Selective activation of heteromeric SK channels contributes to action potential repolarization in mouse atrial myocytes.

BACKGROUND: Activation of small conductance calcium-activated potassium (SK) channels is proposed to contribute to repolarization of the action potential in atrial myocytes. This role is controversial, as these cardiac SK channels appear to exhibit an uncharacteristic pharmacology. OBJECTIVES: The objectives of this study were to resolve whether activation of SK channels contributes to atrial action potential repolarization and to determine the likely subunit composition of the channel. METHODS: The effect of 2 SK channel inhibitors was assessed on outward current evoked in voltage clamp and on action potential duration in perforated patch and whole-cell current clamp recording from acutely isolated mouse atrial myocytes. The presence of SK channel subunits was assessed using immunocytochemistry. RESULTS: A significant component of outward current was reduced by the SK channel blockers apamin and UCL1684. Block by apamin displayed a sensitivity indicating that this current was carried by homomeric SK2 channels. Action potential duration was significantly prolonged by UCL1684, but not by apamin. This effect was accompanied by an increase in beat-to-beat variability and action potential triangulation. This pharmacology was matched by that of expressed heteromeric SK2-SK3 channels in HEK293 cells. Immunocytochemistry showed that atrial myocytes express both SK2 and SK3 channels with an overlapping expression pattern. CONCLUSION: Only proposed heteromeric SK2-SK3 channels are physiologically activated to contribute to action potential repolarization, which is indicated by the difference in pharmacology of evoked outward current and prolongation of atrial action potential duration. The effect of blocking this channel on the action potential suggests that SK channel inhibition during cardiac function has the potential to be proarrhythmic.

Preferential assembly of heteromeric small conductance calcium-activated potassium channels.

The activation of small conductance calcium-dependent (SK) channels regulates membrane excitability by causing membrane hyperpolarization. Three subtypes (SK1-3) have been cloned, with each subtype expressed within the nervous system. The locations of channel subunits overlap, with SK1 and SK2 subunits often expressed in the same brain region. We showed that expressed homomeric rat SK1 subunits did not form functional channels, because subunits accumulated in the Golgi. This raised the question of whether heteromeric channels could form with SK1 subunits. The co-expression of SK1 and SK2 subunits in HEK293 cells preferentially co-assembled to produce heteromeric channels with a fixed stoichiometry of alternating subunits. The expression in hippocampal CA1 neurons of mutant rat SK1 subunits [rat SK1(LV213/4YA)] that produced an apamin-sensitive current changed the amplitude and pharmacology of the medium afterhyperpolarization. The overexpression of rat SK1(LV213/4YA) subunits reduced the sensitivity of the medium afterhyperpolarization to apamin, substantiating the preferential co-assembly of SK1 and SK2 subunits to form heteromeric channels. Species-specific channel assembly occurred as the co-expression of human SK1 with rat SK2 did not form functional heteromeric channels. The replacement of two amino acids within the C-terminus of rat SK2 with those from human SK2 permitted the assembly of heteromeric channels when co-expressed with human SK1. These data showed that species-specific co-assembly was mediated by interaction between the C-termini of SK channel subunits. The finding that SK channels preferentially co-assembled to form heteromeric channels suggested that native heteromeric channels will predominate in cells expressing multiple SK channel subunits.

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.

Allosteric block of KCa2 channels by apamin.

Activation of small conductance calcium-activated potassium (K(Ca)2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin apamin, but the mechanism of block is not understood. For example, apamin binds to both K(Ca)2.2 and K(Ca)2.3 with the same high affinity (K(D) approximately 5 pM for both subtypes) but requires significantly higher concentrations to block functional current (IC(50) values of approximately 100 pM and approximately 5 nM, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that apamin blocks K(Ca)2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.

Small conductance calcium-activated potassium channels: from structure to function.

The cloning of K(Ca)2 channels revealed three subtypes, with each displaying distinct but partially overlapping expression distributions in the mammalian CNS and periphery. Activation of K(Ca)2 channels leads to membrane hyperpolarization and inhibition of action potential firing. Block of K(Ca)2 channels has been suggested as a novel target for cognitive enhancement, depression, myotonic muscular dystrophy and heart arrhythmias. It is clear however, that blockers selective for individual K(Ca)2 channel subtypes would be required to be therapeutically useful. K(Ca)2 channel current is blocked by apamin, with the bee venom toxin being unusual in displaying some selectivity between K(Ca)2 channel subtypes. This suboptimal selectivity is not sufficient to be therapeutically useful and the toxin has been shown in vivo to have a very narrow therapeutic window. Mutational and molecular modelling studies of the K(Ca)2 channels are beginning to determine how selective block might be achieved. Mutagenesis has indicated the importance of the outer pore region and the extracellular loop between transmembrane domains S3 and S4 for block of K(Ca)2 current by apamin. Mapping the sequence of transmembrane domains S5, pore helix and S6 onto the crystal structures of KcsA, MthK and Kv1.2 has provided an approximation of the pore structure. This approach has allowed structural modelling of the interactions between toxins and channel, demonstrating that the toxins that show little discrimination between K(Ca)2 channel subtypes interact with the outer pore and around the K(+) selectivity filter. We present the structural modelling of the interaction of apamin and K(Ca)2.2, which is superimposed onto the crystal structure of Kv1.2. This has shown that apamin interacts only with the outer pore and does not come into contact with channel's selectivity filter. It is clear that by comparing how different toxins interact with each K(Ca)2 channel subtype, a detailed picture will be generated that will aid the development of more specific K(Ca)2 channel blockers.

Vesicular release of glutamate utilizes the proton gradient between the vesicle and synaptic cleft.

Glutamate is released from synaptic vesicles following formation of a fusion pore, connecting the vesicle interior with the synaptic cleft. Release is proposed to result from either full fusion of the vesicle with the terminal membrane or by 'kiss-and-run,' where release occurs through the fusion pore. 'Kiss-and-run' seems implausible as passive diffusion of glutamate through the pore is too slow to account for the rapidity of release. Vesicular accumulation of glutamate is driven by a proton gradient, resulting in the co-release of protons during exocytosis. We tested whether the proton gradient between the vesicle and cleft contributes to glutamate exocytosis. Collapse of the gradient reduced hippocampal glutamatergic transmission, an effect that was not associated with presynaptic changes in excitability, transmitter release probability, or postsynaptic sensitivity. These data indicate that approximately half of glutamate release utilizes the proton gradient between vesicle and cleft, suggesting a significant proportion of release by 'kiss-and-run.'