporal Ca²⁺ dynamics including the negative feedback, to control the arterial excitability and contractility.     

An Extracellular Ion Pathway Plays a Central Role in the Cooperative Gating of a K2P K+ Channel by Extracellular pH

Proton-gated TASK-3 K⁺ channel belongs to the K_2P family of proteins that underlie the K⁺ leak setting the membrane potential in all cells. TASK-3 is under cooperative gating control by extracellular [H⁺]. Use of recently solved K_2P structures allows us to explore the molecular mechanism of TASK-3 cooperative pH gating. Tunnel-like side portals define an extracellular ion pathway to the selectivity filter. We use a combination of molecular modeling and functional assays to show that pH-sensing histidine residues and K⁺ ions mutually interact electrostatically in the confines of the extracellular ion pathway. K⁺ ions modulate the pK_a of sensing histidine side chains whose charge states in turn determine the open/closed transition of the channel pore. Cooperativity, and therefore steep dependence of TASK-3 K⁺ channel activity on extracellular pH, is dependent on an effect of the permeant ion on the channel pH_o sensors.     

Identification of Novel Cholesterol-binding Regions in Kir2 Channels

Inwardly rectifying potassium (Kir) channels play an important role in setting the resting membrane potential and modulating membrane excitability. We have recently shown that cholesterol regulates representative members of the Kir family and that in the majority of the cases, cholesterol suppresses channel function. Furthermore, recent data indicate that cholesterol regulates Kir channels by specific sterol-protein interactions, yet the location of the cholesterol binding site in Kir channels is unknown. Using a combined computational-experimental approach, we show that cholesterol may bind to two nonanular hydrophobic regions in the transmembrane domain of Kir2.1 located between adjacent subunits of the channel. The location of the binding regions suggests that cholesterol modulates channel function by affecting the hinging motion at the center of the pore-lining transmembrane helix that underlies channel gating either directly or through the interface between the N and C termini of the channel.     

Genotypic difference in salinity tolerance in quinoa is determined by differential control of xylem Na loading and stomatal density

Quinoa is regarded as a highly salt tolerant halophyte crop, of great potential for cultivation on saline areas around the world. Fourteen quinoa genotypes of different geographical origin, differing in salinity tolerance, were grown under greenhouse conditions. Salinity treatment started on 10 day old seedlings. Six weeks after the treatment commenced, leaf sap Na and K content and osmolality, stomatal density, chlorophyll fluorescence characteristics, and xylem sap Na and K composition were measured. Responses to salinity differed greatly among the varieties. All cultivars had substantially increased K⁺ concentrations in the leaf sap, but the most tolerant cultivars had lower xylem Na⁺ content at the time of sampling. Most tolerant cultivars had lowest leaf sap osmolality. All varieties reduced stomata density when grown under saline conditions. All varieties clustered into two groups (includers and excluders) depending on their strategy of handling Na⁺ under saline conditions. Under control (non-saline) conditions, a strong positive correlation was observed between salinity tolerance and plants ability to accumulate Na⁺ in the shoot. Increased leaf sap K⁺, controlled Na⁺ loading to the xylem, and reduced stomata density are important physiological traits contributing to genotypic differences in salinity tolerance in quinoa, a halophyte species from Chenopodium family.     

Ammonia emissions and biodegradation of organic carbon during sewage sludge composting with different extra carbon sources

Ammonia emissions during composting result in the reduced value of agronomic production and can also pollute the air. To evaluate the influence of various carbon sources on ammonia emissions, six composting experiments were carried out with different amendments of carbon sources (glucose, sucrose and straw powder). The cumulative ammonia volatilizations were reduced from 3.11 g/kg (R6) to 2.46 g/kg (R1), 2.17 g/kg (R2), 2.23 g/kg (R4) and 1.93 g/kg (R5). Compared to the control, no significant difference of ammonia emissions and carbon degradation was observed for the mixture of R3 (3.15 g/kg), which was amended with straw powder alone. The co-addition of sucrose and straw powder led to the lowest ammonia emissions. According to these results, a higher C/N ratio did not necessarily indicate an effective solution for reducing ammonia emissions, and not all readily available carbon compounds were helpful in reducing ammonia emissions. The addition of sucrose promoted the decomposition of organic carbon during the intensive stage of ammonia emissions, and the combination of straw and sucrose prolonged this promotion. Thus, the co-addition of sucrose and straw powder made it possible to reduce ammonia emissions drastically by nitrogen immobilization.     

