Kinetic modulation of Kv4-mediated A-current by arachidonic acid is dependent on potassium channel interacting proteins.

The Kv4 subfamily of voltage-gated potassium channels is responsible for the transient A-type potassium current that operates at subthreshold membrane potentials to control membrane excitability. Arachidonic acid was shown recently to modulate both the peak amplitude and kinetics of the hippocampal A-current. However, in Xenopus oocytes, arachidonic acid only inhibited the peak amplitude of Kv4 current without modifying its kinetics. These results suggest the existence of Kv4 auxiliary subunit(s) in native cells. We report here a K-channel interacting protein (KChIP)-dependent kinetic modulation of Kv4.2 current in Chinese hamster ovary cells and Kv4.2 and Kv4.3 currents in Xenopus oocytes by arachidonic acid at physiological concentrations. This concentration-dependent effect of arachidonic acid resembled that observed in cerebellar granule neurons and was fully reversible. Other fatty acids, including a nonhydrolyzable inhibitor of both lipooxygenase and cyclooxygenase, 5,8,11,14-eicosatetraynoic acid (ETYA), also mimicked arachidonic acid in modulating Kv4.3 and Kv4.3/KChIP1 currents. Compared with another transient potassium current formed by Kv1.1/Kvbeta1, Kv4.3/KChIP1 current was much more sensitive to arachidonic acid. Association between KChIP1 and Kv4.2 or Kv4.3 was not altered in the presence of 10 microm ETYA as measured by immunoprecipitation and association-dependent growth in yeast. Our data suggest that the KChIP proteins represent a molecular entity for the observed difference between arachidonic acid effects on A-current kinetics in heterologous cells and in native cells and are consistent with the notion that KChIP proteins modulate the subthreshold A-current in neurons.

A novel nervous system beta subunit that downregulates human large conductance calcium-dependent potassium channels.

The pore-forming alpha subunits of many ion channels are associated with auxiliary subunits that influence channel expression, targeting, and function. Several different auxiliary (beta) subunits for large conductance calcium-dependent potassium channels of the Slowpoke family have been reported, but none of these beta subunits is expressed extensively in the nervous system. We describe here the cloning and functional characterization of a novel Slowpoke beta4 auxiliary subunit in human and mouse, which exhibits only limited sequence homology with other beta subunits. This beta4 subunit coimmunoprecipitates with human and mouse Slowpoke. beta4 is expressed highly in human and monkey brain in a pattern that overlaps strikingly with Slowpoke alpha subunit, but in contrast to other Slowpoke beta subunits, it is expressed little (if at all) outside the nervous system. Also in contrast to other beta subunits, beta4 downregulates Slowpoke channel activity by shifting its activation range to more depolarized voltages and slowing its activation kinetics. beta4 may be important for the critical roles played by Slowpoke channels in the regulation of neuronal excitability and neurotransmitter release.

Slob, a novel protein that interacts with the Slowpoke calcium-dependent potassium channel.

Slob, a novel protein that binds to the carboxy-terminal domain of the Drosophila Slowpoke (dSlo) calcium-dependent potassium channel, was identified with a yeast two-hybrid screen. Slob and dSlo coimmunoprecipitate from Drosophila heads and heterologous host cells, suggesting that they interact in vivo. Slob also coimmunoprecipitates with the Drosophila EAG potassium channel but not with Drosophila Shaker, mouse Slowpoke, or rat Kv1.3. Confocal fluorescence microscopy demonstrates that Slob and dSlo redistribute in cotransfected cells and are colocalized in large intracellular structures. Direct application of Slob to the cytoplasmic face of detached membrane patches containing dSlo channels leads to an increase in channel activity. Slob may represent a new class of multi-functional channel-binding proteins.

Characterization of gating and peptide block of mSlo, a cloned calcium-dependent potassium channel.

The 20 amino acid Shaker inactivation peptide blocks mSlo, a cloned calcium-dependent potassium channel. Changing the charge and degree of hydrophobicity of the peptide alters its blocking kinetics. A "triple mutant" mSlo channel was constructed in which three amino acids (T256, S259, and L262), equivalent to those identified as part of the peptide's receptor site in the S4-S5 cytoplasmic loop region of the Shaker channel, were mutated simultaneously to alanines. These mutations produce only limited changes in the channel's susceptibility to block by a series of peptides of varying charge and hydrophobicity but do alter channel gating. The triple mutant channel shows a significant shift in its calcium-activation curve as compared with the wild-type channel. Analysis of the corresponding single amino acid mutations shows that mutation at position L262 causes the most dramatic change in mSlo gating. These results suggest that the three amino acids mutated in the mSlo S4-S5 loop may contribute to, but are not essential for, peptide binding. On the other hand, they do play a critical role in the channel's calcium-sensing mechanism.

