Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ions to flow through the plasma ...

Yale School of Medicine

Visual activity refines developing retinotectal maps and shapes individual retinal arbors via an NMDA receptor-dependent mechanism. As retinal axons grow into tectum, they slow markedly and emit many transient side branches behind the tip, assuming a "bottlebrush" morphology. Some branches are stabilized and branch further, giving rise to a compact arbor. The dynamic rate of branch addition and deletion is increased twofold when MK801 is used to block NMDA receptors, as if this prevents release of a stabilizing signal such as arachidonic acid (AA) from the postsynaptic neuron. In optic tract, AA mediates NCAM and L1 stimulation of axon growth by activating presynaptic protein kinase C (PKC) to phosphorylate GAP-43 and stabilize F-actin, and, if present in tectum, this growth control pathway could be modulated by postsynaptic activation. To test for the effects on arbor morphology of blocking PKC or AA release, we examined DiO-labeled retinal axons of larval zebrafish with time-lapse videomicroscopy. Bath application of the selective PKC inhibitor bisindolylmaleimide from 2 or 3 days onward doubled the rate at which side branches were added and deleted, as seen with MK801, and also prevented maturation of the arbor so that it retained a "bottlebrush" morphology. In order to selectively block the PKC being transported to retinal terminals, we injected the irreversible inhibitor calphostin C into the eye from which the ganglion cells were labeled, and this produced both effects seen with bath application. In contrast, there were no effects of control injections, which included Ringers into the same eye and the same dose into the opposite eye (actually much closer to the tectum of interest), to rule out the possibility that the inhibitor leaked from the eye to act on tectal cells. For comparison, we examined arbors treated with the NMDA blocker MK801 at half-hour time-lapse intervals, and detected the twofold rise in rates of branch addition and deletion previously reported in Xenopus larvae, but not the structural effect seen with the PKC inhibitors. In addition, we could produce both effects seen with PKC inhibitors by using RHC80267 to block AA release from DAG lipase, indicating that AA is the main drive for PKC activation. Thus, the results show a distinct role of AA and presynaptic PKC in both maturation of arbor structure and in the dynamic control of branching. The effects on branch dynamics were present regardless of the level of maturity of arbor structure. The fact that they mimicked those of MK801 suggests that presynaptic PKC may be involved in the NMDA receptor-driven stabilization of developing retinal arbors.

Presynaptic protein kinase C controls maturation and branch dynamics of developing retinotectal arbors: possible role in activity-driven sharpening.

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ion to flow through the plasma membrane. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex. Traditional assays examining the interaction between channels and regulatory proteins generally provide little information on the time-course of interactions in living cells. We have now used a novel label-free technology to detect changes in the distribution of mass close to the plasma membrane following modulation of potassium channels by G protein-coupled receptors (GPCRs). This technology uses optical sensors embedded in microplates to detect changes in the refractive index at the surface of cells. Although the activation of GPCRs has been studied with this system, protein-protein interactions due to modulation of ion channels have not yet been characterized. Here we present data that the characteristic pattern of mass distribution following GPCR activation is significantly modified by the presence of a sodium-activated potassium channel, Slack-B, a channel that is known to be potently modulated by activation of these receptors.

Use of optical biosensors to detect modulation of Slack potassium channels by G protein-coupled receptors.

Malignant migrating partial seizures of infancy (MMPSI) is a rare epileptic encephalopathy of infancy that combines pharmacoresistant seizures with developmental delay. We performed exome sequencing in three probands with MMPSI and identified de novo gain-of-function mutations affecting the C-terminal domain of the KCNT1 potassium channel. We sequenced KCNT1 in 9 additional individuals with MMPSI and identified mutations in 4 of them, in total identifying mutations in 6 out of 12 unrelated affected individuals. Functional studies showed that the mutations led to constitutive activation of the channel, mimicking the effects of phosphorylation of the C-terminal domain by protein kinase C. In addition to regulating ion flux, KCNT1 has a non-conducting function, as its C terminus interacts with cytoplasmic proteins involved in developmental signaling pathways. These results provide a focus for future diagnostic approaches and research for this devastating condition.

De novo gain-of-function KCNT1 channel mutations cause malignant migrating partial seizures of infancy.

Loss of the RNA-binding protein fragile X mental retardation protein (FMRP) represents the most common form of inherited intellectual disability. Studies with heterologous expression systems indicate that FMRP interacts directly with Slack Na(+)-activated K(+) channels (K(Na)), producing an enhancement of channel activity. We have now used Aplysia bag cell (BC) neurons, which regulate reproductive behaviors, to examine the effects of Slack and FMRP on excitability. FMRP and Slack immunoreactivity were colocalized at the periphery of isolated BC neurons, and the two proteins could be reciprocally coimmunoprecipitated. Intracellular injection of FMRP lacking its mRNA binding domain rapidly induced a biphasic outward current, with an early transient tetrodotoxin-sensitive component followed by a slowly activating sustained component. The properties of this current matched that of the native Slack potassium current, which was identified using an siRNA approach. Addition of FMRP to inside-out patches containing native Aplysia Slack channels increased channel opening and, in current-clamp recordings, produced narrowing of action potentials. Suppression of Slack expression did not alter the ability of BC neurons to undergo a characteristic prolonged discharge in response to synaptic stimulation, but prevented recovery from a prolonged inhibitory period that normally follows the discharge. Recovery from the inhibited period was also inhibited by the protein synthesis inhibitor anisomycin. Our studies indicate that, in BC neurons, Slack channels are required for prolonged changes in neuronal excitability that require new protein synthesis, and raise the possibility that channel-FMRP interactions may link changes in neuronal firing to changes in protein translation.

Matthew R. Fleming

Department of Pharmacology

Yale School of Medicine

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Matthew R. Fleming

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Pharmacology

Yale School of Medicine

Use of Label-free Optical Biosensors to Detect Modulation of Potassium Channels by G-protein Coupled Receptors

Department of Pharmacology