Formation Of Heteromeric Channels Research Articles

A single coiled-coil domain mutation in hIKCa channel subunits disrupts preferential formation of heteromeric hSK1:hIKCa channels.

The expression of IKCa (SK4) channel subunits overlaps with that of SK channel subunits, and it has been proposed that the two related subunits prefer to co-assemble to form heteromeric hSK1:hIKCa channels. This implicates hSK1:hIKCa heteromers in physiological roles that might have been attributed to activation of SK channels. We have used a mutation approach to confirm formation of heterometric hSK1:hIKCa channels. Introduction of residues within hSK1 that were predicted to impart sensitivity to the hIKCa current blocker TRAM-34 changed the pharmacology of functional heteromers. Heteromeric channels formed between wildtype hIKCa and mutant hSK1 subunits displayed a significantly higher sensitivity and maximum block to addition of TRAM-34 than heteromers formed between wildtype subunits. Heteromer formation was disrupted by a single point mutation within one COOH-terminal coiled-coil domain of the hIKCa channel subunit. This mutation only disrupted the formation of hSK1:hIKCa heteromeric channels, without affecting the formation of homomeric hIKCa channels. Finally, the Ca2+ gating sensitivity of heteromeric hSK1:hIKCa channels was found to be significantly lower than the Ca2+ gating sensitivity of homomeric hIKCa channels. These data confirmed the preferred formation of heteromeric channels that results from COOH-terminal interactions between subunits. The distinct sensitivity of the heteromer to activation by Ca2+ suggests that heteromeric channels fulfil a distinct function within those neurons that express both subunits.

Modeling the kinetics of heteromeric potassium channels.

Mechanistic mathematical modeling has long been used as a tool for answering questions in cellular physiology. To mathematically describe cellular processes such as cell excitability, volume regulation, neurotransmitter release, and hormone secretion requires accurate descriptions of ion channel kinetics. One class of ion channels currently lacking a physiological model framework is the class of channels built with multiple different potassium protein subunits called heteromeric voltage gated potassium channels. Here we present a novel mathematical model for heteromeric potassium channels that captures both the number and type of protein subunits present in each channel. Key model assumptions are validated by showing our model is the reduction of a Markov model and through observations about voltage clamp data. We then show our model's success in replicating kinetic properties of concatemeric channels with different numbers of K v 1.1 and K v 1.2 subunits. Finally, through comparisons with multiple expression experiments across multiple voltage gated potassium families, we use the model to make predictions about the importance and effect of genetic mutations in heteromeric channel formation.

Cannabidiol counters the effects of a dominant-negative pathogenic Kv7.2 variant

SummaryEpilepsy and neurodevelopmental disorders can arise from pathogenic variants of KCNQ (Kv7) channels. A patient with developmental and epileptic encephalopathy exhibited an in-frame deletion of histidine 260 on Kv7.2. Coexpression of Kv7.2 mutant (mut) subunits with Kv7.3 invoked a decrease in current density, a depolarizing shift in voltage for activation, and a decrease in membrane conductance. Biotinylation revealed an increased level of surface Kv7.2mut compared to Kv7.3 with no change in total membrane protein expression. Super-resolution and FRET imaging confirmed heteromeric channel formation and a higher expression density of Kv7.2mut. Cannabidiol (1 μM) offset the effects of Kv7.2mut by inducing a hyperpolarizing shift in voltage for activation independent of CB1 or CB2 receptors. These data reveal that the ability for cannabidiol to reduce the effects of a pathogenic Kv7.2 variant supports its use as a potential therapeutic to reduce seizure activity.

Investigating oligodendrocyte connexins: Heteromeric interactions between Cx32 and mutant or wild-type forms of Cx47 do not contribute to or modulate gap junction function.