A visually elicited escape response in the fly that does not use the giant fiber pathway.

A housefly elicits an escape in response to an approaching target (Holmqvist & Srinivasan, 1991). This study tests if the giant fiber pathway, which mediates a light-off escape response in a fruitfly (Wyman et al., 1985), also mediates escape to an approaching target in a housefly. Visual stimuli simulating an approaching or receding dark disk were presented to houseflies, Musca domestica, in both behavioral and physiological experiments. Freely behaving flies escaped in response to an expanding dark disk but not to a contracting dark disk. In restrained flies, the giant fiber, here called the giant descending neuron (GDN), was recorded from intracellularly and the tergotrochanteral muscle (TTM), which provides the main thrust in an escape jump, was recorded from extracellularly. During electrical stimulation of the brain, by means of stimulating electrodes inserted into the ventral part of each compound eye, a single spike in the GDN drives the TTM. However, when the TTM responds to visual stimulation that elicits an escape response in a behaving fly, the GDN shows no activity. Similarly to the behavioral results, the TTM of restrained flies showed muscle potentials in response to an expanding dark disk, but not to a contracting disk. However, freely moving flies elicit escapes more than 100 ms on average before the first TTM spike, suggesting that this type of escape does not start with a jump powered by the TTM. In conclusion, this visually evoked escape response in the housefly is not likely to be mediated by the giant fiber pathway. The findings suggest that there exist at least two pathways mediating visually evoked escape responses in flies.

An electronic device for monitoring escape behaviour in Musca and Drosophila.

This paper describes an effective device for detecting the presence of a fly or small insect on a specially constructed detector pad. It was used successfully with Musca domestica (house fly) and Drosophila melanogaster (fruit fly). The detector works by utilising the detector pad as a variable capacitor which forms part of an RC oscillator. Its capacitance changes as the fly comes in contact with it and this change in capacitance is detected by the circuit. The detector uses cheap and readily available components and can be constructed without expert knowledge in electronics. It can be used to detect and determine the timing of the jump of a fly escaping in response to, say, a visual stimulus. It can also be used for screening of mutants of Drosophila which show altered escape responses and for monitoring locomotion of small animals.

A visually evoked escape response of the housefly.

Flies (Musca domestica) avoid danger by initiating a rapid jump followed by flight. To identify the visual cues that trigger the escape response in the housefly, we measured the timing and probability of escapes when the fly was presented with a variety of visual stimuli created by moving targets toward it. Our results show that an escape response is triggered by an approaching dark disk, but not by a receding dark disk. On the other hand, a bright disk elicits escape only when it recedes. A disk with black and white rings is less effective at eliciting escape than is a dark solid disk of the same size. This indicates that the darkening contrast produced by an approaching stimulus is a more crucial parameter than expansion cues contained in the optical flow. Escape is also triggered by a horizontally moving dark edge, but not by a moving bright edge or by a grating. An examination of several visual parameters reveals that the darkening contrast, measured from the onset of stimulation to the start of escape is nearly constant for a variety of stimuli that trigger escape reliably. Thus darkening contrast, coupled with motion may be crucial in eliciting the visually evoked escape response. Other visual parameters such as time-to-contact or target angular velocity seem to be relatively unimportant to the timing of escapes.

Histamine-like immunoreactivity in photoreceptors of the compound eyes and ocelli of the flies Calliphora erythrocephala and Musca domestica.

Antibodies to histamine were used for immunocytochemical studies of the visual system in the flies Calliphora erythrocephala and Musca domestica. Specific immunolabeling of photoreceptors was found both in the compound eyes and ocelli of both species. In the compound eyes histamine-like immunoreactivity (HA-IR) was found in all the short visual fibers (photoreceptors R1-6) and one type of long visual fiber (photoreceptor R8). In addition, the ocellar photoreceptors also show HA-IR. In view of earlier biochemical and pharmacological/physiological findings by Elias and Evans (1983) and Hardie (1987) it thus seems likely that histamine is a neurotransmitter in insect photoreceptors. Interestingly, the second type of long visual fiber (photoreceptor R7) has recently been found to be GABA-immunoreactive (Datum et al. 1986). The two types of long visual fibers may hence use different transmitters which act on different receptors of the postsynaptic neurons in the second visual neuropil, the medulla. In addition to the photoreceptors in the retina and ocelli, we found processes of HA-IR neurons in one of the optic lobe neuropils, the lobula. This finding indicates that histamine may also be a transmitter in certain interneurons in the visual system.