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John P Adelman

Publications and source records attributed to John P Adelman.

At least 19 recordsLinked to original sources
PubMed2006

Regulation of surface localization of the small conductance Ca2+-activated potassium channel, Sk2, through direct phosphorylation by cAMP-dependent protein kinase.

Small conductance, Ca2+-activated voltage-independent potassium channels (SK channels) are widely expressed in diverse tissues; however, little is known about the molecular regulation of SK channel subunits. Direct alteration of ion channel subunits by kinases is a candidate mechanism for functional modulation of these channels. We find that activation of cyclic AMP-dependent protein kinase (PKA) with forskolin (50 microm) causes a dramatic decrease in surface localization of the SK2 channel subunit expressed in COS7 cells due to direct phosphorylation of the SK2 channel subunit. PKA phosphorylation studies using the intracellular domains of the SK2 channel subunit expressed as glutathione S-transferase fusion protein constructs showed that both the amino-terminal and carboxyl-terminal regions are PKA substrates in vitro. Mutational analysis identified a single PKA phosphorylation site within the amino-terminal of the SK2 subunit at serine 136. Mutagenesis and mass spectrometry studies identified four PKA phosphorylation sites: Ser465 (minor site) and three amino acid residues Ser568, Ser569, and Ser570 (major sites) within the carboxyl-terminal region. A mutated SK2 channel subunit, with the three contiguous serines mutated to alanines to block phosphorylation at these sites, shows no decrease in surface expression after PKA stimulation. Thus, our findings suggest that PKA phosphorylation of these three sites is necessary for PKA-mediated reorganization of SK2 surface expression.

Amino Acid Sequence↗
PubMed2006

Small-conductance Ca2+-activated K+ channel type 2 (SK2) modulates hippocampal learning, memory, and synaptic plasticity.

Apamin-sensitive, small-conductance, Ca2+-activated K+ channels (SK channels) modulate neuronal excitability in CA1 neurons. Blocking all SK channel subtypes with apamin facilitates the induction of hippocampal synaptic plasticity and enhances hippocampal learning. In CA1 dendrites, SK channels are activated by Ca2+ through NMDA receptors and restrict glutamate-mediated EPSPs. Studies of SK channel knock-out mice reveal that of the three apamin-sensitive SK channel subunits (SK1-SK3), only SK2 subunits are necessary for the apamin-sensitive currents in CA1 hippocampal neurons. To determine the specific influence of SK2 channels on hippocampal synaptic plasticity, learning, and memory, we used gene targeting through homologous recombination in embryonic stem cells to generate transgenic mice that overexpress SK2 subunits by 10-fold (SK2+/T). In these mice, the apamin-sensitive current in CA1 neurons was increased by approximately fourfold, relative to wild-type (WT) littermates. In addition, the amplitude of synaptically evoked EPSPs recorded from SK2+/T CA1 neurons increased twice as much in response to SK channel blockade relative to EPSPs recorded from WT CA1 neurons. Consistent with this, SK2 overexpression reduced long-term potentiation after high-frequency stimulation compared with WT littermates and severely impaired learning in both hippocampus- and amygdala-dependent tasks. We conclude that SK2 channels regulate hippocampal synaptic plasticity and play a critical role in modulating mechanisms of learning and memory.

Animals↗
PubMed2005

Immunocytochemical localization of small-conductance, calcium-dependent potassium channels in astrocytes of the rat supraoptic nucleus.

Supraoptic nucleus (SON) neurons possess a prominent afterhyperpolarization (AHP) that contributes to spike patterning. This AHP is probably underlain by a small-conductance, CA2+-dependent, K+ type 3 (SK3) channel. To determine the distribution of SK3 channels within the SON, we used immunocytochemistry in rats and in transgenic mice with a regulatory cassette on the SK3 gene, allowing regulated expression with dietary doxycycline (DOX). In rats and wild-type mice, SK3 immunostaining revealed an intense lacy network surrounding SON neurons, with weak staining in neuronal somata and dendrites. In untreated, conditional SK3 knockout mice, SK3 was overexpressed, but the pericellular pattern in the SON was similar to that of rats. DOX-treated transgenic mice exhibited no SK3 staining in the SON. Double staining for oxytocin or vasopressin neurons revealed weak co-localization with SK3 but strong staining surrounding each neuron type. Electron microscopy showed that SK3-like immunoreactivity was intense between neuronal somata and dendrites, in apparent glial processes, but weak in neurons. This was confirmed by using confocal microscopy and double staining for glial fibrillary acidic protein (GFAP) and SK3: many GFAP-positive processes in the SON, and in the ventral dendritic/glial lamina, were shown to contain SK3-like immunoreactivity. These studies suggest a prominent role of SK3 channels in astrocytes. Given the marked plasticity in glial/neuronal relationships, as well as studies suggesting that astrocytes in the central nervous system can generate prominent CA2+ transients to various stimuli, a CA2+-dependent K+ channel may help SON astrocytes with K+ buffering whenever astrocyte intracellular CA2+ is increased.

