Christine Karschin

IRK(1–3) and GIRK(1–4) Inwardly Rectifying K+Channel mRNAs Are Differentially Expressed in the Adult Rat Brain

Molecular cloning together with functional characterization has shown that the newly identified family of inwardly rectifying K+ channels consists of several closely related members encoded by separate genes. In this report we demonstrate the differential mRNA expression and detailed cellular localization in the adult rat brain of seven members of the IRK and GIRK subfamilies. Using both radiolabeled cRNA riboprobes and specific oligonucleotide probes directed to nonconserved regions of both known and newly isolated rat brain cDNAs, in situ hybridization revealed wide distribution with partly overlapping expression of the mRNAs of IRK1–3 and GIRK1–4. Except for the low levels of GIRK4 transcripts observed, the overall distribution patterns of the other GIRK subunits were rather similar, with high levels of expression in the olfactory bulb, hippocampus, cortex, thalamus, and cerebellum. Marked differences in expression levels existed only in some thalamic, brainstem, and midbrain nuclei, e.g., the substantia nigra, superior colliculus, or inferior olive. In contrast, IRK subunits were expressed more differentially: all mRNAs were abundant in dentate gyrus, olfactory bulb, caudate putamen, and piriform cortex. IRK1 and IRK3 were restricted to these regions, but they were absent from most parts of the thalamus, cerebellum, and brainstem, where IRK2 was expressed predominantly. Because channel subunits may assemble as heteromultimers, additional functional characterization based on overlapping expression patterns may help to decipher the native K+ channels in neurons and glial cells.

Overlapping distribution of K(ATP) channel-forming Kir6.2 subunit and the sulfonylurea receptor SUR1 in rodent brain.

ATP‐sensitive K+ channels comprise a complex of at least two proteins: a member of the inwardly rectifying Kir6 family (e.g. Kir6.2) and a sulphonylurea receptor (e.g. SUR1) which belongs to the ATP‐binding cassette (ABC) superfamily. Using specific radiolabeled antisense oligonucleotides, the cellular localization of both mRNAs was investigated in the rodent brain by in situ hybridization. The distribution of both transcripts was widespread throughout the brain and showed a high degree of overlap with peak expression levels in the hippocampus, neocortex, olfactory bulb, cerebellum, and several distinct nuclei of the midbrain and brainstem, indicating their important role in vital brain function.

THIK-1 and THIK-2, a novel subfamily of tandem pore domain K+ channels.

Two cDNAs encoding novel K+channels, THIK-1 and THIK-2 (tandem pore domainhalothane inhibited K +channel), were isolated from rat brain. The proteins of 405 and 430 amino acids were 58% identical to each other. Homology analysis showed that the novel channels form a separate subfamily among tandem pore domain K+ channels. The genes of the human orthologs were identified as human genomic data base entries. They possess one intron each and were assigned to chromosomal region 14q24.1–14q24.3 (human (h) THIK-1) and 2p22–2p21 (hTHIK-2). In rat (r), THIK-1 (rTHIK-1) is expressed ubiquitously; rTHIK-2 expression was found in several tissues including brain and kidney. In situ hybridization of brain slices showed that rTHIK-2 is strongly expressed in most brain regions, whereas rTHIK-1 expression is more restricted. Heterologous expression of rTHIK-1 in Xenopus oocytes revealed a K+channel displaying weak inward rectification in symmetrical K+ solution. The current was enhanced by arachidonic acid and inhibited by halothane. rTHIK-2 did not functionally express. Confocal microscopy of oocytes injected with green fluorescent protein-tagged rTHIK-1 or rTHIK-2 showed that both channel subunits are targeted to the outer membrane. However, coinjection of rTHIK-2 did not affect the currents induced by rTHIK-1, indicating that the two channel subunits do not form heteromers.

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.

Expression pattern and functional characteristics of two novel splice variants of the two-pore-domain potassium channel TREK-2.

Two novel alternatively spliced isoforms of the human two‐pore‐domain potassium channel TREK‐2 were isolated from cDNA libraries of human kidney and fetal brain. The cDNAs of 2438 base pairs (bp) (TREK‐2b) and 2559 bp (TREK‐2c) encode proteins of 508 amino acids each. RT‐PCR showed that TREK‐2b is strongly expressed in kidney (primarily in the proximal tubule) and pancreas, whereas TREK‐2c is abundantly expressed in brain. In situ hybridization revealed a very distinct expression pattern of TREK‐2c in rat brain which partially overlapped with that of TREK‐1. Expression of TREK‐2b and TREK‐2c in human embryonic kidney (HEK) 293 cells showed that their single‐channel characteristics were similar. The slope conductance at negative potentials was 163 ± 5 pS for TREK‐2b and 179 ± 17 pS for TREK‐2c. The mean open and closed times of TREK‐2b at −84 mV were 133 ± 16 and 109 ± 11 μs, respectively. Application of forskolin decreased the whole‐cell current carried by TREK‐2b and TREK‐2c. The sensitivity to forskolin was abolished by mutating a protein kinase A phosphorylation site at position 364 of TREK‐2c (construct S364A). Activation of protein kinase C (PKC) by application of phorbol‐12‐myristate‐13‐acetate (PMA) also reduced whole‐cell current. However, removal of the putative TREK‐2b‐specific PKC phosphorylation site (construct T7A) did not affect inhibition by PMA. Our results suggest that alternative splicing of TREK‐2 contributes to the diversity of two‐pore‐domain K+ channels.