Oligodendrocytes express two gap junction forming connexins, connexin 32 (Cx32) and Cx47; therefore, formation of heteromeric channels containing both Cx47 and Cx32 monomers might occur. Mutations in Cx47 cause both Pelizaeus-Merzbacher-like disease Type 1 (PMLD1) and hereditary spastic paraparesis Type 44 (SPG44) and heteromer formation between these mutants and Cx32 may contribute to the pathogenesis of these disorders. Here, we utilized electrophysiological and antibody-based techniques to examine this possibility. When cells expressing both Cx32 and Cx47 were paired with cells expressing either Cx32 or Cx47, properties were indistinguishable from those produced by cells expressing homotypic Cx32 or Cx47 channels. Similarly, pairing cells expressing both Cx32 and Cx47 with cells expressing Cx30 or Cx43 produced channels indistinguishable from heterotypic Cx32/Cx30 or Cx47/Cx43 channels, respectively. The same assessments were performed on cells expressing Cx32 and four mutant forms of Cx47 (p.I33M associated with SPG44 or p.P87S, p.Y269D or p.M283T associated with PMLD1). None of these mutants showed a functional effect on Cx32. Immunostained cells co-expressing Cx32WT (wild type) and Cx47WT showed a Pearson correlation coefficient close to zero, suggesting that any overlap was due to chance. p.Y269D showed a statistically significant negative correlation with Cx32, suggesting that Cx32 and this mutant overlap less than expected by chance. Co-immunoprecipitation of Cx32 with Cx47WT and mutants show only very low levels of co-immunoprecipitated protein. Overall, our data suggest that interactions between PMLD1 or SPG44 mutants and Cx32 gap junctions do not contribute to the pathogenesis of these disorders.

Preferred Formation of Heteromeric Channels between Coexpressed SK1 and IKCa Channel Subunits Provides a Unique Pharmacological Profile of Ca2+-Activated Potassium Channels.

Three small conductance calcium-activated potassium channel (SK) subunits have been cloned and found to preferentially form heteromeric channels when expressed in a heterologous expression system. The original cloning of the gene encoding the intermediate conductance calcium-activated potassium channel (IKCa) was termed SK4 because of the high homology between channel subtypes. Recent immunovisualization suggests that IKCa is expressed in the same subcellular compartments of some neurons as SK channel subunits. Stochastic optical reconstruction microscopy super-resolution microscopy revealed that coexpressed IKCa and SK1 channel subunits were closely associated, a finding substantiated by measurement of fluorescence resonance energy transfer between coexpressed fluorophore-tagged subunits. Expression of homomeric SK1 channels produced current that displayed typical sensitivity to SK channel inhibitors, while expressed IKCa channel current was inhibited by known IKCa channel blockers. Expression of both SK1 and IKCa subunits gave a current that displayed no sensitivity to SK channel inhibitors and a decreased sensitivity to IKCa current inhibitors. Single channel recording indicated that coexpression of SK1 and IKCa subunits produced channels with properties intermediate between those observed for homomeric channels. These data indicate that SK1 and IKCa channel subunits preferentially combine to form heteromeric channels that display pharmacological and biophysical properties distinct from those seen with homomeric channels.

Structural studies of N-terminal mutants of Connexin 26 and Connexin 32 using 1H NMR spectroscopy

Alterations in gap junctions underlie the etiologies of syndromic deafness (KID) and Charcot-Marie Tooth disease (CMTX). Functional gap junctions are composed of connexin molecules with N-termini containing a flexible turn around G12, inserting the N-termini into the channel pore allowing voltage gating. The loss of this turn correlates with loss of Connexin 32 (Cx32) function by impaired trafficking to the cell membrane. Using 1H NMR we show the N-terminus of a syndromic deafness mutation Cx26G12R, producing “leaky channels”, contains a turn around G12 which is less structured and more flexible than wild-type. In contrast, the N-terminal structure of the same mutation in Cx32 chimera, Cx32*43E1G12R shows a larger constricted turn and no membrane current expression but forms membrane inserted hemichannels. Their function was rescued by formation of heteromeric channels with wild type subunits. We suggest the inflexible Cx32G12R N-terminus blocks ion conduction in homomeric channels and this channel block is relieved by incorporation of wild type subunits. In contrast, the increased open probability of Cx26G12R hemichannels is likely due to the addition of positive charge in the channel pore changing pore electrostatics and impairing hemichannel regulation by Ca2+. These results provide mechanistic information on aberrant channel activity observed in disease.

