Compound heterozygous mutations in the SUR1 (ABCC 8) subunit of pancreatic KATP channels cause neonatal diabetes by perturbing the coupling between Kir6.2 and SUR1 subunits

K(ATP) channels regulate insulin secretion by coupling β-cell metabolism to membrane excitability. These channels are comprised of a pore-forming Kir6.2 tetramer which is enveloped by four regulatory SUR1 subunits. ATP acts on Kir6.2 to stabilize the channel closed state while ADP (coordinated with Mg(2+)) activates channels via the SUR1 domains. Aberrations in nucleotide-binding or in coupling binding to gating can lead to hyperinsulinism or diabetes. Here, we report a case of diabetes in a 7-mo old child with compound heterozygous mutations in ABCC8 (SUR1[A30V] and SUR1[G296R]). In unison, these mutations lead to a gain of K(ATP) channel function, which will attenuate the β-cell response to increased metabolism and will thereby decrease insulin secretion. (86)Rb(+) flux assays on COSm6 cells coexpressing the mutant subunits (to recapitulate the compound heterozygous state) show a 2-fold increase in basal rate of (86)Rb(+) efflux relative to WT channels. Experiments on excised inside-out patches also reveal a slight increase in activity, manifested as an enhancement in stimulation by MgADP in channels expressing the compound heterozygous mutations or homozygous G296R mutation. In addition, the IC(50) for ATP inhibition of homomeric A30V channels was increased ~6-fold, and was increased ~3-fold for both heteromeric A30V+WT channels or compound heterozygous (A30V +G296R) channels. Thus, each mutation makes a mechanistically distinct contribution to the channel gain-of-function that results in neonatal diabetes, and which we predict may contribute to diabetes in related carrier individuals

Novel Mutation in Pancreatic KATP Channels Leads to Inactivation-Induced Congenital Hyperinsulinism

Mentor: Colin G. Nichols From the Washington University Undergraduate Research Digest: WUURD, Volume 5, Issue 1, Fall 2009. Published by the Office of Undergraduate Research. Henry Biggs, Director of Undergraduate Research and Associate Dean in the College of Arts & Sciences; Joy Zalis Kiefer, Undergraduate Research Coordinator, Co-editor, and Assistant Dean in the College of Arts & Sciences; Kristin Sobotka, Editor

Circuit Mechanisms of Spontaneous and Light Evoked Activity in the Retina

The retina is complex neural network that conveys visual information to the brain. It is composed of a wide variety of cell types that establish specific connections to form functional circuits. During development, circuits in the retina generate spontaneous waves of activity that instruct the wiring of the visual system. We study the circuits that produce stage III waves and desynchronize the firing of neighboring ganglion cells with opposite light responses (ON and OFF RGCs), a feature that is thought to help establish parallel ON and OFF pathways in downstream visual areas. We find that intersecting lateral excitatory and vertical inhibitory circuits give rise to precisely patterned stage III retinal waves. The retina can encode a wide range of visual features due to the variety of signals generated by its circuits. Amacrine cells (ACs) are the most diverse class of neurons in the retina yet of the 30 50 AC types in mammalian species, few have been studied in detail. Here, we identify and morphologically characterize three VIP expressing GABAergic AC types (VIP1 , VIP2 and VIP3 ACs) in mice. We find that the somata of VIP1 , VIP2 and VIP3 ACs are asymmetrically distributed along the dorso-ventral axis of the retina and that their neurite arbors differ in size and stratify in distinct sublaminae of the inner plexiform layer. Next, we target VIP ACs under 2 photon guidance for patch clamp recording to analyze light responses and underlying synaptic inputs. We find that VIP1 , VIP2 and VIP3 ACs differ in response polarity and spatial tuning, and contribute to the diversity of inhibitory and neuromodulatory signals in the retina. Finally, to examine the contribution of VIP ACs to visual possessing, we probe how conditional suppression of neurotransmitter release alters circuit function. First, we identify a RGC synaptic pattern using a combination of optogenetics and patch clamp recordings. Then, we chronically silence GABAergic synapses in VIP ACs by genetic deletion of vesicular GABA transporter (VGAT). Preliminary data indicate that this manipulation does not significantly alter downstream light responses. We are currently pursuing additional RGC partners as well as alternative tools to silence VIP ACs

Controlling Visually Guided Behavior by Holographic Recalling of Cortical Ensembles

Neurons in cortical circuits are often coactivated as ensembles, yet it is unclear whether ensembles play a functional role in behavior. Some ensemble neurons have pattern completion properties, triggering the entire ensemble when activated. Using two-photon holographic optogenetics in mouse primary visual cortex, we tested whether recalling ensembles by activating pattern completion neurons alters behavioral performance in a visual task. Disruption of behaviorally relevant ensembles by activation of non-selective neurons decreased performance, whereas activation of only two pattern completion neurons from behaviorally relevant ensembles improved performance, by reliably recalling the whole ensemble. Also, inappropriate behavioral choices were evoked by the mistaken activation of behaviorally relevant ensembles. Finally, in absence of visual stimuli, optogenetic activation of two pattern completion neurons could trigger behaviorally relevant ensembles and correct behavioral responses. Our results demonstrate a causal role of neuronal ensembles in a visually guided behavior and suggest that ensembles implement internal representations of perceptual states

Differential Effects of Axon Initial Segment and Somatodendritic GABAA Receptors on Excitability Measures in Rat Dentate Granule Neurons

GABAA receptors are found on the somatodendritic compartment and on the axon initial segment of many principal neurons. The function of axonal receptors remains obscure, although it is widely assumed that axonal receptors must have a strong effect on excitability. We found that activation of GABAA receptors on the dentate granule neuron axon initial segment altered excitability by depolarizing the voltage threshold for action potential initiation under conditions that minimally affected overall cell input resistance. In contrast, activation of somatic GABAA receptors strongly depressed the input resistance of granule neurons without affecting the voltage threshold of action potential initiation. Although these effects were observed over a range of intracellular chloride concentrations, average voltage threshold was unaffected when ECl rendered GABAA axon initial segment responses explicitly excitatory. A compartment model of a granule neuron confirmed these experimental observations. Low ambient agonist concentrations designed to activate granule neuron tonic currents did not stimulate axonal receptors sufficiently to raise voltage threshold. Using excitatory postsynaptic current (EPSC)-like depolarizations, we show physiological consequences of axonal versus somatic GABAA receptor activation. With axonal inhibition, individual excitatory postsynaptic potentials (EPSPs) largely retained their amplitude and time course, but EPSPs that were suprathreshold under basal conditions failed to reach threshold with GABAA activation. By contrast, somatic inhibition depressed individual EPSPs because of strong shunting. Our results suggest that axonal GABAA receptors have a privileged effect on voltage threshold and that two major measures of neuronal excitability, voltage threshold and rheobase, are differentially affected by axonal and somatic GABAA receptor activation