James Maylie

Small-conductance, calcium-activated potassium channels from mammalian brain

Members of a previously unidentified family of potassium channel subunits were cloned from rat and human brain. The messenger RNAs encoding these subunits were widely expressed in brain with distinct yet overlapping patterns, as well as in several peripheral tissues. Expression of the messenger RNAs in Xenopus oocytes resulted in calcium-activated, voltage-independent potassium channels. The channels that formed from the various subunits displayed differential sensitivity to apamin and tubocurare. The distribution, function, and pharmacology of these channels are consistent with the SK class of small-conductance,calcium-activated potassium channels, which contribute to the afterhyperpolarization in central neurons and other cell types.

Mechanism of calcium gating in small-conductance calcium-activated potassium channels

The slow afterhyperpolarization that follows an action potential is generated by the activation of small-conductance calcium-activated potassium channels (SK channels). The slow afterhyperpolarization limits the firing frequency of repetitive action potentials (spike-frequency adaption) and is essential for normal neurotransmission. SK channels are voltage-independent and activated by submicromolar concentrations of intracellular calcium. They are high-affinity calcium sensors that transduce fluctuations in intracellular calcium concentrations into changes in membrane potential. Here we study the mechanism of calcium gating and find that SK channels are not gated by calcium binding directly to the channel α-subunits. Instead, the functional SK channels are heteromeric complexes with calmodulin, which is constitutively associated with the α-subunits in a calcium-independent manner. Our data support a model in which calcium gating of SK channels is mediated by binding of calcium to calmodulin and subsequent conformational alterations in the channel protein.

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

Small-conductance Ca2+-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 Ca2+ influx within individual spines. SK channels are tightly coupled to synaptically activated Ca2+ sources, and their activity reduces the amplitude of NMDAR-dependent Ca2+ transients. These effects are mediated by a feedback loop within the spine head; during an excitatory postsynaptic potential (EPSP), Ca2+ influx opens SK channels that provide a local shunting current to reduce the EPSP and promote rapid Mg2+ block of the NMDAR. Thus, blocking SK channels facilitates the induction of long-term potentiation by enhancing NMDAR-dependent Ca2+ signals within dendritic spines.

Small-Conductance Ca2+-Activated K+ Channels: Form and Function

Small-conductance Ca(2+)-activated K(+) channels (SK channels) are widely expressed throughout the central nervous system. These channels are activated solely by increases in intracellular Ca(2+). SK channels are stable macromolecular complexes of the ion pore-forming subunits with calmodulin, which serves as the intrinsic Ca(2+) gating subunit, as well as with protein kinase CK2 and protein phosphatase 2A, which modulate Ca(2+) sensitivity. Well-known for their roles in regulating somatic excitability in central neurons, SK channels are also expressed in the postsynaptic membrane of glutamatergic synapses, where their activation and regulated trafficking modulate synaptic transmission and the induction and expression of synaptic plasticity, thereby affecting learning and memory. In this review we discuss the molecular and functional properties of SK channels and their physiological roles in central neurons.

Determinants of Apamin and d-Tubocurarine Block in SK Potassium Channels

Small conductance calcium-activated potassium channels show a distinct pharmacology. Some, but not all, are blocked by the peptide toxin apamin, and apamin-sensitive channels are also blocked by d-tubocurarine. Cloned SK channels (small conductance calcium-activated potassium channel) recapitulate these properties. We have investigated the structural basis for these differences and found that two amino acid residues on either side of the deep pore are the primary determinants of sensitivity to apamin and differential block by d-tubocurarine. Therefore, the pharmacology of SK channels compared with other potassium channels correlates with structural differences in the outer pore region. However, introduction of a tyrosine residue in the position analogous to that which determines sensitivity to external tetraethylammonium for voltage-gated potassium channels endows SK channels with an equivalent tetraethylammonium sensitivity, indicating that the outer vestibules of the pores are similar. The pharmacology of channels formed in oocytes coinjected with SK1 and SK2 mRNAs, or with SK1-SK2 dimer mRNA, show that SK subunits may form heteromeric channels.

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.

Mice deficient in Six5 develop cataracts : implications for myotonic dystrophy

Expansion of a CTG trinucleotide repeat in the 3′ UTR of the gene DMPK at the DM1 locus on chromosome 19 causes myotonic dystrophy, a dominantly inherited disease characterized by skeletal muscle dystrophy and myotonia, cataracts and cardiac conduction defects. Targeted deletion of Dm15, the mouse orthologue of human DMPK, produced mice with a mild myopathy and cardiac conduction abnormalities, but without other features of myotonic dystrophy, such as myotonia and cataracts. We, and others, have demonstrated that repeat expansion decreases expression of the adjacent gene SIX5 (refs 7,8), which encodes a homeodomain transcription factor. To determine whether SIX5 deficiency contributes to the myotonic dystrophy phenotype, we disrupted mouse Six5 by replacing the first exon with a β-galactosidase reporter. Six5-mutant mice showed reporter expression in multiple tissues, including the developing lens. Homozygous mutant mice had no apparent abnormalities of skeletal muscle function, but developed lenticular opacities at a higher rate than controls. Our results suggest that SIX5 deficiency contributes to the cataract phenotype in myotonic dystrophy, and that myotonic dystrophy represents a multigenic disorder.

Small-conductance calcium-activated potassium channels.

ABSTRACT: SK channels play a fundamental role in all excitable cells. SK channels are potassium selective and are activated by an increase in the level of intracellular calcium, such as occurs during an action potential. Their activation causes membrane hyperpolarization, which inhibits cell firing and limits the firing frequency of repetetive action potentials. The intracellular calcium increase evoked by action potential firing decays slowly, allowing SK channel activation to generate a long‐lasting hyperpolarization termed the slow afterhyperpolarization (sAHP). This spike‐frequency adaptation protects the cell from the deleterious effects of continuous tetanic activity and is essential for normal neurotransmission. Slow AHPs can be classified into two groups, based on sensitivity to the bee venom toxin apamin. In general, apamin‐sensitive sAHPs activate rapidly following a single action potential and decay with a time constant of approximately 150 ms. In contrast, apamin‐insensitive sAHPs rise slowly and decay with a time constant of approximately 1.5 s. The basis for this kinetic difference is not yet understood. Apamin‐sensitive and apamin‐insensitive SK channels have recently been cloned. This chapter will compare with different classes of sAHPs, discuss the cloned SK channels and how they are gated by calcium ions, describe the molecular basis for their different pharmacologies, and review the possible role of SK channels in several pathological conditions.

SK2 channel plasticity contributes to LTP at Schaffer collateral–CA1 synapses

Long-term potentiation (LTP) of synaptic strength at Schaffer collateral synapses has largely been attributed to changes in the number and biophysical properties of AMPA receptors (AMPARs). Small-conductance Ca2+-activated K+ channels (SK2 channels) are functionally coupled with NMDA receptors (NMDARs) in CA1 spines such that their activity modulates the shape of excitatory postsynaptic potentials (EPSPs) and increases the threshold for induction of LTP. Here we show that LTP induction in mouse hippocampus abolishes SK2 channel activity in the potentiated synapses. This effect is due to SK2 channel internalization from the postsynaptic density (PSD) into the spine. Blocking PKA or cell dialysis with a peptide representing the C-terminal domain of SK2 that contains three known PKA phosphorylation sites blocks the internalization of SK2 channels after LTP induction. Thus the increase in AMPARs and the decrease in SK2 channels combine to produce the increased EPSP underlying LTP.

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.