Expression Of Channel Subtypes Research Articles

K V 7 Channel Subtype Expression and Physiological Roles in Rat Urinary Bladder Smooth Muscle Excitability and Contractility

Introduction Voltage-gated K+ (KV) channels, specifically type 7 (KV7), are emerging key regulators of urinary bladder smooth muscle (UBSM) excitability and contractility. The KV7 channel family consists of 5 pore-forming α-subunits (KV7.1–KV7.5) capable of assembling as homotetrameric (e.g. KV7.4) or heterotetrameric (e.g. KV7.4/KV7.5) channels; thus, further increasing species/tissue expression diversity and regulatory potential. However, much remains unknown about KV7 channel properties in UBSM and the translational value of the common experimental animal model - the rat. Hypothesis KV7 channels, especially homomeric or heteromeric KV7.4 and KV7.5 subtypes, regulate rat UBSM excitability and contractility. Methods UBSM tissue without mucosa was obtained from adult male Sprague Dawley rats. UBSM homogenates were used for Western blots to detect KV7 channel subtypes with or without surface biotinylation. UBSM isometric tension recordings were carried out in the presence of tetrodotoxin for spontaneous, 20 mM K+-induced and 1 µM carbachol-induced contractions or in its absence for the electrical field stimulated (EFS) contractions. For electrophysiological amphotericin-B perforated patch-clamp studies, single UBSM cells were prepared using enzymatic dissociation. UBSM cell membrane potential was recorded in current-clamp (I=0) mode at room temperature. The following KV7 channel modulators were tested: XE991 (KV7.1-KV7.5 subtype blocker), retigabine (KV7.2-KV7.5 activator), and ICA-069673 (selective activator for KV7.2/KV7.3, which at higher concentrations preferentially enhances KV7.4 and KV7.4/KV7.5 but not KV7.5 channels). Results Western blot experiments detected KV7.4 and KV7.5 channels in UBSM under both non- and surface-biotinylation assay conditions. The latter condition revealed that for both KV7.4 and KV7.5 subtypes surface localization predominated (>75 %). In isometric tension experiments, retigabine attenuated contractility in a concentration-dependent manner for all four contraction protocols (EC50 values: 2.6-58.7 µM for the amplitude and force, with complete inhibition of contractility at 100 µM, n=3-10). XE991 (10 µM) depolarized the membrane potential of isolated UBSM cells (Δ10.5±3.6 mV, n=5, p<0.05). In contrast, retigabine (10 µM, Δ-6.5±1.8 mV, n=4, p<0.04) and ICA-069673 (10 µM, Δ-11.5±2.4 mV, n=4, p<0.02), when applied separately, induced UBSM cell hyperpolarization. In the presence of XE991, ICA-069673 (10 µM) did not significantly change the membrane potential of isolated UBSM cells (Δ0.4±1.2 mV, n=4, p>0.05). Conclusions Our data show that both KV7.4 and KV7.5 channel subtypes are expressed in rat UBSM and display preferential cell membrane localization. Pharmacological modulation of KV7 channels can control UBSM cell excitability and tissue contractility. The hyperpolarizing effect of ICA-069673, which is blocked by XE991, revealed a critical role of KV7.4 homomeric and/or KV7.4/KV7.5 heteromeric channels in UBSM function.

First Evidence of Kv3.1b Potassium Channel Subtype Expression during Neuronal Serotonergic 1C11 Cell Line Development.