Animals↗
PubMed2005

SK channels and NMDA receptors form a Ca2+-mediated feedback loop in dendritic spines.

Small-conductance Ca(2+)-activated K(+) channels (SK channels) influence the induction of synaptic plasticity at hippocampal CA3-CA1 synapses. We find that in mice, SK channels are localized to dendritic spines, and their activity reduces the amplitude of evoked synaptic potentials in an NMDA receptor (NMDAR)-dependent manner. Using combined two-photon laser scanning microscopy and two-photon laser uncaging of glutamate, we show that SK channels regulate NMDAR-dependent Ca(2+) influx within individual spines. SK channels are tightly coupled to synaptically activated Ca(2+) sources, and their activity reduces the amplitude of NMDAR-dependent Ca(2+) transients. These effects are mediated by a feedback loop within the spine head; during an excitatory postsynaptic potential (EPSP), Ca(2+) influx opens SK channels that provide a local shunting current to reduce the EPSP and promote rapid Mg(2+) block of the NMDAR. Thus, blocking SK channels facilitates the induction of long-term potentiation by enhancing NMDAR-dependent Ca(2+) signals within dendritic spines.

Animals↗
PubMed2005

A novel isoform of SK2 assembles with other SK subunits in mouse brain.

The SK2 subtype of small conductance Ca2+-activated K+ channels is widely distributed throughout the central nervous system and modulates neuronal excitability by contributing to the afterhyperpolarization that follows an action potential. Western blots of brain membrane proteins prepared from wild type and SK2-null mice reveal two isoforms of SK2, a 49-kDa band corresponding to the previously reported SK2 protein (SK2-S) and a novel 78-kDa form. Complementary DNA clones from brain and Western blots probed with an antibody specific for the longer form, SK2-L, identified the larger molecular weight isoform as an N-terminally extended SK2 protein. The N-terminal extension of SK2-L is cysteine-rich and mediates disulfide bond formation between SK2-L subunits or with heterologous proteins. Immunohistochemistry revealed that in brain SK2-L and SK2-S are expressed in similar but not identical patterns. Heterologous expression of SK2-L results in functional homomeric channels with Ca2+ sensitivity similar to that of SK2-S, consistent with their shared core and intracellular C-terminal domains. In contrast to the diffuse, uniform surface distribution of SK2-S, SK2-L channels cluster into sharply defined, distinct puncta suggesting that the extended cysteine-rich N-terminal domain mediates this process. Immunoprecipitations from transfected cells and mouse brain demonstrate that SK2-L co-assembles with the other SK subunits. Taken together, the results show that the SK2 gene encodes two subunit proteins and suggest that native SK2-L subunits may preferentially partition into heteromeric channel complexes with other SK subunits.

Amino Acid Sequence↗
PubMed2005

SK channels in excitability, pacemaking and synaptic integration.

Small conductance calcium-activated potassium channels link elevations of intracellular calcium ions to membrane potential, exerting a hyperpolarizing influence when activated. The consequences of SK channel activity have been revealed by the specific blocker apamin, a peptide toxin from honeybee venom. Recent studies have revealed unexpected roles for SK channels in fine-tuning intrinsic cell firing properties and in responsiveness to synaptic input. They have also identified specific roles for different SK channel subtypes. A host of Ca2+ sources, including distinct subtypes of voltage-dependent calcium channels, intracellular Ca2+ stores and Ca2+-permeable ionotropic neurotransmitter receptors, activate SK channels. The macromolecular complex in which the Ca2+ source, SK channels and various modulators are assembled determines the kinetics and consequences of SK channel activation.

