The research program of the Shapiro laboratory centers on the physiology, structure and regulation of voltage-gated K+ and Ca2+, TRP and Cl- ion channels that serve multiple roles in nerve and muscle, as well as their roles as novel therapeutic targets in myriad diseases of the nervous system. The K+ channels are called “M-type”, originally called such from their inhibition by stimulation of muscarinic acetylcholine receptors in sympathetic neurons. These K+ channels are widely expressed throughout the peripheral and central nervous system. We study the physiology, modulation and functional role of these channels in peripheral and central neurons, in smooth muscle, in heterologous expression systems, and in the brain slice from the hippocampus and other brain regions. M channels, composed of subunits encoded by the KCNQ (Kv7) family of genes, play fundamental roles in the nervous and cardiovascular systems, inner ear and epithelia. Those roles include regulation of neuronal excitability, control over neurotransmitter release, pacemaking and K+ transport. We study regulation of M-channels by a variety of receptors coupled to Gq/11 G proteins, via numerous cytoplasmic 2nd-messingers, including lipids, protein kinases and phosphatases, and intracellular Ca2+ ions acting through proteins such as calmodulin. Thus, the Shapiro laboratory performs neuroscience research using Biophysical, Molecular, Cellular, Genetic and Behavioral approaches. Our work is very collaborative in nature, involving investigators in other departments at UT Health SA, other institutions in the USA, and many around the world.
In the past several years, we have also focused on transcriptional regulation of expression of the channels, both in normal and pathophysiological states, such as during epilepsy, stroke and traumatic brain injury. We are probing the mechanisms of epileptogenesis, and how to prevent its development, focusing on the temporal lobe, particularly circuits in the hippocampus. We also study the regulation of N- and P/Q- and L-type voltage-gated Ca2+ channels, which drive exocytosis, release of neurotransmitter at nerve terminals, and transcriptional regulation, and whose modulation is a prime mechanism of synaptic plasticity. Our novel imaging and electrophysiology studies have broadened to include TRP and ASIC cation channels, Ano Ca2+-activated Cl- channels, and others.
Several neurophysiological approaches are used, including preparations of primary neurons from the peripheral and central nervous systems and heterologous systems in which channels, receptors, and signaling molecules are transfected into mammalian tissue-culture cells. Our work is greatly enhanced by the ability to manipulate expression of exogenous genes, or to “knock down” endogenous ones, using transgenic mice. The experimental techniques include patch-clamp electrophysiology, cutting-edge molecular biology, advanced imaging techniques such as total internal reflection fluorescence (TIRF) microscopy, Förster resonance energy transfer (FRET), confocal and “super-resolution” microscopy, molecular and cellular modeling, biophysical chemical analysis of channel complexes and structural biology.
Our recent discoveries include findings that ion channels, receptors and signaling proteins are clustered into discrete “nanodomains” that orchestrate directed signals in ganglia and brain, nanodomains which we have visualized using visible light at 5 nm resolution via STORM “nanoscopy.”. Such nanodomain complexes of proteins direct neuronal excitability in distinct and purposeful ways, guide transcriptional expression, and underlie plasticity, cognition and circuit development in brain. We have carefully mapped out the multiple domains of KCNQ ion channels involved in interactions with phosphatidylinositol 4,5-bisphosphate (PIP2), a lipid signaling molecule of critical importance. Moving into structural biological techniques of X-ray crystallography and NMR spectroscopy and biophysical chemical analysis, we have obtained the structure of Ca2+/calmodulin bound to a KCNQ channel, yielding insights into how diverse intracellular signals interact on a molecular level. Finally, our multiple translational neuroscience projects have unveiled novel therapeutic interventions that seem likely to prevent epilepsy, cognitive dysfunction and mood disorders after traumatic brain injury and stroke, and novel approaches to prevent epilepsy from developing, or new approaches to counter-acting this disorder in people.
Carver, C.C. and M.S. Shapiro (2018). Divergent region-specific regulation of neuronal excitability by muscarinic Gq-coupled receptors via M-type K+ channels and TRPC cation channels in the hippocampus (Submitted to Neuron).
Choveau, F., Bierbower, S.M, De la Rosa, V.J., Hernandez, C., C., and M. S. Shapiro (2018). Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates KCNQ3 K+ channels through multiple sites of action. (submitted to Journal of General Physiology).
Zhang, J., Carver, C.C., Choveau F., and M.S. Shapiro. (2016). Clustering and functional coupling of diverse ion channels and signaling proteins revealed by super-resolution STORM microscopy in neurons. Neuron. Oct 19;92(2):461-478.
Bierbower, S.M., Choveau, F., Lechleiter, J.D. and M.S. Shapiro (2014). Augmentation of M-type (KCNQ) potassium channels reduces stroke-induced brain injury Journal of Neuroscience. 35:2101-2111.
Bierbower, S.M. and M.S. Shapiro (2013). Förster resonance energy transfer-based imaging at the cell surface of live cells. Methods Mol. Biol. 998:209-16.
Zhang, J. and M. S. Shapiro (2012). Activity-dependent Transcriptional Regulation of M-type K+ Channels by AKAP79/150-mediated NFAT Actions. Neuron 76:1133-46.
Choveau, F.S., Hernandez, C.C., Bierbower, S.M. and M. S. Shapiro (2012). Pore determinants of KCNQ3 K+ current expression. Biophysical Journal 102:2489-2498.
Choveau, F.S., Bierbower, S.M. and M. S. Shapiro (2012). Pore helix-S6 interactions are critical in governing current amplitudes of KCNQ3 K+ Channels. Biophysical Journal 102: 2499-2509.