The University of Texas Health Science Center - Department of Physiology

Education

B.A., Physics, University of Chicago, 1984
Ph.D., Physiology, Rush University Medical Center, 1991

Research

Physiology and regulation of ion channels of excitable cells.

What are ion channels? - The nervous system is a vast array of connected cells, called neurons, which are responsible for organizing and directing the activities of all animals. The heart is composed of cardiomyocytes that are electrically connected to control and sustain the cardiovascular system. These neurons and cardiomyocytes work by using a combination of electrical and chemical signals.

The electrical signals do not use electrons but rather the charged salt atoms (ions) of body fluids that are our heritage of evolving from the sea. The movement of ions in and out of cells is mediated by a class of proteins called ion channels, macromolecular pores that span the lipid membranes of cells, and selectively permit the passage of particular ions. The ionic currents carried by ion channels underlie the basic electrical signals that allow the nervous system to function. We are interested in the physiology and regulation of these ion channel proteins. With the evolution of increasingly sophisticated electrical and computer technologies, highly-sensitive techniques have been developed to directly observe these very tiny electric currents in living cells. In particular, the patch-clamp technique allows us to electrically record the ionic currents from individual cells, or even from individual ion channel molecules, and these minute signals are then studied using computers.

Many signaling pathways modulate ion channels.
Since ion channels are so critical to brain and heart function, a vast system of neurotransmitters and hormones regulate the physiology of excitable cells by acting on specific receptors in the cell membrane. The intracellular actions of these receptors are often mediated by a ubiquitous family of signal-transducing proteins called G proteins, biological molecular switches that transduce the extracellular presence of a neurotransmitter into the modulation of some intracellular target. In neurons and cardiomyocytes, as well as many other types of cells, these G protein-mediated signaling pathways frequently act on ion channels. Our laboratory focuses on these G-protein signaling pathways that act on two important types of ion channels. The first makes an important neuronal K⁺current called the M current, called such because it is strongly modulated by muscarinic acetylcholine agonists. The second are Ca²⁺ channels, whose currents drive exocytosis and the release of neurotransmitter at nerve terminals. By modulating Ca²⁺ channel activity, synaptic transmission is thus directly regulated. Modulation of the M-type K⁺ current plays a strong role in regulating the overall excitability of neurons, and on the firing of action potentials. Several different receptors act via G proteins to modulate the M current and Ca²⁺ currents, including muscarinic acetylcholine, angiotensin II, bradykinin and purinergic receptors. We know that these G-protein pathways use intracellular second messengers, and that the intracellular mechanisms they use are often distinct. One goal of the lab is to elucidate the mechanisms of this receptor-specificity of action.

The genes underlying "M-type" currents have been identified, and now know that several different subunits of the KCNQ (Kv7) K⁺ channel family, consisting of KCNQ1-5 (Kv7.1-7.5), come together to form the M-type K⁺ channels. Ca²⁺ channels are composed of pore-forming α-subunits, along with auxiliary β, α2δ and γ subunits. With these K⁺ and Ca²⁺ channel clones, we reconstitute modulatory pathways acting on the channels in a heterologous mammalian expression system. Using this reconstituted system, as well as preparations of primary sympathetic neurons, this lab seeks to use the biophysical technique of patch clamp electrophysiology, along with techniques of molecular biology and biochemistry, to probe the molecular mechanism of G-protein modulation of the M current. Intracellular Ca²⁺ ions also play a role in modulation of the channels, and we also use the technique of Ca²⁺ imaging, using fluorescent probes and very sensitive cameras, to directly observe intracellular Ca²⁺ in individual cells and determine how Ca²⁺and other putative second messengers, interact to regulate neuronal electrical signaling.

Using the patch clamp technique, we can record the currents through individual ion channel proteins. We have recently analyzed Kv7 (KCNQ) channels at the single-channel level, and discovered that they have dramatically different open probabilities.

Shown above are recordings from cell-attached patches containing a single Kv7.2, 7.3, 7.4 or 7.5 channel using our CHO cell heterologous expression system. Openings are upward. We are currently investigating the structural mechanisms underlying the very different activities of Kv7 (KCNQ) channels, using patch-clamp and biochemical and molecular biological techniques.

Many channels are regulated by the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP₂), including M-type K⁺ channels and N-type Ca²⁺ channels. Some receptors coupled to the G_q/11 family of G proteins can deplete the PIP₂ abundance in the membrane by strong activation of phospholipase C (PLC), thus suppressing the channels. Other G_q/11-coupled receptors do not deplete PIP₂ and we hypothesize that they stimulate PIP₂ synthesis at the same time as PIP₂ hydrolysis, compensating for any PIP₂ depletion that might occur. Shown below is this receptor-specific scenario in sympathetic neurons.

Stimulation of the M₁ muscarinic receptor activates PLCβ, which hydrolyzes much PIP₂, depleting its abundance and inhibiting the N-type Ca²⁺ channel. Intracellular Ca²⁺ signals are shown not produced because the IP₃ receptor is too far away from the M₁ receptor. Stimulation of the B2 bradykinin receptor also hydrolyzes much PIP₂, but its co-localization with the IP₃ receptor permits Ca²⁺ signals, which stimulates PIP₂ synthesis via neuronal calcium sensor-1 (NCS-1)-mediated stimulation of PI4-kinase. We continue to investigate the molecular mechanism by which PIP₂ interacts with K⁺ and Ca²⁺ channels, and the molecules involved in receptor-specific lipid signals.

With the combined use of biophysics, molecular biology. biochemistry and single-cell imaging, this lab works toward the identification of the relevant signaling molecules in modulation of ion channels and the understanding of the precise mechanisms they use.

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