M.D.-Ph.D. Student Examines Functional Effects of Dual Ion Channel Regulation in the Brain

BY JOHN KOESTER, PH.D., Professor of Clinical Neurobiology and Behavior and Acting Director, Center for Neurobiology & Behavior

FOR MORE THAN 50 YEARS NEUROLOGISTS HAVE USED THE various rhythms embedded in the cortical EEG for diagnostic purposes. But throughout most of that period the mechanisms that underlie these rhythms and their functional significance remained a mystery. Consider the cortical “spindles” that occur in packets of 7-14 hz bursts of activity that last a few seconds and are separated by intervals of several seconds. These spindles occur during slow wave (non-REM) sleep. Recent evidence suggests they may be involved in the consolidation of long-term memory.

A parallel line of research focuses on the mechanisms that generate spindle activity. The spindles have been shown to be an emergent property of a network of neurons distributed in the thalamus, reticular formation, and the cerebral cortex. Bursts of action potentials in thalamic neurons that project to the cortex drive the cortical rhythm. It is now possible to account for many of the features of this rhythm by examining in molecular detail the functional properties of neuronal ion channels. A recent contribution to this effort comes from the work of an M.D.-Ph.D. student, Jing Wang, in the laboratory of Dr. Steven Siegelbaum, professor of pharmacology. Mr. Wang analyzed the functional properties of the voltage-gated HCN ion channels found in thalamocortical neurons.

Ion channels are proteins that control the electrical activity of nerve and muscle cells by allowing ions, such as sodium and potassium, to cross the cell membrane. Most channels have gates that control their opening and closing. Some channels are gated by the binding of ligands in the extracellular or intracellular environment. Other channels are gated by the voltage gradient across the cell membrane. The HCN channels are of particular interest because they are gated by both voltage and the intracellular metabolite, cyclic-adenosinemonophosphate (cAMP). In the absence of cAMP, the HCN channels open when the intracellular potential becomes more negative (termed hyperpolarization). When cAMP binds to the HCN channels, it enhances the rate and extent to which these channels open. This effect underlies the ability of sympathetic transmitters, such as epinephrine and norepinephrine, to accelerate the heart rate. The regulation of the channel by cAMP also causes changes in the firing pattern of the thalamocortical neurons during the sleep-wake cycle that are believed to regulate consciousness.

Previous studies emphasized the importance of transient rises in cAMP levels in cells resulting from the transient release of hormones and neurotransmitters. For example, during the “fight or flight” reflex, release of epinephrine and norepinephrine produces a relatively brief rise in intracellular cAMP levels that modulates the electrical excitability of neurons and muscle cells to alter the state of vigilance and ability to respond. Mr. Wang found that not only can a transient rise in cAMP change the state of electrical firing of a neuron but also, conversely, a change in the electrical activity of a neuron can influence the efficacy of cAMP. The key to this novel type of cAMP signaling lies in the dual regulation of the HCN channels by membrane potential and by cAMP.

LEFT: Jing Wang, left, and Dr. Steven Siegelbaum. RIGHT: Figure demonstrating likely role of cAMP regulation of Ih current in spindle formation. The EEG recording at top (A) shows spindling during slow wave sleep. In B, intracellular recording from thalamic slice shows spindling: bursts of activity (action potentials) followed by silent periods. During activity, Ih increases, causing a prolonged afterdepolarization (ADP) that silences activity. When the ADP decays, activity resumes. Figure taken from Luthi A, McCormick DA. (1998). H-current: properties of a neuronal and network pacemaker. Neuron 21: 9-12.
Nearly 40 years ago, Monod, Wyman, and Changeux proposed an influential theory for how ligand-binding might lead to receptor activation. Their theory, applied to ion channels, proposes that a channel undergoes a conformational change between a resting (closed) state and an active (open) state. The conformational change that gates the channel open is tightly coupled to a conformational change in the ligand-binding site that enhances the affinity of the open channel for ligand. For HCN channels, this means that a hyperpolarizing voltage change will do two things. First, it will open the channel. Second, the change in configuration that underlies the opening process also will enhance the affinity of the channel for cAMP. Mr. Wang showed that this effect could account for a puzzling slow phase of HCN channel opening.

Nearly all cells have a very low level of basal cAMP, even in the absence of surface receptor stimulation. This level of cAMP is normally too low to bind to the HCN channels when closed, due to their low affinity for cAMP. However, when the membrane voltage is hyperpolarized, as occurs during a burst of inhibitory input to a thalamocortical neuron, the HCN channels will begin to open. Once open, the channels will then bind basal cAMP due to their enhanced affinity. This binding reaction will slowly draw channels from the closed-unliganded pool to the open- unliganded pool, to the open-cAMP-bound pool, resulting in a very slow phase of channel opening that can take tens of seconds to reach completion, a process termed dynamic allosteric regulation.

What is the physiological relevance of dynamic allosteric regulation? Might it play a role in certain diseases? Mr. Wang, in collaboration with Matthew Nolan, a postdoctoral fellow, showed that the long quiet periods during spindling in thalamocortical neurons, which are triggered by inhibitory synaptic input, could be explained by the prolonged activation of the HCN channels due to cAMP binding to the open channel. During epilepsy, the pattern of thalamocortical neuron firing is disrupted. Recent studies indicate changes in expression of HCN channels associated with febrile seizures, possibly resulting in altered excitability.

The concept of dynamic allosteric coupling is also important for its more general implications. Since the 1950s, when the second messenger role of cAMP in the liver was first elucidated, its ability to modulate enzyme or channel function was thought to be strictly a function of its cytoplasmic concentration. Mr. Wang’s results demonstrate for the first time that the dynamics of enyzme or channel regulation by cAMP is a function not only of cAMP concentration in the cytoplasm, but also the state of the channel. Other channel types are likewise dually regulated by voltage and ligand binding. It will be important to reexamine them, and allosterically regulated enzymes as well, for evidence that this allosteric coupling may extend the dynamic range of their functions.

| Top |