Book of abstracts - British Neuroscience Association

12.01
Use of capillary chip based patch-clamp platform to study
molecular details of Kir channel gating
Kurata H T, Nichols C G
Dept of Cell Biology and Physiology,, Washington University School of
Medicine, , 660 South Euclid Avenue, Saint Louis, MO 63110, USA
Intracellularly applied blockers can be ‘trapped’ by voltage-dependent
channel closure of voltage-gated (Kv) potassium channels; seminal
evidence that the voltage-operated gate lies near the intracellular
opening of the channel, and closes an inner cavity around the
blockers. Similar ‘blocker-trapping’ experiments on ligand-gated Kir
channels are less straightforward, due to difficulties in achieving rapid
control of channel gating. To overcome these difficulties we used the
rapid solution-switching capabilities of the Dynaflow system
(Cellectricon Inc.), which requires very small solution volumes, and
allows full temporal control of both solution switching and voltage. We
have demonstrated that ATP-induced closure of KATP channels
prevents the dissociation of spermine from the pore, showing that
closure of the ligand-operated gate similarly isolates an inner cavity
that can accommodate a spermine molecule. We have examined the
trapping of a panel of compounds of differing lengths, and now report
that longer ‘extended’ synthetic polyamines, which share the same
voltage-dependence of block as spermine, are not trapped by ATPinduced
channel closure. These data suggest that extended
polyamines prevent channel closure in the presence of ATP. The
demonstrated length-dependence for trapping allows us to use
extended polyamines as coarse-scale molecular calipers of channel
dimensions. Computational simulations of polyamine blockade, in
models of inward rectifier channels based on crystal structures of
KirBac1.1, are consistent with the constraints on the inner cavity
dimensions imposed by this analysis. These findings illustrate the
essential similarity of ligand-dependent and voltage-dependent gating
in Kir and Kv channels, respectively.
12.02
Two-pore domain potassium channels enable sustained highfrequency
firing.
Brickley S G, Aller M I, Veale E L, Sandu C, Alder F G
Biophysics Group, Division of Cell and Molecular Biology, Imperial College
London, UK.
The ability of neurons to fire at high frequencies during sustained
depolarization is generally explained in terms of the properties of voltagegated
ion channels. By contrast, two-pore domain potassium (K2P)
channels impart a voltage-independent, non-inactivating, leak conductance
that hyperpolarizes the resting membrane potential (RMP) by increasing
the membranes potassium permeability. In addition to a depolarised resting
membrane potential, we find that cerebellar granule neurons (CGNs)
lacking the TASK-3 type K2P channel exhibit marked accommodation of
action potential firing. The accommodation phenotype was not associated
with any change in the functional properties of the underlying sodium
channels, nor could it simply be explained by the more depolarized resting
membrane potential that resulted from this K2P channel deletion. A
functional rescue experiment utilizing a dynamic current clamp approach to
mimic a non-linear conductance of the type lost in these mice was able to
restore wild-type firing properties to adult TASK-3 KO CGNs. We propose
that the TASK-3 conductance enables more sodium channels to be
available for activation, and so helps CGNs to fire at high frequencies in
response to sustained depolarization. This represents an unappreciated
contribution of K2P channels to neuronal function as TASK-3 channels
serve to both select and support high frequency firing.
12.03
Modulation of functional L-Type calcium channels by the
microtubule associated protein tau.
Montgomery J R, Marrion N V
Department of Pharmacology, School of Medical Sciences, University
Walk, Bristol, BS8 1TD
L-type Ca2+ channels are a subtype of voltage-gated Ca2+ channels
characterised by pharmacological sensitivity to dihydropyridines.
Cav1.2 and Cav1.3 pore forming subunits produce L-type currents
when expressed with auxiliary subunits. Both channel isoforms are
present in a number of neurons, including hippocampal neurons,
where they regulate many processes including activation of Ca2+activated
channels and gene transcription.
The cytoskeletal protein α-actinin2 is involved in association of L-type
channels with the Ca2+-activated K+ channel SK2 in cardiac
myocytes. It is proposed that functional coupling of L and SK channels
in hippocampus may also require interaction with a cytoskeletal
protein. One such protein is the microtubule associated protein tau,
which is abundant in hippocampus, with Cav1.3 and phosphorylated
tau sharing somatic subcellular localisation in hippocampal neurons.
Full length human tau (4R2N) altered function of L-type channels
composed of Cav1.3, Cavß3 and Caα2δ1subunits. The activation
curve for this channel complex was shifted to more depolarised
potentials in the presence of hTau4R2N, with no effect on current
amplitude. The effect of hTau4R2N was subunit specific because no
effect was observed on currents composed of Cav1.3, Cavß2A and
Cavα2δ1 or Cav1.2, Cavß3 and Cavα2δ1. A second human tau
isoform, 4R1N, which lacks an amino-terminal insert, was found to
have no effect on activity of any of the subunit combinations above.
13.01
Changes in protein phosphorylation reveal that different pools of
synaptic vesicles can release by distinct exocytotic modes
Green J E (1), Sihra T S (2), Ashton A C (1)
(1) Biological Sciences, University of Central Lancashire, Preston, UNITED
KINGDOM, (2) Pharmacology, University College London, London,
UNITED KINGDOM.
Exocytosis was studied in rat cerebrocortical synaptosomes maximally
loaded with FM2-10 styryl dye under conditions that labelled all releasable
synaptic vesicles (SVs). Greater FM2-10 release was induced upon
stimulation with various stimuli after pre-treatment with the protein
phosphatase inhibitor okadaic acid (OA) than in untreated controls. FM dye
release from individual synaptosomes was measured by confocal
microscopy, which demonstrated that OA changes the release kinetics of
individual terminals, and does not merely induce the exocytosis of some
non-releasing synaptosomes. However, release of ATP (a neurotransmitter
present in many terminal types) evoked by these same stimuli showed no
enhancement by OA. This indicates that FM2-10 dye release does not
accurately reflect the true amount of neurotransmitter secretion, as
maximum dye release relies on exocytosis occurring by full fusion (FF),
rather than “kiss-and-run” (KR) in which SVs release via a transiently open
fusion pore. Bromophenol blue (BPB) quenching of styryl dyes also reveals
the KR component of release; with BPB, evoked release was quantitatively
similar in control and OA pre-treated terminals. Intriguingly, studying the
release of the readily releasable pool (RRP) and reserve pool (RP) of
vesicles revealed that only the former pool normally undergo KR
exocytosis. Furthermore, the use of the above paradigms has revealed that
the RRP can be spontaneously released at 37°C. However, this RRP was
distinct from a spontaneously loaded pool that showed different
characteristics from both the RRP and RP.
These data suggest that tau is not acting as a chaperone to aid
surface expression, but interacts with Cav1.3 and/or Cavß3 subunits
to affect function by utilising the first amino-terminal insert of tau.
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