Sindhu Rajan

International Union of Pharmacology. LV. Nomenclature and Molecular Relationships of Two-P Potassium Channels

In less than a decade since their discovery, the study of K2P channels has revealed that background leak of potassium ions via dedicated pathways is a highly regulated mechanism to control cellular excitability. Potassium leak pathways, active at rest, stabilize membrane potential below firing

Sumoylation Silences the Plasma Membrane Leak K+ Channel K2P1

Reversible, covalent modification with small ubiquitin-related modifier proteins (SUMOs) is known to mediate nuclear import/export and activity of transcription factors. Here, the SUMO pathway is shown to operate at the plasma membrane to control ion channel function. SUMO-conjugating enzyme is seen to be resident in plasma membrane, to assemble with K2P1, and to modify K2P1 lysine 274. K2P1 had not previously shown function despite mRNA expression in heart, brain, and kidney and sequence features like other two-P loop K+ leak (K2P) pores that control activity of excitable cells. Removal of the peptide adduct by SUMO protease reveals K2P1 to be a K+-selective, pH-sensitive, openly rectifying channel regulated by reversible peptide linkage.

Heteromerization of Kir2.x potassium channels contributes to the phenotype of Andersen's syndrome.

Andersens syndrome, an autosomal dominant disorder related to mutations of the potassium channel Kir2.1, is characterized by cardiac arrhythmias, periodic paralysis, and dysmorphic bone structure. The aim of our study was to find out whether heteromerization of Kir2.1 channels with wild-type Kir2.2 and Kir2.3 channels contributes to the phenotype of Andersens syndrome. The following results show that Kir2.x channels can form functional heteromers: (i) HEK293 cells transfected with Kir2.x–Kir2.y concatemers expressed inwardly rectifying K+ channels with a conductance of 28–30 pS. (ii) Expression of Kir2.x–Kir2.y concatemers in Xenopus oocytes produced inwardly rectifying, Ba2+ sensitive currents. (iii) When Kir2.1 and Kir2.2 channels were coexpressed in Xenopus oocytes the IC50 for Ba2+ block of the inward rectifier current differed substantially from the value expected for independent expression of homomeric channels. (iv) Coexpression of nonfunctional Kir2.x constructs, in which the GYG region of the pore region was replaced by AAA, with wild-type Kir2.x channels produced both homomeric and heteromeric dominant-negative effects. (v) Kir2.1 and Kir2.3 channels could be coimmunoprecipitated in membrane extracts from isolated guinea pig cardiomyocytes. (vi) Yeast two-hybrid analysis showed interaction between the N- and C-terminal intracellular domains of different Kir2.x subunits. Coexpression of Kir2.1 mutants related to Andersens syndrome with wild-type Kir2.x channels showed a dominant negative effect, the extent of which varied between different mutants. Our results suggest that differential tetramerization of the mutant allele of Kir2.1 with wild-type Kir2.1, Kir2.2, and Kir2.3 channels represents the molecular basis of the extraordinary pleiotropy of Andersens syndrome.

Interaction with 14-3-3 proteins promotes functional expression of the potassium channels TASK-1 and TASK-3.

The two‐pore‐domain potassium channels TASK‐1, TASK‐3 and TASK‐5 possess a conserved C‐terminal motif of five amino acids. Truncation of the C‐terminus of TASK‐1 strongly reduced the currents measured after heterologous expression in Xenopus oocytes or HEK293 cells and decreased surface membrane expression of GFP‐tagged channel proteins. Two‐hybrid analysis showed that the C‐terminal domain of TASK‐1, TASK‐3 and TASK‐5, but not TASK‐4, interacts with isoforms of the adapter protein 14‐3‐3. A pentapeptide motif at the extreme C‐terminus of TASK‐1, RRx(S/T)x, was found to be sufficient for weak but significant interaction with 14‐3‐3, whereas the last 40 amino acids of TASK‐1 were required for strong binding. Deletion of a single amino acid at the C‐terminal end of TASK‐1 or TASK‐3 abolished binding of 14‐3‐3 and strongly reduced the macroscopic currents observed in Xenopus oocytes. TASK‐1 mutants that failed to interact with 14‐3‐3 isoforms (V411*, S410A, S410D) also produced only very weak macroscopic currents. In contrast, the mutant TASK‐1 S409A, which interacts with 14‐3‐3‐like wild‐type channels, displayed normal macroscopic currents. Co‐injection of 14‐3‐3ζ cRNA increased TASK‐1 current in Xenopus oocytes by about 70 %. After co‐transfection in HEK293 cells, TASK‐1 and 14‐3‐3ζ (but not TASK‐1ΔC5 and 14‐3‐3ζ) could be co‐immunoprecipitated. Furthermore, TASK‐1 and 14‐3‐3 could be co‐immunoprecipitated in synaptic membrane extracts and postsynaptic density membranes. Our findings suggest that interaction of 14‐3‐3 with TASK‐1 or TASK‐3 may promote the trafficking of the channels to the surface membrane.

Charybdotoxin binding in the IKs pore demonstrates two MinK subunits in each channel complex

I(Ks) voltage-gated K(+) channels contain four pore-forming KCNQ1 subunits and MinK accessory subunits in a number that has been controversial. Here, I(Ks) channels assembled naturally by monomer subunits are compared to those with linked subunits that force defined stoichiometries. Two strategies that exploit charybdotoxin (CTX)-sensitive subunit variants are applied. First, CTX on rate, off rate, and equilibrium affinity are found to be the same for channels of monomers and those with a fixed 2:4 MinK:KCNQ1 valence. Second, 3H-CTX and an antibody are used to directly quantify channels and MinK subunits, respectively, showing 1.97 +/- 0.07 MinK per I(Ks) channel. Additional MinK subunits do not enter channels of monomeric subunits or those with fixed 2:4 valence. We conclude that two MinK subunits are necessary, sufficient, and the norm in I(Ks) channels. This stoichiometry is expected for other K(+) channels that contain MinK or MinK-related peptides (MiRPs).