Evolutionary Origins of the Shaker Family of Voltage-Gated Potassium Channels

The Shaker family of voltage-gated K+ channels in bilaterians consists of four closely related gene subfamilies (Shaker, Shab, Shaw and Shal) that regulate neuronal excitability in diverse ways. We examined the evolutionary origins of the gene family by characterizing Shaker channels in early branching metazoan lineages. Shaker family channels can be definitively identified by the combination of a voltage-dependent potassium channel core and a cytosolic domain (T1) that mediates subfamily-specific tetrameric assembly. The Shaker, Shab, Shaw and Shal subfamilies are present in cnidarians, a close sister group to the bilaterians. We show that all four subfamilies are highly conserved on a functional level in the sea anemone Nematostella vectensis, an emerging cnidarian model organism. The Nematostella Shaker, Shal and Shaw subfamilies include cnidarian-specific expansions of regulatory subunits that extend functional diversity through the formation of heteromeric channels. We were unable to identify Shaker family genes in choanoflagellates or sponges, but found over 40 Shaker family genes in a ctenophore (Mnemiopsis leidyi). Ctenophores are the earliest branching metazoans with a nervous system. Phylogenetic analysis indicates that the ctenophore Shaker family expanded independently and diverged prior to emergence of the Shaker, Shab, Shal and Shaw subfamilies. The phylogeny and functional analysis support Shaker as the oldest of these subfamilies. Two Mnemiopsis Shaker family subunits heteromerize with Nematostella and mouse Shaker subfamily channels, but not with Shab, Shaw or Shal. Thus the subunit interface of Shaker subfamily may retain ancestral features. Our results indicate that the Shaker family is metazoan-specific and was present by the time neurons evolved. However, the Shaker, Shab, Shal and Shaw subfamilies evolved in the cnidarian/bilaterian lineage long after the evolution of the first nervous systems.

Functional Analysis of Missense Mutations in Kv8.2 Causing Cone Dystrophy with Supernormal Rod Electroretinogram

Mutations in KCNV2 have been proposed as the molecular basis for cone dystrophy with supernormal rod electroretinogram. KCNV2 codes for the modulatory voltage-gated potassium channel α-subunit, Kv8.2, which is incapable of forming functional channels on its own. Functional heteromeric channels are however formed with Kv2.1 in heterologous expression systems, with both α-subunit genes expressed in rod and cone photoreceptors. Of the 30 mutations identified in the KCNV2 gene, we have selected three missense mutations localized in the potassium channel pore and two missense mutations localized in the tetramerization domain for analysis. We characterized the differences between homomeric Kv2.1 and heteromeric Kv2.1/Kv8.2 channels and investigated the influence of the selected mutations on the function of heteromeric channels. We found that two pore mutations (W467G and G478R) led to the formation of nonconducting heteromeric Kv2.1/Kv8.2 channels, whereas the mutations localized in the tetramerization domain prevented heteromer generation and resulted in the formation of homomeric Kv2.1 channels only. Consequently, our study suggests the existence of two distinct molecular mechanisms involved in the disease pathology.

Characterization of novel Pannexin 1 isoforms from rat pituitary cells and their association with ATP-gated P2X channels

Our previous studies have showed that Pannexin 1 (Panx1), a member of a recently discovered family of gap junction proteins, is expressed in the pituitary gland. Here we investigated the presence and expression pattern of Panx1 isoforms in pituitary cells, their roles in ATP release, and their association with purinergic P2X receptor subtypes that are native to pituitary cells. In addition to the full-size Panx1, termed Panx1a, pituitary cells also express two novel shorter isoforms, termed Panx1c and Panx1d, which formation reflects the existence of alternative splicing sites in exons 2 and 4, respectively. Panx1c is lacking the Phe108-Gln180 sequence and P2X1d is missing the Val307-Cys426 C-terminal end sequence. Confocal microscopy and biotin labeling revealed that Panx1a is expressed in the plasma membrane, whereas Panx1c and Panx1d show the cytoplasmic localization when expressed as homomeric proteins. The three Panx1 isoforms and Panx2 form homomeric and heteromeric complexes in any combination. These splice forms can also physically associate with ATP-gated P2X2, P2X3, P2X4, and P2X7 receptor channels. The Panx1a-mediated ATP release in AtT-20 immortalized pituitary cells is attenuated when co-expressed with Panx1c or Panx1d. These results suggest that Panx1c and Panx1d may serve as dominant-negative effectors to modulate the functions of Panx1a through formation of heteromeric channels. The complex patterns of Panx1 expression and association could also define the P2X-dependent roles of these channels in cell types co-expressing both proteins.