Animals↗
PubMed2004

Protein kinase CK2 is coassembled with small conductance Ca(2+)-activated K+ channels and regulates channel gating.

Small conductance Ca(2+)-activated K+ channels (SK channels) couple the membrane potential to fluctuations in intracellular Ca2+ concentration in many types of cells. SK channels are gated by Ca2+ ions via calmodulin that is constitutively bound to the intracellular C terminus of the channels and serves as the Ca2+ sensor. Here we show that, in addition, the cytoplasmic N and C termini of the channel protein form a polyprotein complex with the catalytic and regulatory subunits of protein kinase CK2 and protein phosphatase 2A. Within this complex, CK2 phosphorylates calmodulin at threonine 80, reducing by 5-fold the apparent Ca2+ sensitivity and accelerating channel deactivation. The results show that native SK channels are polyprotein complexes and demonstrate that the balance between kinase and phosphatase activities within the protein complex shapes the hyperpolarizing response mediated by SK channels.

Adenosine Triphosphate↗
PubMed2004

Small conductance Ca2+-activated K+ channel knock-out mice reveal the identity of calcium-dependent afterhyperpolarization currents.

Action potentials in many central neurons are followed by a prolonged afterhyperpolarization (AHP) that influences firing frequency and affects neuronal integration. In hippocampal CA1 pyramidal neurons, the current ascribed to the AHP (IAHP) has three kinetic components. The IfastAHP is predominantly attributable to voltage-dependent K+ channels, whereas Ca2+-dependent and voltage-independent K+channels contribute to the ImediumAHP (ImAHP) and IslowAHP (IsAHP). Apamin, which selectively suppresses a component of the mAHP, increases neuronal excitability and facilitates the induction of synaptic plasticity at Schaffer collateral synapses and hippocampal-dependent learning. The Ca2+-dependent components of the AHP have been attributed to the activity of small conductance Ca2+-activated K+ (SK) channels. Examination of transgenic mice, each lacking one of the three SK channel genes expressed in the CNS, reveals that mice without the SK2 subunit completely lack the apamin-sensitive component of the ImAHP in CA1 neurons, whereas the IsAHP is not different in any of the SK transgenic mice. In each of the transgenic lines, the expression levels of the remaining SK genes are not changed. The results demonstrate that only SK2 channels are necessary for the ImAHP, and none of the SK channels underlie the IsAHP.

Animals↗
PubMed2004

Activation kinetics of the slow afterhyperpolarization in hippocampal CA1 neurons.

The activation of the slow afterhyperpolarization (sAHP) in CA1 neurons was studied using whole-cell recordings in the presence of inhibitors of the fast and medium-duration AHPs. The amplitude of the slow afterhyperpolarization current (IsAHP) increased as a function of duration and magnitude of the depolarizing voltage pulse reflecting graded increases in Ca(2+) influx through voltage-dependent Ca(2+) channels. Therefore, the time constant for activation, tau(max), determined from a family of IsAHPs as a function of pulse duration, was voltage dependent decreasing several-fold within the range of -20 to 20 mV and was dependent on extracellular [Ca(2+)]. The IsAHP displayed a pronounced rising phase that was well fit by a single exponential with a time constant, tau(rise), that was invariant of pulse duration, voltage, IsAHP amplitude, or external [Ca(2+)] and was significantly slower than the tau(max). In current clamp, the magnitude of the sAHP increased with the number of evoked action potentials, yet tau(rise) of the sAHP was invariant of action potential number and was similar to the tau(rise) of the IsAHP recorded in voltage-clamp. The results suggest that there are two components to the development of the IsAHP, a rapid, voltage- and Ca(2+)-dependent step, the magnitude and rate of which reflects the voltage dependence of the Ca(2+) channels, that triggers a second rate-limiting, voltage-independent process that dictates the slow IsAHP rise kinetics.

Animals↗
PubMed2004

Regional distribution of SK3 mRNA-containing neurons in the adult and adolescent rat ventral midbrain and their relationship to dopamine-containing cells.