Kir6.1 is the principal pore-forming subunit of astrocyte but not neuronal plasma membrane K-ATP channels.

ATP-sensitive potassium channels (K-ATP channels) directly couple the energy state of a cell to its excitability, are activated by hypoxia, and have been suggested to protect neurons during disturbances of energy metabolism such as transient ischemic attacks or stroke. Molecular studies have demonstrated that functional K-ATP channels are octameric protein complexes, consisting of four sulfonylurea receptor proteins and four pore-forming subunits which are members of the Kir6 family of inwardly rectifying potassium channels. Here we show, using specific antibodies against the two known pore-forming subunits (Kir6.1 and Kir6.2) of K-ATP channels, that only Kir6.1 and not Kir6.2 subunits are expressed in astrocytes. In addition to a minority of neurons, Kir6.1 protein is present on hippocampal, cortical, and cerebellar astrocytes, tanycytes, and Bergmann glial cells. We also provide ultrastructural evidence that Kir6.1 immunoreactivity is primarily localized to distal perisynaptic and peridendritic astrocyte plasma membrane processes, and we confirm the presence of functional K-ATP channels in Bergmann glial cells by slice-patch-clamp experiments. The identification of Kir6.1 as the principal pore-forming subunit of plasma membrane K-ATP channels in astrocytes suggests that these glial K-ATP channels act in synergy with neuronal Kir6.2-mediated K-ATP channels during metabolic challenges in the brain.

Distribution and localization of a G protein-coupled inwardly rectifying K+ channel in the rat

The cellular distribution of the mRNA of the inwardly rectifying K+ channel KGA (GIRK1) was investigated in rat tissue by in situ hybridization. KGA was originally cloned from the heart and represents the first G protein‐activated K+ channel identified. It is expressed in peripheral tissue solely in the atrium, but not in the ventricle, skeletal muscle, lung and kidney. In the central nervous system KGA is most prominently expressed in the Ammons horn and dentate gyrus of the hippocampus, neocortical layers II–VI, cerebellar granular layer, olfactory bulb, anterior pituitary, thalamic nuclei and several distinct nuclei of the lower brainstem. The abundant expression of KGA in many CNS neurons support its important role as a major target channel for G protein mediated receptor function.

Genetic and functional linkage of Kir5.1 and Kir2.1 channel subunits

We have identified several cDNAs for the human Kir5.1 subunit of inwardly rectifying K+ channels. Alternative splicing of exon 3 and the usage of two alternative polyadenylation sites contribute to cDNA diversity. The hKir5.1 gene KCNJ16 is assigned to chromosomal region 17q23.1–24.2, and is separated by only 34 kb from the hKir2.1 gene (KCNJ2). In the brain, Kir5.1 mRNA is restricted to the evolutionary older parts of the hindbrain, midbrain and diencephalon and overlaps with Kir2.1 in the superior/inferior colliculus and the pontine region. In the kidney Kir5.1 and Kir2.1 are colocalized in the proximal tubule. When expressed in Xenopus oocytes, Kir5.1 is efficiently targeted to the cell surface and forms electrically silent channels together with Kir2.1, thus negatively controlling Kir2.1 channel activity in native cells.

Distribution of inwardly rectifying potassium channels in the brain.

Publisher Summary This chapter discusses the distribution of inwardly rectifying potassium (kir) channel subunits in the brain. Expression and localization of Kir subunit mRNAs in the mammalian brain has mainly been demonstrated using in situ hybridization. Because mRNAs are predominantly found in somata, this analysis defines brain nuclei and cell populations expressing a given channel subunit. Relative amounts of observed hybridization signals also reflect strong or weak expression in these brain nuclei. More recent immunocytochemical studies that evaluate the localization of Kir2.1, Kir3.1, Kir3.2, and Kir3.4 proteins using specific antibodies reach beyond the information provided by mRNA localization. Both light and electron microscopic studies have identified Kir subunits at high cellular resolution, allowing localization at pre- or postsynaptic sites, or defining neuronal compartments with several subunits coexpressed.

Kalium-Einwärtsgleichrichter - ursprüngliche Kanalstrukturen als Basis funktioneller Vielfalt

lnwardly Rectifying Kir ChannelsPrimitive Channel Structures as Basis for Functional Diversity. With the cloning and characterization of the genesthat code for inwardly rectifying K+ (Kir) channels many neuroscientists have become interested in a family of K+ channels whose members are structurally and functionally different from voltage-dependent (Kv) channels. Various Kir channel subtypes with primitive structures have evolved for different cell types to specifically regulate membrane excitability a~d K+ homeostasis. The high degree of functional variability is based on -20 different genes, but is generated also by other mechanisms of protein and gene regulation. Channel isoforms originated from alternative splicing, targeted assembly of different subunits into heteromeric channel structures and association with other membrane proteins have been identified as means to modulate channel open probability and thus neuronal excitability. In addition to the cell-specific and differential regulation of Kir channels by G protein-mediated signaling cascades these mechanisms may play an important rote in the fine-tuning of slow synaptic transmission in the centrat nervous system.