Bertil Hille

Modulation of ion-channel function by G-protein-coupled receptors

Neurotransmitters acting through G-protein-coupled receptors change the electrical excitability of neurons. Activation of receptors can affect the voltage dependence, the speed of gating, and the probability of opening of various ion channels, thus changing the computational state and outputs of a neuron. Each cell expresses many kinds of receptors, and uses several intracellular signaling pathways to modulate channel function in different ways. It has become possible to dissect these pathways by combining pharmacological and biophysical experiments. Recent patch-clamp work in sympathetic neurons will be summarized to illustrate the mechanisms underlying modulation and its significance.

G protein-coupled mechanisms and nervous signaling

Bertil Hille Department of Physiology and Biophysics University of Washington Seattle, Washington, 98195 Signaling systems using receptors coupled to GTP- binding regulatory proteins (C proteins) are an essen- tial component of animal nervous systems. We know of hundredsof homologous receptorsand manyintra- cellular biochemical cascades that they control. This review considers the significance for nervous signal- ing of this diversity and of the branching intracellular events set in motion. Many of these questions have been reviewed before (Ross, 1989). My intention, how- ever, is to describe how molecular aspects of this sys- tem may bear on the neuroanatomy and integrative functions of the nervous system. Signaling via a Cascade The first G protein-coupled signaling pathway to be understood in any depth was the 8-adrenergic path- way, which leads to CAMP-dependent phosphoryla- tion of many target proteins. All the molecules car- rying the signal have been identified, purified, and sequenced. The canonical pathway starts with three membrane proteins (Figure IA): the B-adrenergic receptor (an integral membrane protein), the GTP- binding regulatory protein C, (a membrane-associ- ated protein), and a primary effector, the enzyme ade- nylyl cyclase (another integral protein). Binding of the extracellular agonist turns the intracellular face of the receptor into a catalyst that can dock with G, and promote the exchange of cytoplasmic GTP for the GDP normally bound to G, at rest. The unstimulated C&GDP is a stable heterotrimer,

PIP2 is a necessary cofactor for ion channel function: How and why?

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a minority phospholipid of the inner leaflet of plasma membranes. Many plasma membrane ion channels and ion transporters require PIP2 to function and can be turned off by signaling pathways that deplete PIP2. This review discusses the dependence of ion channels on phosphoinositides and considers possible mechanisms by which PIP2 and analogues regulate ion channel activity.

Recovery from muscarinic modulation of M current channels requires phosphatidylinositol 4,5-bisphosphate synthesis.

Suppression of M current channels by muscarinic receptors enhances neuronal excitability. Little is known about the molecular mechanism of this inhibition except the requirement for a specific G protein and the involvement of an unidentified diffusible second messenger. We demonstrate here that intracellular ATP is required for recovery of KCNQ2/KCNQ3 current from muscarinic suppression, with an EC(50) of approximately 0.5 mM. Substitution of nonhydrolyzable ATP analogs for ATP slowed or prevented recovery. ADPbetaS but not ADP also prevented the recovery. Receptor-mediated inhibition was irreversible when recycling of agonist-sensitive pools of phosphatidylinositol-4,5-bisphosphate (PIP(2)) was blocked by lipid kinase inhibitors. Lipid phosphorylation by PI 4-kinase is required for recovery from muscarinic modulation of M current.

GPR55 is a cannabinoid receptor that increases intracellular calcium and inhibits M current.

The CB1 cannabinoid receptor mediates many of the psychoactive effects of Δ9THC, the principal active component of cannabis. However, ample evidence suggests that additional non-CB1/CB2 receptors may contribute to the behavioral, vascular, and immunological actions of Δ9THC and endogenous cannabinoids. Here, we provide further evidence that GPR55, a G protein-coupled receptor, is a cannabinoid receptor. GPR55 is highly expressed in large dorsal root ganglion neurons and, upon activation by various cannabinoids (Δ9THC, the anandamide analog methanandamide, and JWH015) increases intracellular calcium in these neurons. Examination of its signaling pathway in HEK293 cells transiently expressing GPR55 found the calcium increase to involve Gq, G12, RhoA, actin, phospholipase C, and calcium release from IP3R-gated stores. GPR55 activation also inhibits M current. These results establish GPR55 as a cannabinoid receptor with signaling distinct from CB1 and CB2.

Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate

Phosphatidylinositol 4,5-bisphosphate is a signaling phospholipid of the plasma membrane that has a dynamically changing concentration. In addition to being the precursor of inositol trisphosphate and diacylglycerol, it complexes with and regulates many cytoplasmic and membrane proteins. Recent work has characterized the regulation of a wide range of ion channels by phosphatidylinositol 4,5-bisphosphate, helping to redefine the role of this lipid in cells and in neurobiology. In most cases, phosphatidylinositol 4,5-bisphosphate increases channel activity, and its hydrolysis by phospholipase C reduces channel activity.

Rapid Chemically Induced Changes of PtdIns(4,5)P2 Gate KCNQ Ion Channels

To resolve the controversy about messengers regulating KCNQ ion channels during phospholipase C–mediated suppression of current, we designed translocatable enzymes that quickly alter the phosphoinositide composition of the plasma membrane after application of a chemical cue. The KCNQ current falls rapidly to zero when phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 or PI(4,5)P2] is depleted without changing Ca2+, diacylglycerol, or inositol 1,4,5-trisphosphate. Current rises by 30% when PI(4,5)P2 is overproduced and does not change when phosphatidylinositol 3,4,5-trisphosphate is raised. Hence, the depletion of PI(4,5)P2 suffices to suppress current fully, and other second messengers are not needed. Our approach is ideally suited to study biological signaling networks involving membrane phosphoinositides.

Modulation of Ca2+ channels by G-protein beta gamma subunits.

CALCIUM ions entering cells through voltage-gated Ca2+ channels initiate rapid release of neurotransmitters and secretion of hormones. Ca2+ currents can be inhibited in many cell types by neurotransmitters acting through G proteins via a membrane-delimited pathway independently of soluble intracellular messengers1–4. Inhibition is typically caused by a positive shift in the voltage dependence and a slowing of channel activation and is relieved by strong depolarization resulting in facilitation of Ca2+currents1,4–6. This pathway regulates the activity of N-type and P/ Q-type Ca2+ channels1,2,7, which are localized in presynaptic terminals8,9 and participate in neurotransmitter release10–13. Synaptic transmission is inhibited by neurotransmitters through this mechanism1,4. G-protein a subunits confer specificity in receptor coupling1–4,14–17, but it is not known whether the Gα or Gβγ subunits are responsible for modulation of Ca2+channels. Here we report that Gβγ subunits can modulate Ca2+ channels. Transfection of Gβγ into cells expressing P/Q-type Ca2+ channels induces modulation like that caused by activation of G protein-coupled receptors, but Gα subunits do not. Similarly, injection or expression of Gβγ subunits in sympathetic ganglion neurons induces facilitation and occludes modulation of N-type channels by noradrenaline, but Gα subunits do not. In both cases, the Gγ subunit is ineffective by itself, but overexpression of exogenous Gβ subunits is sufficient to cause channel modulation.

Light scattering and birefringence changes during nerve activity

Two optical techniques—light scattering and birefringence—have been used to detect rapid structural changes accompanying the action potentials in two types of non-myelinated nerve fibre. Changes in light scattering originate from at least two different phenomena, while a large part of the birefringence change seems to be directly dependent on the potential difference across the axon membrane, and arises in radially oriented molecules associated with the membrane.

Ionic channels in nerve membranes

Abstract In summary a wide variety of experiments with axons provides kinetic, electrochemical, and pharmacological evidence that three types of channels through the membrane contribute to the ionic permeability changes underlying action potentials. In normal function one channel accounts for most of the movements of Na ions, another accounts for most of the movements of K ions, and the last accounts for the remaining “leakage” fluxes. The Na, K, and leakage channels seem to be independent specializations of the membrane. Theoretical, electrical, and pharmacological evidence suggest that Na channels are short narrow pores that open and close in an all-or-nothing fashion. An open Na channel in a squid giant axon may have a conductance of about 0.5 nmho. Very little is known about K and leakage channels except that K channels may be longer pores with a lower conductance than Na channels.