SK3 small conductance, calcium-activated potassium channels play an important role in regulating the activity of mesencephalic dopamine (DA) neurons. In the present series of experiments, in situ hybridization techniques were used to compare SK3 and tyrosine hydroxylase (TH) mRNA expression throughout the rostrocaudal extent of the ventral midbrain in juvenile and adult rats. SK3 mRNA was found exclusively in areas that also contained large numbers of DA neurons including the substantia nigra (SN), the ventral tegmental area, and related cell groups (VTA-A10). An anteroposterior and mediolateral gradient in SK3 mRNA hybridization was apparent in the VTA-A10 but not in the SN. Younger rats appeared to possess higher levels and less regional variation in TH and SK3 transcripts. These results are consistent with previous studies reporting differential expression of SK3 protein within the midbrain and suggest that variations in SK3 channel distribution could contribute to differences in dopamine-related functions in the rat.

Age Factors↗
PubMed2003

Small conductance Ca2+-activated K+ channels and calmodulin.

Small conductance Ca(2+)-activated K(+) channels (SK channels) contribute to the long lasting afterhyperpolarization (AHP) that follows an action potential in many central neurones. The biophysical and pharmacological attributes of cloned SK channels strongly suggest that one or more of them underlie the medium component of the AHP that regulates interspike interval and plays an important role in setting tonic firing frequency. The cloned SK channels comprise a distinct subfamily of K(+) channels. Heterologously expressed SK channels recapitulate the biophysical and pharmacological hallmarks of native SK channels, being gated solely by intracellular Ca(2+) ions with no voltage dependence to their gating, small unitary conductance values and sensitivity to the bee venom peptide toxin, apamin. Molecular, biochemical and electrophysiological studies have revealed that Ca(2+) gating in SK channels is due to heteromeric assembly of the SK alpha pore-forming subunits with calmodulin (CaM). Ca(2+) binding to the N-terminal E-F hands of CaM is responsible for SK channel gating. Crystallographic studies suggest that SK channels gate as a dimer-of-dimers, and that the physical gate of SK channels resides at or near the selectivity filter of the channels. In addition, Ca(2+)-independent interactions between the SK channel alpha subunits and CaM are necessary for proper membrane trafficking.

Action Potentials↗
PubMed2003

Urinary bladder instability induced by selective suppression of the murine small conductance calcium-activated potassium (SK3) channel.

Small conductance, calcium-activated potassium (SK) channels have an important role in determining the excitability and contractility of urinary bladder smooth muscle. Here, the role of the SK isoform SK3 was examined by altering expression levels of the SK3 gene using a mouse model that conditionally overexpresses SK3 channels (SK3T/T). Prominent SK3 immunostaining was found in both the smooth muscle (detrusor) and urothelium layers of the urinary bladder. SK currents were elevated 2.4-fold in isolated myocytes from SK3T/T mice. Selective suppression of SK3 expression by dietary doxycycline (DOX) decreased SK current density in isolated myocytes, increased phasic contractions of isolated urinary bladder smooth muscle strips and exposed high affinity effects of the blocker apamin of the SK isoforms (SK1-3), suggesting an additional participation from SK2 channels. The role of SK3 channels in urinary bladder function was assessed using cystometry in conscious, freely moving mice. The urinary bladders of SK3T/T had significantly greater bladder capacity, and urine output exceeded the infused saline volume. Suppression of SK3 channel expression did not alter filling pressure, threshold pressure or bladder capacity, but micturition pressure was elevated compared to control mice. However, SK3 suppression did eliminate excess urine production and caused a marked increase in non-voiding contractions. The ability to examine bladder function in mice in which SK3 channel expression is selectively altered reveals that these channels have a significant role in the control of non-voiding contractions in vivo. Activation of these channels may be a therapeutic approach for management of non-voiding contractions, a condition which characterizes many types of urinary bladder dysfunctions including urinary incontinence.

Animals↗
PubMed2003

Altered expression of small-conductance Ca2+-activated K+ (SK3) channels modulates arterial tone and blood pressure.