Lateral habenular neurons projecting to reward-processing monoaminergic nuclei express hyperpolarization-activated cyclic nucleotid-gated cation channels

The lateral habenular complex (LHb) is a key signal integrator between limbic forebrain regions and monoaminergic hindbrain nuclei. Major projections of LHb neurons target the dopaminergic ventral tegmental area (VTA) and the serotonergic dorsal (DR) and median raphe nuclei (MnR). Both monoaminergic neurotransmitter systems play a central role in reward processing and reward-related decision-making. Glutamatergic LHb efferents terminate on GABAergic neurons in the VTA, the rostromedial tegmental nucleus (RMTg), and the raphe nuclei, thereby suppressing monoamine release when required by the present behavioral context. Recent studies suggest that the LHb exerts a strong tonic inhibition on monoamine release when no reward is to be obtained. It is yet unknown whether this inhibition is the result of a continuous external activation by other brain areas, or if it is intrinsically generated by LHb projection neurons. To analyze whether the tonic inhibition may be the result of a hyperpolarization-activated cyclic nucleotid-gated cation channel (HCN)-mediated pacemaker activity of LHb projection neurons, we combined retrograde tracing in rats with in situ hybridization of HCN1 to HCN4 mRNAs. In fact, close to all LHb neurons targeting VTA or raphe nuclei are equipped with HCN subunit mRNAs. While HCN1 mRNA is scarce, most neurons display strong expression of HCN2 to HCN4 mRNAs, in line with the potential formation of heteromeric channels. These results are supported by quantitative PCR and immunocytochemical analyses. Thus, our data suggest that the tonic inhibition of monoamine release is intrinsically generated in LHb projection neurons and that their activity may only be modulated by synaptic inputs to the LHb.

Novel mutation p.Gly59Arg inGJB6encoding connexin 30 underlies palmoplantar keratoderma with pseudoainhum, knuckle pads and hearing loss

Connexins, components of the gap junction, are expressed in several organs including the skin and the cochlea. Mutations in connexin genes including GJB2 (Cx26), GJB3 (Cx31), GJB4 (Cx30.3), GJB6 (Cx30) and GJA1 (Cx43) are responsible for various dermatological syndromes and/or inherited hearing loss, frequently showing overlapping phenotypes. To clarify the spectrum of clinical phenotypes caused by connexin mutations. We report a 32-year-old Japanese woman with mild palmoplantar keratoderma (PPK) with severe sensorineural hearing loss, knuckle pads and pseudoainhum of her toes. Direct sequencing revealed no mutation in GJB2, but a novel heterozygous missense mutation p.Gly59Arg in GJB6. Electron microscopy revealed no apparent morphological abnormality of gap junctions in the patient's lesional epidermis. The patient harboured the novel GJB6 missense mutation p.Gly59Arg in the first extracellular loop of Cx30. Mutations in glycine 59 of Cx26 are associated with PPK-deafness syndrome, and the similar phenotype here supports the observed heteromeric channel formation; the dominant nature of the mutation suggests an effect on gap junctions similar to that of the comparable mutation in Cx26.

