Current Research Projects - Catterall Lab
Structural Basis for the Physiological Function of Nav and Cav Channels
Understanding the structural basis for voltage-dependent gating and selective ion conductance of sodium and calcium channels is a major goal of research in molecular neuroscience and structural biology. Determination of the three-dimensional structures of ancestral bacterial Nav channels by x-ray crystallography in pre-open and inactivated states at atomic resolution (Payandeh et al., Nature 2011, 2012) now allows us to probe the structural basis for physiological function of these channel proteins at the atomic level. Our overall strategy is to introduce structural elements of the complex mammalian Nav and Cav channels into the simple structure of the bacterial sodium channel NavAb, demonstrate that the functional properties of mammalian sodium channels are reconstituted in the bacterial channel, and determine the structural basis for the functions of mammalian sodium and calcium channels. A major success with this experimental approach was determination of the structural basis for calcium conductance and selectivity in Cav channels (Tang et al., Nature, 2014). Current experiments are aimed at understanding the ion selectivity for Na+ vs. K+ , the mechanism of voltage-dependent activation, and the mechanisms of fast and slow inactivation using this experimental strategy. These experiments use a combination of high-resolution structural biology, molecular modeling, structure-guided site-directed mutagenesis, and high-resolution electrophysiological recording methods.
Structural Basis for Pharmacology of Nav and Cav Channels
Voltage-gated sodium and calcium channels are the molecular targets for drugs used to treat pain, epilepsy, bipolar disorder, cardiac arrhythmia, and hypertension, but the structural basis for the complex actions of these drugs remain unknown. In previous structure-function studies, we defined the amino acid residues that form the receptor sites for these major classes of drugs: local anesthetics, antiarrhythmic, and antiepileptic drugs on Na channels and both antiarrhythmic and antihypertensive drugs on Cav channels. Remarkably, the bacterial Nav and Cav channels retain substantial binding affinity and conserved mechanisms of action of these drugs. We are now using a combination of high-resolution structure determination of model Nav and Cav channels with drugs bound plus high-resolution electrophysiological analysis of mutations in the drug receptor sites to define drug action at the atomic level. We are interacting with pharmaceutical companies to study new generations of these drugs and to provide structural information for structure-based drug design.
Sodium Channels in Epilepsy and Autism
A major research direction in the laboratory aims to understand the dysfunction of Nav channels in inherited diseases. Using a mouse genetic model of Dravet Syndrome, which is caused by heterozygous loss-of-function mutations in the brain sodium channel Nav1.1, we found that selective loss of firing of GABAergic inhibitory neurons caused by loss-of-function of Nav1.1 is the cause for epilepsy and premature death in this intractable form of childhood epilepsy (Yu et al., Nature Neurosci. 2006; Cheah et al., PNAS 2012; Rubinstein et al., Brain 2015). Our mouse model of Dravet Syndrome recapitulates the cognitive deficit and autistic-like behaviors of individuals with this disease, and these disease phenotypes are also caused by failure of firing of GABAergic interneurons (Han et al., Nature 2012; Rubinstein et al., Brain 2015). Our current experiments are dissecting the roles of different classes of interneurons in epilepsy, cognitive deficit, and autistic-like behaviors using traditional mouse genetics and optogenetics. Our goals are to fully understand the pathogenesis of each of the major facets of this complex disease and explore novel therapeutic approaches to this devastating disease that are based on understanding of its pathophysiology and pharmacology. Our experimental approaches combine mouse genetics, electrophysiological recording at single cell, brain slice, and whole animal levels, and behavioral analyses.
Calcium Channel Regulation and Synaptic Plasticity
Voltage-gated calcium channels in nerve terminals are activated by action potentials and conduct calcium (Ca) into the nerve terminal at active zones to initiate neurotransmitter release and synaptic transmission. Cav2.1 channels conduct P/Q-type Ca current that triggers release of the neurotransmitter glutamate at synapses in the central nervous system. We have defined a presynaptic Ca channel signaling complex containing SNARE proteins, G proteins, Ca sensor proteins, and Ca/calmodulin-dependent protein kinase II, which all serve as regulators of Cav2.1 channel activity and effectors of local Ca signaling. Regulation of presynaptic Ca channels by calmodulin and related Ca sensor proteins contribute in an important way to short-term synaptic plasticity, including short-term synaptic facilitation and the rapid phase of synaptic depression. Calmodulin and Ca sensor proteins bind to a bipartite regulatory site in the proximal C-terminal domain of Cav2.1 channels. Differential expression of these proteins controls the balance of facilitation and rapid depression of synaptic transmission. Mice with mutations that prevent facilitation of Cav2.1 channels have reduced short-term synaptic facilitation, slowed synaptic depression, and dramatically impaired spatial learning and memory. We are now probing the contribution of regulation of Cav2.1 channels by Ca sensor proteins to short-term synaptic plasticity in different types of synapses, and we are studying how defects in short-term and long-term synaptic plasticity caused by mutations of Cav2.1 channels impair encoding of spatial information, spatial learning, and memory for spatial context. Our experimental approaches combine mouse genetics, electrophysiological recording of synaptic transmission in cell culture, brain slices, and other ex vivo synaptic preparations, and both physiological and behavioral analyses in genetically modified mice.
Calcium Channel Regulation in the Fight-or-Flight Response
The fight-or-flight response is a conserved behavioral response of all vertebrates to fear, stress, and exercise. The increase in contractile force of cardiac muscle during fear, stress, and exercise is mediated by the sympathetic nervous system through beta-adrenergic/PKA phosphorylation and up-regulation of cardiac Cav1.2 channels, which causes increased Ca entry into ventricular myocytes. Our studies of cardiac Cav1.2 channels have defined a signaling complex of the proteolytically processed, C-terminal-truncated form of the Cav1.2 channel with its noncovalently associated distal C-terminal and A Kinase Anchoring Protein 15 as the substrate for up-regulation by the beta-adrenergic/cAMP/PKA signaling pathway in the fight-or-flight response. Phosphorylation at Ser1700 and Thr1704 in the interface between the distal and proximal C-terminal domains relieves autoinhibition of the Cav1.2 channel and increases its activity. Mutation of these phosphorylation sites causes reduced beta-adrenergic regulation, impaired fight-or-flight response, cardiac hypertrophy, and fatal heart failure. We are now studying the molecular changes in the calcium channel signaling complex that accompany hypertrophy and heart failure in order to understand the pathogenic process and develop new approaches for therapeutic intervention. Our experimental approaches combine biochemical and structural studies of the Cav1.2 signaling complex, physiological analysis of cardiac function in vivo, and electrophysiological studies of regulation of Cav1.2 channels in dissociated cardiac myocytes and transfected non-muscle cells using whole-cell and single-channel patch clamp recording methods.