Movement of the S4 segment in the hERG potassium channel during membrane depolarization

Abstract

The hERG potassium channel is a member of the voltage gated potassium (Kv) channel family, comprising a pore domain and four voltage sensing domains (VSDs). Like other Kv channels, the VSD senses changes in membrane voltage and transmits the signal to gates located in the pore domain; the gates open at positive potentials (activation) and close at negative potentials, thereby controlling the ion flux. hERG, however, differs from other Kv channels in that it is activated slowly but inactivated rapidly – a property that is crucial for the role it plays in the repolarization of the cardiac action potential. Voltage-gating requires movement of gating charges across the membrane electric field, which is accomplished by the transmembrane movement of the fourth transmembrane segment, S4, of the VSD containing the positively charged arginine or lysine residues. Here we ask if the functional differences between hERG and other Kv channels could arise from differences in the transmembrane movement of S4. To address this, we have introduced single cysteine residues into the S4 region of the VSD, expressed the mutant channels in Xenopus oocytes and examined the effect of membrane impermeable para-chloromercuribenzene sulphonate on function by the two-electrode voltage clamp technique. Our results show that depolarization results in the accessibility of seven consecutive S4 residues, including the first two charged residues, K525 and R528, to extracellularly applied reagent. These data indicate that the extent of S4 movement in hERG is similar to other Kv channels, including the archabacterial KvAP and the Shaker channel of Drosophila.     

Ion selectivity of the Kat1 K+ channel pore

Kat1 is a highly selective inward-rectifying K⁺ channel that opens for extended periods under conditions of extreme hyperpolarization. Over 200 point mutants in the pore region of the Kat1 K⁺ channel were generated and examined in the yeast Saccharomyces cerevisiae and Xenopus oocytes to assess the effect of the mutations on ion selectivity. Substitutions at the tyrosine of the signature sequence G-Y-G resulted in the most significant alterations in ion selectivity, consistent with its role in the selectivity filter. However, greater than 80% of the mutations throughout the greater pore region also conferred a defect in selectivity demonstrating that the entire pore of Kat1 contributes to the ion selectivity of this channel. Surprisingly, we identified a novel class of mutant channel that conferred enhanced selectivity of K⁺ over Na⁺. Mutants of this class frequently displayed sensitivity to the competing ion Cs⁺. This finding has led us to speculate that the Kat1 channel pore has evolved to balance not only K⁺/Na⁺ selectivity, but selectivity over Cs⁺, and possibly a wide spectrum of potential competing ions.     

N type rapid inactivation in human Kv1.4 channels: functional role of a putative C-terminal helix

Voltage gated potassium channels are tetrameric membrane proteins, which have a central role in cellular excitability. Human Kv1.4 channels open on membrane depolarization and inactivate rapidly by a ‘ball and chain’ mechanism whose molecular determinants have been mapped to the cytoplasmic N terminus of the channel. Here we show that the other terminal end of the channel also plays a role in channel inactivation. Swapping the C-terminal residues of hKv1.4 with those from two non-inactivating channels (hKv1.1 and hKv1.2) affects the rates of inactivation, as well as the recovery of the channel from the inactivated state. Secondary structure predictions of the hKv1.4 sequence reveal a helical structure at its distal C-terminal. Complete removal or partial disruption of this helical region results in channels with remarkably slowed inactivation kinetics. The ionic selectivity and voltage-dependence of channel opening were similar to hKv1.4, indicative of an unperturbed channel pore. These results demonstrate that fast inactivation is modulated by structural elements in the C-terminus, suggesting that the process involves the concerted action of the N- and C-termini.