INVOLVEMENT OF THE HABENULAR COMPLEX IN THE COGNITIVE DEFICITS OF SCHIZOPHRENIA

The lateral habenular complex (LHb) is a key signal integrator between limbic forebrain regions and monoaminergic hindbrain nuclei. Major projections of LHb neurons target the dopaminergic ventral tegmental area (VTA) and the serotonergic dorsal (DR) and median raphe nuclei (MnR). Both monoaminergic neurotransmitter systems play a central role in reward processing and reward-related decision-making. Glutamatergic LHb efferents terminate on GABAergic neurons in the VTA, the rostromedial tegmental nucleus (RMTg), and the raphe nuclei, thereby suppressing monoamine release when required by the present behavioral context. Recent studies suggest that the LHb exerts a strong tonic inhibition on monoamine release when no reward is to be obtained. It is yet unknown whether this inhibition is the result of a continuous external activation by other brain areas, or if it is intrinsically generated by LHb projection neurons. To analyze whether the tonic inhibition may be the result of a hyperpolarization-activated cyclic nucleotid-gated cation channel (HCN)-mediated pacemaker activity of LHb projection neurons, we combined retrograde tracing in rats with in situ hybridization of HCN1 to HCN4 mRNAs. In fact, close to all LHb neurons targeting VTA or raphe nuclei are equipped with HCN subunit mRNAs. While HCN1 mRNA is scarce, most neurons display strong expression of HCN2 to HCN4 mRNAs, in line with the potential formation of heteromeric channels. These results are supported by quantitative PCR and immunocytochemical analyses. Thus, our data suggest that the tonic inhibition of monoamine release is intrinsically generated in LHb projection neurons and that their activity may only be modulated by synaptic inputs to the LHb.

Acid‐Sensing Ion Channel 2 modulates the current properties and neuropeptide potentiation of proton‐gated currents

Acid‐sensing ion channels (ASICs) are proton‐gated channels required for normal behavior and sensory transduction. ASICs may also play an important role in injuries where prolonged acidosis occurs including stroke and inflammation. The specific biophysical properties of ASIC channels are dependent on their subunit composition and the presence of modulatory compounds such as RFamide neuropeptides. ASIC2 subunits regulate ASIC1a current via the formation of heteromeric channels, but the specific properties modulated by these subunits are not entirely understood. In this study, we examined the biophysical properties of ASIC1/2 heteromeric channels expressed in Xenopus oocytes. We determined that ASIC response to RFamide neuropeptides is dramatically altered by the presence of ASIC2 subunits. Surprisingly, we found that steady‐state desensitization of both heteromeric and ASIC2a homomeric channels was inhibited by RFamide peptides. Furthermore, heteromeric channels were responsive to a larger number of peptides compared to ASIC1a homomultimers. Our data suggest that in addition to affecting primary current properties, the ASIC2 subunits may also confer enhanced peptide sensitivity to proton‐gated currents in vivo, which may be important for the physiological and pathological roles of ASICs.

Zebrafish Acid-sensing Ion Channel (ASIC) 4, Characterization of Homo- and Heteromeric Channels, and Identification of Regions Important for Activation by H+

There are four genes for acid-sensing ion channels (ASICs) in the genome of mammalian species. Whereas ASIC1 to ASIC3 form functional H+-gated Na+ channels, ASIC4 is not gated by H+, and its function is unknown. Zebrafish has two ASIC4 paralogs: zASIC4.1 and zASIC4.2. Whereas zASIC4.1 is gated by extracellular H+, zASIC4.2 is not. This differential response to H+ makes zASIC4 paralogs a good model to study the properties of this ion channel. In this study, we found that surface expression of homomeric zASIC4.2 is higher than that of zASIC4.1. Surface expression of zASIC4.1 was much increased by formation of heteromeric channels, suggesting that zASIC4.1 contributes to heteromeric ASICs in zebrafish neurons. Robust surface expression of H+-insensitive zASIC4.2 suggests that zASIC4.2 functions as a homomer and is gated by an as yet unknown stimulus, different from H+. Moreover, we identified a small region just distal to the first transmembrane domain that is crucial for the differential H+ response of the two paralogs. This post-TM1 domain may have a general role in gating of members of this gene family.

Thermosensitive TRPV Channel Subunits Coassemble into Heteromeric Channels with Intermediate Conductance and Gating Properties

