Regulation of Ca currents and ECC by CaV1.1 voltage-sensors

Voltage-gated calcium channels control numerous important functions of excitable cells, like muscle contraction, hormone and neurotransmitter secretion, and activity-dependent gene regulation. To activate calcium currents in response to membrane depolarization calcium channels comprise four voltage-sensing domains symmetrically arranged around a common channel pore. In mammals, the four voltage-sensing domains of a calcium channels are structurally and functionally distinct from each other. In the skeletal muscle channel isoform, the voltage-sensing domains differentially regulate two separate functions: calcium influx through the channel pore and calcium release from cytoplasmic stores, i.e. excitation-contraction (EC) coupling. However, which of the four voltage-sensing domains regulate which of the two functions, and what are the molecular mechanisms underlying their specific activation properties is not known. Here we propose experiments to elucidate how the concerted action of the four voltage-sensing domains regulates opening and closing of the channel, and to address the long-standing conundrum as to how the skeletal muscle calcium channel can activate two functions, channel gating and EC coupling, simultaneously, but at distinct voltages and with distinct kinetics. We hypothesize that voltage-sensing domains I and IV control channel gating, whereas domains II and/or VSD III control EC coupling, and that specific ionic interactions between oppositely charged amino acids in each voltage-sensing domain determine its unique voltage-sensing properties and activation of its corresponding function. We will address these questions with three complementary approaches: (1) Structure-guided site-directed mutagenesis and functional analysis in the native environment of cultured skeletal muscle cells. (2) Computer-based molecular dynamics simulation of the channel structure in its resting and activated states. (3) High-resolution functional and structural analysis of channel chimeras comprised of a homo-tetrameric bacterial channel carrying voltage-sensing domains of the mammalian channel. This structure-function analysis addresses prominent biophysical problems regarding the mechanisms of voltage-sensing and regulation of ion channels, as well as the mechanism of skeletal muscle EC-coupling. We strive to generate the first in silico structure model of a functional mammalian (hetero-tetrameric) voltage-gated ion channel, and to create an innovative chimeric channel model system for high fidelity structural and functional analysis of individual VSDs.

Differential regulation of Ca currents and EC-coupling by CaV1.1 voltage-sensors In this FWF project, the Innsbruck physiologists led by Prof. Bernhard Flucher have deciphered a key mechanism by which the contraction of our voluntary muscles is controlled. The contraction of our skeletal muscles is voluntarily activated by the motoric nervous system. An electrical signal reaches the muscle. In the muscle cell, the rapid change in the concentration of calcium ions then leads to contraction. The higher the calcium concentration, the stronger the muscle contraction. How is the electrical signal at the cell surface translated into the increase in calcium concentration inside the cell? Although all molecules essential to this process are now known, the mechanism of this signal transduction process -called excitation-contraction coupling- still not understood. The change in voltage at the cell membrane is sensed by a voltage-activated calcium channel, whose activation initiates the contraction. In cardiac muscle and the smooth muscles of internal organs, activation of the calcium channel leads to the opening of its channel pore, allowing calcium to flow in from the extracellular space. This is not the case in skeletal muscle, where all the calcium required for contraction is released from internal stores. Here, the calcium channel activates the opening of a calcium release channel inside the muscle cell. Thus, the voltage-activated calcium channel functions merely as a voltage sensor. From the protein structure of the calcium channel, we know that this modular membrane protein has a total of four voltage sensors arranged around a common channel pore. It was previously unknown whether all of these are involved in activating contraction. A division of functions among the four voltage sensors is suggested by the fact that muscle contraction is activated very rapidly with small changes in voltage, while the opening of the channel pore occurs slowly and only with significantly larger changes in voltage. In the present project, the Innsbruck researchers combined state-of-the-art molecular genetic, microscopic, and electrophysiological methods with computer-assisted structural analysis to answer this question. The research results clearly demonstrate that a single, fast-acting and sensitive voltage sensor is responsible for controlling contraction. However, all four voltage sensors are required to open the channel pore, and the speed and sensitivity of this process are limited by one slow and one relatively insensitive voltage sensor. These new findings represent an important advance in muscle physiology and provide fascinating insights into the structure and function of an important group of proteins, the voltage-activated calcium channels.