Kv3.1 channel is abundantly expressed in neurons and its dysfunction causes sleep loss, neurodegenerative diseases and depression. Fluoxetine, a serotonin selective reuptake inhibitor commonly used to treat depression, acts also on Kv3.1. To define the relationship between Kv3.1 and serotonin receptors (SR) pharmacological modulation, we showed that 1C11, a serotonergic cell line, expresses different voltage gated potassium (VGK) channels subtypes in the presence (differentiated cells (1C11D)) or absence (not differentiated cells (1C11ND)) of induction. Only Kv1.2 and Kv3.1 transcripts increase even if the level of Kv3.1b transcripts is highest in 1C11D and, after fluoxetine, in 1C11ND but decreases in 1C11D. The Kv3.1 channel protein is expressed in 1C11ND and 1C11D but is enhanced by fluoxetine only in 1C11D. Whole cell measurements confirm that 1C11 cells express (VGK) currents, increasing sequentially as a function of cell development. Moreover, SR 5HT1b is highly expressed in 1C11D but fluoxetine increases the level of transcript in 1C11ND and significantly decreases it in 1C11D. Serotonin dosage shows that fluoxetine at 10 nM blocks serotonin reuptake in 1C11ND but slows down its release when cells are differentiated through a decrease of 5HT1b receptors density. We provide the first experimental evidence that 1C11 expresses Kv3.1b, which confirms its major role during differentiation. Cells respond to the fluoxetine effect by upregulating Kv3.1b expression. On the other hand, the possible relationship between the fluoxetine effect on the kinetics of 5HT1b differentiation and Kv3.1bexpression, would suggest the Kv3.1b channel as a target of an antidepressant drug as well as it was suggested for 5HT1b.

CaV2 channel subtype expression in rat sympathetic neurons is selectively regulated by α2δ subunits

ABSTRACTType two voltage gated calcium (CaV2) channels are the primary mediators of neurotransmission at neuronal presynapses, but their function at neural soma is also important in regulating excitability.1 Mechanisms that regulate CaV2 channel expression at synapses have been studied extensively, which motivated us to perform similar studies in the soma. Rat sympathetic neurons from the superior cervical ganglion (SCG) natively express CaV2.2 and CaV2.3.2 We noted previously that heterologous expression of CaV2.1 but not CaV2.2 results in increased calcium current in SCG neurons.3 In the present study, we extended these observations to show that both CaV2.1 and CaV2.3 expression resulted in increased calcium currents while CaV2.2 expression did not. Further, CaV2.1 could displace native CaV2.2 channels, but CaV2.3 expression could not. Heterologous expression of the individual accessory subunits α2δ-1, α2δ-2, α2δ-3, or β4 alone failed to increase current density, suggesting that the calcium current ceiling when CaV2.2 was over-expressed was not due to lack of these subunits. Interestingly, introduction of recombinant α2δ subunits produced surprising effects on displacement of native CaV2.2 by recombinant channels. Both α2δ-1 and α2δ-2 seemed to promote CaV2.2 displacement by recombinant channel expression, while α2δ-3 appeared to protect CaV2.2 from displacement. Thus, we observe a selective prioritization of CaV channel functional expression in neurons by specific α2δ subunits. These data highlight a new function for α2δ subtypes that could shed light on subtype selectivity of CaV2 membrane expression.

Subarachnoid Hemorrhage Suppresses KV1 and KV2 Currents via Different Mechanisms in Rat Parenchymal Arteriolar Myocytes

Subarachnoid hemorrhage (SAH) leads to membrane potential depolarization in arteriolar myocytes and enhanced arteriolar tone in brain parenchymal arterioles (Nystoriak et al, 2011). However, the mechanism underlying this augmented constriction is currently unknown. Here, we studied the impact of SAH on voltage‐gated potassium (KV) currents. In rat parenchymal arterioles from control animals, KV1.2, 1.5, 2.1 and 2.2 mRNA was expressed, and KV1 and KV2 currents were detected by conventional whole cell patch clamp electrophysiology. Both KV1 and KV2 currents as well as total KV (4‐aminopyridine‐sensitive) currents were significantly suppressed in arteriolar myocytes after SAH, however KV channel subtype expression was not changed. Our previous work demonstrated that a blood component oxyhemoglobin causes KV current suppression through heparin binding EGF‐like growth factor (HB‐EGF). HB‐EGF caused significant K+ current suppression in myocytes from control animals, but not after SAH. Further, HB‐EGF suppressed K+ currents in the absence and presence of KV2 channel inhibition, but failed to alter currents in the presence of 4‐aminopyridine or during KV1 channel blockade. These data suggest SAH causes KV1 current suppression through HB‐EGF shedding, while KV2 current suppression is independent of the HB‐EGF pathway. Supported by NIH P01 HL095488, Totman Medical Research Trust and the Peter Martin Fund.