Heat-sensitive transient receptor potential (TRP) channels (TRPV1–4) form the major cellular sensors for detecting temperature increases. Homomeric channels formed by thermosensitive TRPV subunits exhibit distinct temperature thresholds. While these subunits do share significant sequence similarity, whether they can coassemble into heteromeric channels has been controversial. In the present study we investigated the coassembly of TRPV subunits using both spectroscopy-based fluorescence resonance energy transfer (FRET) and single-channel recordings. Fluorescent protein–tagged TRPV subunits were coexpressed in HEK 293 cells; FRET between different subunits was measured as an indication of the formation of heteromeric channels. We observed strong FRET when fluorescence signals were collected selectively from the plasma membrane using a “spectra FRET” approach but much weaker or no FRET from intracellular fluorescence. In addition, no FRET was detected when TRPV subunits were coexpressed with members of the TRPM subfamily or CLC-0 chloride channel subunits. These results indicate that a substantial fraction of TRP channels in the plasma membrane of cotransfected cells were heteromeric. Single-channel recordings confirmed the existence of multiple heteromeric channel forms. Interestingly, heteromeric TRPV channels exhibit intermediate conductance levels and gating kinetic properties. As these subunits coexpress both in sensory neurons and in other tissues, including heart and brain, coassembly between TRPV subunits may contribute to greater functional diversity.

Tissue Distribution Profiles of the Human TRPM Cation Channel Family

Eight members of the TRP-melastatin (TRPM) subfamily have been identified, whose physiological functions and distribution are poorly characterized. Although tissue expression and distribution patterns have been reported for individual TRPM channels, comparisons between individual studies are not possible because of variations in analysis techniques and tissue selection. We report here a comparative analysis of the expression patterns of all of the human TRPM channels in selected peripheral tissues and the central nervous system (CNS) using two distinct but complimentary approaches: TaqMan and SYBR Green real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). These techniques generated comparative distribution profiles and demonstrated tissue-specific co-expression of TRPM mRNA species, indicating significant potential for the formation of heteromeric channels. TRPM channels 2, 4, 5, 6, and 7 in contrast to 1, 3, and 8 are widely distributed in the CNS and periphery. The tissues demonstrating highest expression for individual family members were brain (TRPM1), brain and bone marrow (TRPM2), brain and pituitary (TRPM3), intestine and prostate (TRPM4), intestine, pancreas, and prostate (TRPM5), intestine and brain (TRPM6), heart, pituitary, bone, and adipose tissue (TRPM7), and prostate and liver (TRPM8). The data reported here will guide the elucidation of TRPM channel physiological functions.

Epithelial Na+ Channel Subunit Stoichiometry

Ion channels, including the epithelial Na(+) channel (ENaC), are intrinsic membrane proteins comprised of component subunits. Proper subunit assembly and stoichiometry are essential for normal physiological function of the channel protein. ENaC comprises three subunits, alpha, beta, and gamma, that have common tertiary structures and much amino acid sequence identity. For maximal ENaC activity, each subunit is required. The subunit stoichiometry of functional ENaC within the membrane remains uncertain. We combined a biophysical approach, fluorescence intensity ratio analysis, used to assess relative subunit stoichiometry with total internal reflection fluorescence microscopy, which enables isolation of plasma membrane fluorescence signals, to determine the limiting subunit stoichiometry of ENaC within the plasma membrane. Our results demonstrate that membrane ENaC contains equal numbers of each type of subunit and that at steady state, subunit stoichiometry is fixed. Moreover, we find that when all three ENaC subunits are coexpressed, heteromeric channel formation is favored over homomeric channels. Electrophysiological results testing effects of ENaC subunit dose on channel activity were consistent with total internal reflection fluorescence/fluorescence intensity ratio findings and confirmed preferential formation of heteromeric channels containing equal numbers of each subunit.

PH Dependency and Desensitization Kinetics of Heterologously Expressed Combinations of Acid-sensing Ion Channel Subunits

The exact subunit combinations of functional native acid-sensing ion channels (ASICs) have not been established yet, but both homomeric and heteromeric channels are likely to exist. To determine the ability of different subunits to assemble into heteromeric channels, a number of ASIC1a-, ASIC1b-, ASIC2a-, ASIC2b-, and ASIC3-containing homo- and heteromeric channels were studied by whole-cell patch clamp recordings with respect to pH sensitivity, desensitization kinetics, and level of sustained current normalized to peak current. Analyzing and comparing data for these three features demonstrated unique heteromeric channels in a number of co-expression experiments. Formation of heteromeric ASIC1a+2a and ASIC1b+2a channels was foremost supported by the desensitization characteristics that were independent of proton concentration, a feature none of the respective homomeric channels has. Several lines of evidence supported formation of ASIC1a+3, ASIC1b+3, and ASIC2a+3 heteromeric channels. The most compelling was the desensitization characteristics, which, besides being proton-independent, were faster than those of any of the respective homomeric channels. ASIC2b, which homomerically expressed is not activated by protons per se, did not appear to form unique heteromeric combinations with other subunits and in fact appeared to suppress the function of ASIC1b. Co-expression of three subunits such as ASIC1a+2a+3 and ASIC1b+2a+3 resulted in data that could best be explained by coexistence of multiple channel populations within the same cell. This observation seems to be in good agreement with the fact that ASIC-expressing sensory neurons display a variety of acid-evoked currents.