Expression Pattern in Brain of TASK-1, TASK-3, and a Tandem Pore Domain K+ Channel Subunit, TASK-5, Associated with the Central Auditory Nervous System

TWIK-related acid-sensitive K(+) (TASK) channels contribute to setting the resting potential of mammalian neurons and have recently been defined as molecular targets for extracellular protons and volatile anesthetics. We have isolated a novel member of this subfamily, hTASK-5, from a human genomic library and mapped it to chromosomal region 20q12-20q13. hTASK-5 did not functionally express in Xenopus oocytes, whereas chimeric TASK-5/TASK-3 constructs containing the region between M1 and M3 of TASK-3 produced K(+) selective currents. To better correlate TASK subunits with native K(+) currents in neurons the precise cellular distribution of all TASK family members was elucidated in rat brain. A comprehensive in situ hybridization analysis revealed that both TASK-1 and TASK-3 transcripts are most strongly expressed in many neurons likely to be cholinergic, serotonergic, or noradrenergic. In contrast, TASK-5 expression is found in olfactory bulb mitral cells and Purkinje cells, but predominantly associated with the central auditory pathway. Thus, TASK-5 K(+) channels, possibly in conjunction with auxiliary proteins, may play a role in the transmission of temporal information in the auditory system.

Aspartate 112 is the selectivity filter of the human voltage-gated proton channel.

The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage-gated proton channel, HV1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes, histamine secretion by basophils, sperm capacitation, and airway pH. The most selective ion channel known, HV1 shows no detectable permeability to other ions. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity, or (2) water molecules ‘frozen’ in a narrow pore conduct protons while excluding other ions. Here we identify aspartate 112 as a crucial component of the selectivity filter of HV1. When a neutral amino acid replaced Asp 112, the mutant channel lost proton specificity and became anion-selective or did not conduct. Only the glutamate mutant remained proton-specific. Mutation of the nearby Asp 185 did not impair proton selectivity, indicating that Asp 112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp 112, the mutant channel was still anion-permeable. Evidently, the proton specificity of HV1 requires an acidic group at the selectivity filter.

Zinc inhibition of monomeric and dimeric proton channels suggests cooperative gating

Voltage‐gated proton channels are strongly inhibited by Zn2+, which binds to His residues. However, in a molecular model, the two externally accessible His are too far apart to coordinate Zn2+. We hypothesize that high‐affinity Zn2+ binding occurs at the dimer interface between pairs of His residues from both monomers. Consistent with this idea, Zn2+ effects were weaker in monomeric channels. Mutation of His193 and His140 in various combinations and in tandem dimers revealed that channel opening was slowed by Zn2+ only when at least one His was present in each monomer, suggesting that in wild‐type (WT) HV1, Zn2+ binding between His of both monomers inhibits channel opening. In addition, monomeric channels opened exponentially, and dimeric channels opened sigmoidally. Monomeric channel gating had weaker temperature dependence than dimeric channels. Finally, monomeric channels opened 6.6 times faster than dimeric channels. Together, these observations suggest that in the proton channel dimer, the two monomers are closely apposed and interact during a cooperative gating process. Zn2+ appears to slow opening by preventing movement of the monomers relative to each other that is prerequisite to opening. These data also suggest that the association of the monomers is tenuous and allows substantial freedom of movement. The data support the idea that native proton channels are dimeric. Finally, the idea that monomer–dimer interconversion occurs during activation of phagocytes appears to be ruled out.

K2P channels and their protein partners

A decade since their discovery, the K2P channels are recognized as pathways dedicated to regulated background leakage of potassium ions that serve to control neuronal excitability. The recent identification of protein partners that directly interact with K2P channels (SUMO, 14-3-3 and Vpu1) has exposed new regulatory pathways. Reversible linkage to SUMO silences K2P1 plasma membrane channels; phosphorylation of K2P3 enables 14-3-3 binding to affect forward trafficking, whereas it decreases open probability of K2P2; and, Vpu1, an HIV encoded partner, mediates assembly-dependent degradation of K2P3. An operational strategy has emerged: tonic inhibition of K2P channels allows baseline neuronal activity until enhanced potassium leak is required to suppress excitability.

In vitro processing and secretion of mutant insulin proteins that cause permanent neonatal diabetes

Permanent neonatal diabetes mellitus is a rare form of insulin-requiring diabetes presenting within the first few weeks or months of life. Mutations in the insulin gene are the second most common cause of this form of diabetes. These mutations are located in critical regions of preproinsulin and are likely to prevent normal processing or folding of the preproinsulin/proinsulin molecule. To characterize these mutations, we transiently expressed proinsulin-GFP fusion proteins in MIN6 mouse insulinoma cells. Our study revealed three groups of mutant proteins: 1) mutations that result in retention of proinsulin in the endoplasmic reticulum (ER) and attenuation of secretion of cotransfected wild-type insulin: C43G, F48C, and C96Y; 2) mutations with partial ER retention, partial recruitment to granules, and attenuation of secretion of wild-type insulin: G32R, G32S, G47V, G90C, and Y108C; and 3) similar to (2) but with no significant attenuation of wild-type insulin secretion: A24D and R89C. The mutant insulin proteins do not prevent targeting of wild-type insulin to secretory granules, but most appear to lead to decreased secretion of wild-type insulin. Each of the mutants triggers the expression of the proapoptotic gene Chop, indicating the presence of ER stress.