Crucial role of a shared extracellular loop in apamin sensitivity and maintenance of pore shape of small-conductance calcium-activated potassium (SK) channels

Activation of small-conductance calcium (Ca(2+))-dependent potassium (K(Ca)2) channels (herein called "SK") produces membrane hyperpolarization to regulate membrane excitability. Three subtypes (SK1-3) have been cloned and are distributed throughout the nervous system, smooth muscle, and heart. It is difficult to discern the physiological role of individual channel subtypes as most blockers or enhancers do not discriminate between subtypes. The archetypical blocker apamin displays some selectivity between SK channel subtypes, with SK2 being the most sensitive, followed by SK3 and then SK1. Sensitivity of SK1 is species specific, with the human isoform being blocked by the toxin, whereas the rat is not. Mutation studies have identified residues within the outer pore that suggest apamin blocks by an allosteric mechanism. Apamin also uses a residue within the S3-S4 extracellular loop to produce a high-sensitivity block. We have identified that a 3-amino acid motif within this loop regulates the shape of the channel pore. This motif is required for binding and block by apamin, suggesting that a change in pore shape underlies allosteric block. This motif is absent in rat SK1, explaining why it is insensitive to block by apamin. The overlapping distribution of SK channel subtype expression suggests that native heteromeric channels may be common. We show that the S3-S4 loop of one subunit overlaps the outer pore of the adjacent subunit, with apamin interacting with both regions. This arrangement provides a unique binding site for each combination of SK subunits within a coassembled channel that may be targeted to produce blockers specific for heteromeric SK channels.

An Inducible Expression System of the Calcium-Activated Potassium Channel 4 to Study the Differential Impact on Embryonic Stem Cells

Rationale. The family of calcium-activated potassium channels consists of four members with varying biological functions and conductances. Besides membrane potential modulation, SK channels have been found to be involved in cardiac pacemaker cell development from ES cells and morphological shaping of neural stem cells. Objective. Distinct SK channel subtype expression in ES cells might elucidate their precise impact during cardiac development. We chose SK channel subtype 4 as a potential candidate influencing embryonic stem cell differentiation. Methods. We generated a doxycycline inducible mouse ES cell line via targeted homologous recombination of a cassette expressing a bicistronic construct encoding SK4 and a fluorophore from the murine HPRT locus. Conclusion. We characterized the mouse ES cell line iSK4-AcGFP. The cassette is readily expressed under the control of doxycycline, and the overexpression of SK4 led to an increase in cardiac and pacemaker cell differentiation thereby serving as a unique tool to characterize the cell biological variances due to specific SK channel overexpression.

Regional differences in nerve terminal Na+ channel subtype expression and Na+ channel‐dependent glutamate and GABA release in rat CNS

We tested the hypothesis that expression of pre-synaptic voltage-gated sodium channel (Na(v)) subtypes coupled to neurotransmitter release differs between transmitter types and CNS regions in a nerve terminal-specific manner. Na(v) coupling to transmitter release was determined by measuring the sensitivity of 4-aminopyridine (4AP)-evoked [(3)H]glutamate and [(14)C]GABA release to the specific Na(v) blocker tetrodotoxin (TTX) for nerve terminals isolated from rat cerebral cortex, hippocampus, striatum and spinal cord. Expression of various Na(v) subtypes was measured by immunoblotting using subtype-specific antibodies. Potencies of TTX for inhibition of glutamate and GABA release varied between CNS regions. However, the efficacies of TTX for inhibition of 4AP-evoked glutamate release were greater than for inhibition of GABA release in all regions except spinal cord. The relative nerve terminal expression of total Na(v) subtypes as well as of specific subtypes varied considerably between CNS regions. The region-specific potencies of TTX for inhibition of 4AP-evoked glutamate release correlated with greater relative expression of total nerve terminal Na(v) and Na(v)1.2. Nerve terminal-specific differences in the expression of specific Na(v) subtypes contribute to transmitter-specific and regional differences in pharmacological sensitivities of transmitter release.