Cloning and developmental expression of Shaker potassium channels in the cochlea of the chicken

Signal coding by the receptor and neuronal cells of the auditory system involves various ion channels that modulate a sound stimulus. The genes that encode a number of these ion channels and their accessory subunits are presently unknown for channels found in the sensory epithelium and cochlear nerve. Among these genes are those that encode delayed rectifier and transient type potassium channels found in both the sensory cells and the ganglion. Here, we report the cloning and developmental expression of Shaker family members that include cKv1.2, cKv1.3, cKv1.5, and the Shaker-related cGMP-gated potassium channel cKCNA10. Clones were obtained by screening a chicken embryonic cochlea cDNA library using, as a probe, a mixture of two DNA fragments of cKv1.2 and cKv1.3 obtained by the reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis revealed chicken homologues of Kv1.2, Kv1.3, Kv1.5 and cGMP-gated potassium channels with a deduced amino acid homology of 96–98%, 82–84%, 67–71% and 67–79% to correspondent mammalian homologues. During development of chicken inner ear, RT-PCR studies show expression of cKv1.2, cKv1.3 and cKv1.5 as early as Embryonic Day (ED) 3, while cKCNA10 was detected at low levels beginning on ED6 and was highly expressed by ED9. Additionally, analysis of expression in different parts of the cochlea showed that these genes were co-expressed in different regions of the cochlea, including the cochlear ganglion, sensory epithelium, lagena, and tegmentum. This expression pattern suggests the potential for the formation of heteromeric channels from the corresponding α-subunits in these various tissues.

MRNA distribution analysis of human TRPC family in CNS and peripheral tissues

The mammalian homologues of the Drosophila transient receptor potential (TRP) channel are plasma membrane proteins involved in the regulation of cellular Ca 2+ influx. These ion channels can be activated subsequent to either depletion of Ca 2+ from internal stores or through receptor-mediated processes. The mRNA expression patterns of several individual mammalian short transient receptor potential channels (TRPCs) have been described. Cross-comparisons between these data, however, are at best difficult predominantly due to the non-quantitative methods used. Furthermore there is limited data on the expression of TRPC family members in human tissues. In the present study we used a single technique, namely TaqMan real-time quantitative RT-PCR, to investigate the mRNA distribution of human TRPC1, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 (hTRPCs) in discrete human brain areas, peripheral tissues as well as a panel of cell-lines. All hTRPCs studied were widely expressed within CNS and significant peripheral expression was often observed. Despite this, each channel exhibited a distinctive hallmark distribution profile. hTRPC1 was widely expressed in CNS and peripheral tissues, whereas hTRPC3 and hTRPC5 were predominantly expressed in tissues of CNS. hTRPC4 mRNA was detected in CNS and certain peripheral tissues such as bone, heart and prostate. hTRPC6 was homogeneously expressed throughout the CNS and peripheral tissues with the highest levels in placenta and lung. hTRPC7 mRNA was also broadly expressed in CNS as well as some peripheral tissues. The pattern of expression of the TRPCs was quite different in the various cell lines examined. TRPC3 and TRPC6 were selectively present in HEK-293 cells whilst TRPC1 was broadly distributed in the cell lines analyzed. In contrast TRPC4 and TRPC5 mRNAs were predominantly expressed in HK-2 and HEK-293 cell lines respectively. TRPC7 was selectively expressed in COS-1, COS-7 and HK-2 cell lines. These results show tissue- and cell-specific co-expression of multiple TRPC forms indicating widespread potential for formation of heteromeric channels. These data will be useful in the complex task of relating channel subunit composition to function in native cells.