Calcium as a Second Messenger

Calcium plays a pivotal role in various cell signaling events, ranging from muscle contraction to apoptosis. Calcium is employed as a second messenger in electrically excitable cells such as neurons or endocrine cells. Non-excitable cells such as blood cells or cells from the immune system also depend on this ion to be readily available.

A typical electropysiological patch-clamp setup as used in the LCMS

The rig consists of a Zeiss microscope with a CCD camera and avideo monitor attached for observation. A pre-amplifier and electrode holder are mounted to a motorized Eppendorf XYZ micromanipulator. The bath chamber itselfis motorized (Luigs & Neumann) and controlled by the Eppendorf micromanipulator. The pre-amplifier is connected to an EPC-9 patch clamp amplifier (HEKA) which, in turn, is controlled by the acquisition program Pulse (HEKA) running on a Mac. In addition, the setup features a multi-wavelength photometer (T.I.L.L. Photonics) and photo-multiplier setup for fluoremetric measurements. The software for these measurements is the charting program X-Chart Standalone (HEKA) running on a Mac via an ITC-16 interface (Instrutech).

Neher's Lab

From left to right:
Erwin Neher, Fred Sigworth, Alain Marty, Bert Sakman, Owen Hamill - Max-Planck-Institute for biophysical Chemistry, 1981.

Nobel Prize for Physiology or Medicine in 1991

Bert Sakman and Erwin Neher in Stockholm, 1991

The Whole-Cell Patch-Clamp Technique

A fine-tipped glass electrode (to the right) filled with solutions mimicking cytosolic conditions is lowered onto an isolated cell (in yellow). A high-resistance seal between the plasma membrane and the pipette tip is formed through gentle suction applied to the pipette. After seal formation the patch enclosed by the pipette tip is broken and provides access to the cell interior. With this, control is gained over the cytosolic milieu and the the electrical potential of the cell. The extracellular milieu can be changed rapidly by microperfusion of the patched cell using a second glass pipette (to the left). Examples of the actual experimental setup while patching mast cells or RBL-1 cells are show in black and white.

Calcium Imaging

The movie shows pseudo-color images of several rat basophilic cells (RBL) loaded with Fura-2. The cells are stimulated with antigen to release calcium into the cytosol. The blue color indicates low levels of calcium. The amount of calcium release is coded by color from yellow to red.

The mother of all cells

A schematic representation of all currently known calcium channel proteins. Voltage-operated channels (VOCs) open and close following membrane voltage changes, whereas receptor-operated channels (ROCs) are activated by extracellular ligands (A). Second-messenger operated channels (SMOCs) can be activated either by G-proteins (G) or by second messengers produced by effector enzymes (E). VOCs can be functionally coupled to ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) either by calcium itself (CICR) or by voltage (DICR). Store-operated channels (SOCs) cause calcium influx by interaction of STIM (S) and CRACM (Orai). SOCs can reliably be activated by IP3-induced depletion of intracellular calcium stores, the endoplasmic reticulum (ER). IP3 production is induced by the receptor (R) stimulation and phospholipase C (PLC) activation. The calcium concentration in the ER is measured via STIM (S) communicating with CRACM in the plasma membrane. Smooth endoplasmic reticulum calcium ATPases (SERCA) and calcium pumps in the plasma membrane maintain the calcium homeostasis of the cell.

Introduction

The Laboratory of Cell and Molecular Signaling was established in July 1998 through a collaborative effort of The Queen's Medical Center and the University of Hawaii. The LCMS is headed by Andrea Fleig, Ph.D. and Reinhold Penner, M.D. Ph.D. and located within the University of Hawaii Tower on the campus of The Queen's Medical Center.

The Science

Practically every cell type uses calcium to control a plethora of vital cellular responses. The LCMS focuses on various aspects of calcium signaling in electrically excitable as well as non-excitable cells using electrophysiology, fluorimetry, flow cytometry, molecular biology, and pharmacology.

Our approach to study signaling events is mainly at the single-cell level. The patch-clamp technique has revolutionized cellular physiology since its introduction in the early 1980's (it earned its developers Erwin Neher and Bert Sakmann the Nobel Prize for Physiology or Medicine in 1991).

This electrophysiological technique allows investigators to assess important parameters such as ionic currents of a whole cell or even down to the molecular level of single ion channels, cell membrane potential, secretory mechanisms resolving even the fusion of a single secretory vesicle.

When combined with microfluorimetry and digital imaging techniques, the method can be used to measure the spatio-temporal aspects of intracellular levels and distribution of ions such as calcium, sodium, or chloride, changes in pH, and even production and movement of signaling molecules.

In the LCMS, these techniques are complemented by additional methods from areas such as biochemistry and genetics. Together, these techniques and tools provide the methodological framework to investigate signaling pathways at the cellular or molecular level in native systems (cell lines, primary and tissue cultures, brain slice preparations), in genetically modified systems (transient and/or stable transfections of cells), and in primary cells derived from transgenic animals.