The endothelium is a critical regulator of vascular tone, and dysfunction of the endothelium contributes to numerous cardiovascular pathologies. Recent studies suggest that apamin-sensitive, small-conductance, Ca2+-activated K+ channels may play an important role in active endothelium-dependent vasodilations, and expression of these channels may be altered in disease states characterized by vascular dysfunction. Here, we used a transgenic mouse (SK3T/T) in which SK3 expression levels can be manipulated with dietary doxycycline (DOX) to test the hypothesis that the level of expression of the SK subunit, SK3, in endothelial cells alters arterial function and blood pressure. SK3 protein was elevated in small mesenteric arteries from SK3T/T mice compared with wild-type mice and was greatly suppressed by dietary DOX. SK3 was detected in the endothelium and not in the smooth muscle by immunohistochemistry. In whole-cell patch-clamp experiments, SK currents in endothelial cells from SK3T/T mice were almost completely suppressed by dietary DOX. In intact arteries, SK3 channels contributed to sustained hyperpolarization of the endothelial membrane potential, which was communicated to the arterial smooth muscle. Pressure- and phenylephrine-induced constrictions of SK3T/T arteries were substantially enhanced by treatment with apamin, suppression of SK3 expression with DOX, or removal of the endothelium. In addition, suppression of SK3 expression caused a pronounced and reversible elevation of blood pressure. These results indicate that endothelial SK3 channels exert a profound, tonic, hyperpolarizing influence in resistance arteries and suggest that the level of SK3 channel expression in endothelial cells is a fundamental determinant of vascular tone and blood pressure.

Animals↗
PubMed2003

Small conductance Ca2+-activated K+ channels and calmodulin: cell surface expression and gating.

Small conductance Ca2+-activated K+ channels (SK channels) are heteromeric complexes of pore-forming alpha subunits and constitutively bound calmodulin (CaM). The binding of CaM is mediated in part by the electrostatic interaction between residues Arg-464 and Lys-467 of SK2 and Glu-84 and Glu-87 of CaM. Heterologous expression of the double charge reversal in SK2, SK2 R464E/K467E (SK2:64/67), did not yield detectable surface expression or channel activity in whole cell or inside-out patch recordings. Coexpression of SK2:64/67 with wild type CaM or CaM1,2,3,4, a mutant lacking the ability to bind Ca2+, rescued surface expression. In patches from cells coexpressing SK2:64/67 and wild type CaM, currents were recorded immediately following excision into Ca2+-containing solution but disappeared within minutes after excision or immediately upon exposure to Ca2+-free solution and were not reactivated upon reapplication of Ca2+-containing solution. Channel activity was restored by application of purified recombinant Ca2+-CaM or exposure to Ca2+-free CaM followed by application of Ca2+-containing solution. Coexpression of the double charge reversal E84R/E87K in CaM (CaM:84/87) with SK2:64/67 reconstituted stable Ca2+-dependent channel activity that was not lost with exposure to Ca2+-free solution. Therefore, Ca2+-independent interactions with CaM are required for surface expression of SK channels, whereas the constitutive association between the two channel subunits is not an essential requirement for gating.

Animals↗
PubMed2003

Determinants contributing to estrogen-regulated expression of SK3.

The rSK3 gene encodes a small conductance Ca(2+)-activated K(+) channel that is transcriptionally regulated by estrogen. To examine determinants of rSK3 gene expression, the CAP site was defined and the promoter was identified. A 33 bp sequence adjacent to the promoter was shown to act as an enhancer in L6 cells that express SK3 and estrogen receptor alpha (ER alpha). The 33 bp enhancer was unable to stimulate transcription in Cos7 cells that do not express SK3 or ER alpha. However when cotransfected with ER alpha and stimulated with 17 beta-estradiol (E2) the enhancer was activated. Interestingly, expression of ER alpha in Cos7 cells and E2 treatment was sufficient to induce expression of the endogenous SK3 gene. Only Sp1 and Sp3 specifically bound to the enhancer from Cos7 and L6 nuclear extracts, however, ER alpha increased Sp1s affinity for the enhancer. The data suggest that estrogen regulates SK3 gene expression through interactions between ER alpha and Sp1.

Animals↗
PubMed2003

It takes two to tango, but three to ISA.

Rapidly inactivating A-type potassium channels are important determinants of firing frequency in many excitable cells. Nadal et al. (in this issue of Neuron) purified A-type potassium (I(SA)) channels from rat cerebellum and identified a novel beta subunit. This protein, DPPX, associates with the pore-forming subunits and endows previously elusive kinetic properties on A-type channels formed from cloned subunits.

Animals↗