Biomedical subjects

James Maylie

Publications and source records attributed to James Maylie.

9 recordsLinked to original sources

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↗

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

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

A mouse model of episodic ataxia type-1.

Episodic ataxia type-1 (EA1) is a dominant human neurological disorder characterized by stress-induced attacks of ataxia. EA1 is caused by mutations in the voltage-gated potassium channel Kv1.1, and affected individuals are heterozygous. Here we introduced the V408A EA1 mutation into mice using homologous recombination. In contrast to Kv1.1 null mice, homozygous V408A/V408A mice died after embryonic day 3 (E3). V408A/+ mice showed stress-induced loss of motor coordination that was ameliorated by acetazolamide, a carbonic anhydrase inhibitor that minimizes EA1 symptoms in human patients. We made electrophysiological recordings from cerebellar Purkinje cells in both V408A/+ mice and their wild-type littermates. V408A/+ mice showed a greater frequency and amplitude of spontaneous GABAergic inhibitory postsynaptic currents (IPSCs) than did wild type; however, the amplitude or frequency of miniature IPSCs and the basket cell firing frequency did not differ between groups. The stress-induced motor dysfunction in V408A mice is similar to that of family members harboring the EA1 allele, and our findings suggest that these behavioral changes are linked to changes in GABA release.

Acetazolamide↗

PubMed2002

Small conductance Ca2+-activated K+ channels modulate synaptic plasticity and memory encoding.

Activity-dependent changes in neuronal excitability and synaptic strength are thought to underlie memory encoding. In hippocampal CA1 neurons, small conductance Ca2+-activated K+ (SK) channels contribute to the afterhyperpolarization, affecting neuronal excitability. In the present study, we examined the effect of apamin-sensitive SK channels on the induction of hippocampal synaptic plasticity in response to a range of stimulation frequencies. In addition, the role of apamin-sensitive SK channels on hippocampal-dependent memory encoding and retention was also tested. The results show that blocking SK channels with apamin increased the excitability of hippocampal neurons and facilitated the induction of synaptic plasticity by shifting the modification threshold to lower frequencies. This facilitation was NMDA receptor (NMDAR) dependent and appeared to be postsynaptic. Mice treated with apamin demonstrated accelerated hippocampal-dependent spatial and nonspatial memory encoding. They required fewer trials to learn the location of a hidden platform in the Morris water maze and less time to encode object memory in an object-recognition task compared with saline-treated mice. Apamin did not influence long-term retention of spatial or nonspatial memory. These data support a role for SK channels in the modulation of hippocampal synaptic plasticity and hippocampal-dependent memory encoding.

Action Potentials↗

PubMed2002

Localization of the activation gate for small conductance Ca2+-activated K+ channels.

Small conductance Ca2+-activated K+ (SK) channels open in response to increased cytosolic Ca2+ and contribute to the afterhyperpolarization in many excitable cell types. Opening of SK channels is initiated by Ca2+ binding to calmodulin that is bound to the C terminus of the channel. Based on structural information, a chemomechanical gating model has been proposed in which the chemical energy derived from Ca2+ binding is transduced into a mechanical force that restructures the protein to allow K+ ion conduction through the pore. However, the residues that comprise the physical gate of the SK channels have not been identified. In voltage-gated K+ (Kv) channels, access to the inner vestibule is controlled by a bundle crossing formed by the intracellular end of the sixth transmembrane domain (S6) of each of the four channel subunits. Probing SK channels with internally applied quaternary amines suggests that the inner vestibules of Kv and SK channels share structural similarity. Using substituted cysteine accessibility mutagenesis, the relatively large molecule [2-(trimethylammonium)] methanethiosulfonate accessed positions near the putative bundle crossing more rapidly in the open than the closed state but did not modify S6 positions closer to the selectivity filter. In contrast, the smaller compound, 2-(aminoethyl) methanethiosulfonate (MTSEA), modified a position predicted to lie in the lumen immediately intracellular to the selectivity filter equivalently in the open and closed states. The pore blocker tetrabutylammonium impeded MTSEA access to this position in both open and closed channels. The results suggest that the SK channel gate is not formed by the cytoplasmic end of S6 but resides deep in the channel pore in or near the selectivity filter.

Animals↗

PubMed2002

SK channels are necessary but not sufficient for denervation-induced hyperexcitability.

Skeletal muscle hyperexcitability is characteristically associated with denervation. Expression of SK3, a small conductance Ca(2+)-activated K(+) channel (SK channel) in skeletal muscle is induced by denervation, and direct application of apamin, a peptide blocker of SK channels, dramatically reduces hyperexcitability. To investigate the role of SK3 channels in denervation- induced hyperexcitability, SK3 expression was manipulated using a transgenic mouse that harbors a tetracycline-regulated SK3 gene. Electromyographic (EMG) recordings from anterior tibial (AT) muscle showed that denervated muscle from transgenic or wild-type animals had equivalent hyperexcitability that was blocked by apamin. In contrast, denervated skeletal muscle from SK3tTA mice lacking SK3 channels showed little or no hyperexcitability, similar to results from wild-type innervated skeletal muscle. However, innervated skeletal muscle from SK3tTA mice containing SK3 channels did not show hyperexcitability. The results demonstrate that SK3 channels are necessary but not sufficient for denervation-induced skeletal muscle hyperexcitability.

Action Potentials↗