THE STRUCTURE, FUNCTION AND MODULATION OF VOLTAGE-GATED CALCIUM CHANNELS

Voltage-gated calcium channels are important therapeutic targets

In nerve, muscle and secretory cells, voltage-gated calcium channels transduce membrane

electrical signals into cytosolic calcium signals that drive a variety of cellular processes, including neuroransmitter

release, muscle contraction and gene expression. Calcium channels trigger these processes by opening in

response to electrical stimulation, thus allowing calcium ions to flow into the cell where they act as signaling

molecules that are detected by calcium binding proteins.

In humans, the dysregulation of calcium channels underlies life-threatening cardiac arrhythmias,

neurological disorders including epilepsy, ataxia and migraine headaches, muscular disorders such as

hypokalemic periodic paralysis and malignant hyperthermia, and congenital stationary night blindness. We are

using a broad combination of approaches to develop an in-depth understanding of how the structure of the

calcium channel is related to its function, and how these channels are modulated by intracellular signaling

molecules, calium channel blockers, venoms, toxins, and engineered proteins.

Our long-term goal is to develop novel therapeutic strategies to treat human disorders that are either directly

linked to defective calcium channels or to signaling pathways that lie upstream or downstream of these

channels. We are pursuing two projects to accomplish this goal:

Calcium channel gating is modulated by intracellular calcium

Ion channel gating is a complex process involving numerous structural determinants. Calcium-dependent

inactivation is one form of gating that was first described in 1978, but clues regarding the mechanism by which

this important form of gating occurs was not described until very recently. In 1999, we found that the

ubiquitous calcium signaling molecule, calmodulin, is anchored to the channel in the absence and presence of

calcium. Membrane depolarization causes the calcium channel to open, resulting in an influx of calcium.

Calcium ions bind to the anchored calmodulin, the Ca/calmodulin complex associates with a site on the

C-terminus of the channel, and the resulting conformational change induces channel inactivation. An EF-hand

motif that is crucial for calcium-dependent inactivation lies upstream of the Ca/calmodulin binding site, yet this

segment does not appear to bind calcium. This is surprising, given that EF-hands typically do bind calcium.

Therefore, it is likely this segment functions as a signal transducer that couples calmodulin binding to channel

gating. We are using a number of molecular approaches including yeast 2-hybrid, site-directed mutagenesis,

Fluorescence Resonance Energy Transfer (FRET) and patch-clamp electrophysiology to investigate how

Ca/calmodulin binding is coupled to the channel's inactivation-gating machinery, and what role the EF-hand

motif plays in transducing this signal.

Drugs and toxins modulate calcium channel gating by inducing structural rearrangements in the pore

L-type calcium channels, one subset of the calcium channel superfamily, are the target proteins for a

number of drugs including the dihydropyridines (DHPs). DHPs are an important class of drugs, used extensively

in the treatment of angina, hypertension and stroke. The mechanism by which DHPs modulate calcium

channel behavior is not known. We have found that the DHP receptor site is allosterically coupled to the

channel's selectivity filter, a region in the pore that can bind calcium witha high affinity, thus enabling the

channel to select calcium (and exclude other ions) as its permeant ion. This finding is intriguing, given that ion

channel gating is frequently linked to dynamic rearrangements in the pores of various ion channels. We

hypothesize that the conformational changes that occur upon DHP binding are transduced to the pore where

gating is altered and that, by studying the allosteric interactions between calcium and DHP binding, we are

actually studying conformational changes essential for coupling DHP binding to gating.

Toxins from the venoms of a wide range of organisms bind to voltage-gated calcium channels. We have identified a subset of toxins that function as allosteric modulators of DHP binding. We are using these toxins as molecular probes to study the structural rearrangements that link DHP binding to changes in channel gating. By identifying the binding sites for the toxins, and simultaneously developing structural models of the channel on the computer, one can generate a topographical map of the channel. This topographical map will be used to identify pharmacological "hot spots" on the channel that can be targeted by new classes of drugs. We are addressing this project using several techniques, including

whole-cell patch-clamp electrophysiology, site-directed mutagenesis, homology modeling and radioligand binding.