Expression of voltage-gated potassium channels Kv1.3 and Kv1.5 in human gliomas

K + channels play an important role in glial cell proliferation and are functionally expressed in glial tumors. Because voltage-gated K + channel (Kv) subtypes Kv1.3 and Kv1.5 have been shown to contribute to growth-related properties of normal glia rather specifically, we investigated different human glioma samples for the expression of these channel subtypes using reverse transcriptase-PCR. Kv1.5 expression correlated with glioma entities and malignancy grades, i.e. expression was high in astrocytomas, moderate in oligodendrogliomas, and low in glioblastomas. No such correlation was evident for Kv1.3 expression. This study shows a clear differential expression of Kv1.5 in gliomas according to subtype and malignancy grade. This result corresponds to previous data on the expression of voltage-gated sodium channels in gliomas, which likewise showed a low or absent expression of channel subtypes in high-grade tumors.

Molecular characterization of voltage-gated sodium channels in human gliomas.

Voltage-sensitive sodium channels appear to be an electrophysiological hallmark of gliomas. However, the expression of channel subtypes is unclear in these tumors. In this study different gliomas were investigated for the expression of sodium channel subtypes Na(v)1.1, Na(v)1.2, Na(v)1.3, Na(v)1.4, Na(v)1.6, and Na(x)(Na(v)2.1) using RT-PCR. At least one subtype of channels could be detected in each tumor. High-grade gliomas expressed fewer sodium channel subtypes and these at weaker levels than low-grade tumors. Expression of Na(v)1.6, the most abundant isoform in the CNS, was almost absent in the gliomas except the pilocytic variant. Our study gives clear evidence for a differential expression of sodium channel subtypes in gliomas and indicates a predominant expression of channels related to malignancy grades.

Effect of prolonged hypoxia on Na + channel mRNA subtypes in the developing rat cortex

Voltage-gated Na + channels are regulated in response to oxygen deprivation in the mammalian cortex. Past investigations have demonstrated that Na + channel protein expression is up-regulated in the immature brain exposed to prolonged hypoxia. Since it is unknown as to which Na + channel subtype(s) is involved in this regulation, we used RT-PCR to assess the effect of hypoxia on Na + channel I, II and III α-subunit mRNA expression in the developing rat cortex. Na + channel II mRNA tended to increase during early development, whereas Na + channel I and III did not change or slightly decreased with age. Hypoxic exposure for 1-day had no effect on Na + channel expression, while 5-day hypoxia significantly increased Na + channel III density, with a slight increase in Na + channel I and no appreciable change in Na + channel II. These results suggest that Na + channel subtype expression in the developing cortex is differentially regulated in response to prolonged hypoxic exposure.

Isolation and heterologous expression of two genomic clones encoding Shaker-related potassium channels of trout CNS.

Two Shaker-related potassium channel genes (termed tsha1 and tsha2) expressed in the CNS of trout were cloned and sequenced. The coding regions of both genes were not interrupted by introns and exhibited a high overall sequence similarity to other members of the Shaker subfamily. By computer-assisted sequence alignments, tsha1 was identified as a fish homologue to the mammalian Kv1.2 subtype of potassium channels, whereas tsha2 did not show a preferential sequence homology but shared a uniform similarity to Kv1.1, Kv1.2, and Kv1.3. Upon heterologous expression in a mammalian glial cell line, both channels exhibited delayed rectifier current properties that differed from each other by their threshold potentials of activation and their pharamcological features: wheras the tsha1-mediated current was efficiently blocked by submicromolar concentrations of alpha-DTX but not by TEA, tsha2 was highly TEA-sensitive, correlating well with differences in the amino acid structure of the pore outer mouth region. As revealed by RT-PCR, Shaker-related potassium channels were sequentially expressed during trout brain development: tsha2 was found initially at stage 34, followed by tsha1 at stage 36, whereas two other members of the Shaker family (tsha3 and tsha4) were detectable much earlier (stage 30, hatching). In the mature brain tissue, no regional specialization of Shaker channel subtype expression was noted.