General Overview

The laboratory’s work has concentrated on various aspects of calcium signaling in electrically excitable as well as non-excitable cells. Its mainstay is the elucidation of calcium signaling and Calcium-Release-Activated-Calcium channels (CRAC) in immune responses. This line of work has been expanded to microglia, the immune cells of the brain, which play an important role in neurodegenerative diseases. Another focus investigates the regulation of inositol-signaling pathways. We have also started to correlate calcium signaling and cell proliferation, which may lead to identify a central role of this ion in apoptotic cell death and tumorigenesis. Recently, we have initiated a project concerning the physiological function of four novel ion channels of the Transient-Receptor Potential (TRP)-family involved in calcium homeostasis, specifically TRPM2, TRPM4, TRPM5 and TRPM7.

Current Projects

Functional expression of novel proteins

Our laboratory has been collaborating with researchers at University of Washington, Harvard Medical School and University of Mainz to identify new ion channels. Several human DNA sequences have been cloned and transformed into cells so that they over-express the proteins encoded by these genes on the surface of the cell. The functional characterization of these proteins resulted in the discovery of four novel ion channels of the TRP-family.

TRPM2 (LTRPC2) is a calcium-permeable cation channel that has a novel gating mechanism mediated by ADP-ribose and facilitated by calcium.

TRPM4 (LTRPC4) is the first calcium-activated cation channel whose molecular structure has been identified.

TRPM5 (LTRPC5) is the second calcium-activated cation channel whose molecular structure has been identified. Unlike TRPM4, TRPM5 is sensitive to the rate at which calcium changes occur rather than to the overall calcium concentration.

TRPM7 (LTRPC7) is a constitutively active divalent ion channel. It is the first ion channel identified to conduct calcium and magnesium equally well. The activity of TRPM7 is regulated by intracellular Mg•ATP and magnesium concentrations. We therefore coined endogenous TRPM7-like currents MagNuM (magnesium-nucleotide regulated metal current).

CRACM (Orai): The prototypical store-operated calcium-influx pathway Icrac (for "calcium-release activated calcium current") originally was identified in our laboratory in 1992. Since then, we and others have acquired substantial information about ICRAC’s physiological and clinical importance. A recent finding established that a protein called stromal interaction molecule (STIM1) acts as the sensor for store calcium content and is required for functional store-operated calcium influx. This finding was followed by the identification of another essential protein for store-operated calcium entry called CRAC Modulator 1 (CRACM1) by our group and Orai1 by an independent study from the Rao and Lewis laboratories. While neither STIM1 nor CRACM1 overexpression significantly affect ICRAC, the combined overexpression of STIM1 and CRACM1 greatly amplifies store-operated currents and these currents possess the most defining characteristics of Icrac.

Regulation of store-operated calcium influx

The predominant mechanism for calcium influx in immune cells such as mast cells, lymphocytes and macrophages involves cross-talk between intracellular calcium stores and plasma membrane calcium channels. Calcium influx is triggered by release of calcium from the endoplasmic reticulum stores by an as yet unidentified mechanism. The laboratory is investigating the molecular components and physiological importance of this signal transduction pathway. The goal is to identify the messengers in the signaling cascade at the molecular level and develop pharmacological tools to modulate this ubiquitous signaling mechanism in a therapeutically meaningful manner.

Calcium signaling in the activation process of microglia

Microglia represent approximately 5 - 12 % of the cells in a given brain region, a number that closely resembles that of neurons. Functionally, microglia cells participate in the immune response within the CNS by phagocytosis of bacteria and debris, production of cytokines, and initiation of subsequent growth factor cascades. They are also involved in neurodegeneration under pathological conditions. The laboratory is investigating the electrophysiological characteristics and calcium signaling events in resting and activated microglia cells and the role they play in neurodegenerative diseases.

Calcium signaling in cell growth and proliferation

All cells must choose from three possible fates: live and reproduce, live without reproduction, or die. Cell division, differentiation, senescence, and apoptotic cell death are tightly controlled events in the cell’s life. Deregulation of these processes can lead to pathological conditions, such as malignancy and autoimmune disorders. Despite intensive research on numerous molecular events coordinating cell cycle control and cell division, the role of calcium in the mitotic cell cycle process remains elusive. We have started to address this central question and the results will, for the first time, define the role of calcium channels in controlling cell growth and proliferation.

Regulation of calcium influx by inositol phosphate metabolism

Up- and down-regulation of intracellular calcium levels initiate many calcium-dependent cellular responses. Such intracellular calcium oscillations are observed in many cell types following receptor stimulation and exhibit characteristic patterns in a given cell type. They are initiated by inositol 1,4,5-trisphosphate (InsP3), a second messenger that triggers calcium release from intracellular stores and subsequent activation of store-operated calcium influx, CRAC. Cellular InsP3 is metabolized within seconds by two enzymes: 5-phosphatase [yielding Ins(1,4)P2] and 3-kinase, which generates inositol 1,3,4,5-tetrakisphosphate (InsP4). We presently investigate the effects of InsP4 in enhancing and shaping the time course of calcium influx. This line of research may reveal feedback mechanisms between inositol phosphate and calcium pathways, ultimately determining the regulation of cellular calcium oscillations.


Patch-Clamp Movie


LCMS Patch-Clamp Movie. Calcium Influx in RBL cells