How do we know what type of membranes we are studying?

Intracellular membranes and their channels must be carefully extracted from the interior of the cell. To obtain heavy sarcoplasmic reticulum membranes enrichedd in RyR2, mice hearts are homogenized and fractionated by differential centrifugation under a sucrose gradient. Marcia Cortés-Gutiérrez has developed simple methods to quickly separate SR membranes with functionaly intact RyR2 channels. Biochemical and electrophysiological analysis reveal that a microsomal fraction can be obtained which contains RyR2 and its anxiliary proteins.

Does the proapototic protein BAX form a pore for cytochrome c translocation?

Bax is a protein normally residing in the cytosol. Upon stimulation, Bax translocates to the outer mitochondrial membrane triggering cytochrome c release and initiating the apoptotic death of the cell. The exact molecular mechanism by which Bax promotes cytochrome c release from the mitochondrial compartment is unknown. One hypothesis is that in response to the apoptotic stimulus, Bax changes its conformation oligomerizing in the outer mitochondrial membrane forming a large pore of sufficient diameter to permit cytochrome c release from the mitochondria. To investigate this possibility, recombinant human Bax is added to a planar lipid bilayer and the electrophysiological activity is recorded. We find that, under oligomerization promoting conditions, Bax forms characteristicaly large pores with high open probabilities. Kris Sheets, a graduate student from the LSU Health Science Center is studying the interactions of Bax pores with cytochome c.

What methods are used to study regulation of RyR2 by lumenal Ca2+?

Most of known and well studied ion channels are surface (plasma membrane) channels. Their study has been possible because of the spectacular development of the patch clamp technique. With this method, a glass pipette can be approached from the exterior and landed on the plasma membrane to record ion channel currents under conditions of voltage control. Intracellular membranes are normally unavailable for the patch clamp technique. The alternative strategy is to extract the intracellualr membranes we are interested in (subcellular fractionantion and differential centrifugation) and then incorporate vesicles containing the channel into an artificial planar lipid bilayer. The technique is one of the first methods developed to study ion channels and is still the best method to study single ion channels from intracellular membranes. Planar lipid bilayers allow full manipulation of ionic and pharmacological conditions around the ion channel. Thus, we study how the open probability of RyR2 incorporated in a planar lipid bilayer is affected when changing the Ca2+ concentration in the face that noramally is exposed to the interior of the SR.

Why are we interested in lumenal regulation of RyR2?

Ryanodine Receptors from cardiac muscle open in response to a small depolariztion-induced entry of Ca2+ ("trigger Ca2+"). Thus, the RyR it is a calcium-sensitive channel. Its opening allows a massive release of Ca2+ stored inside the sarcoplasmic reticulum. Released Ca2+ induces further opening of RyR initiating an intrinsically positive feedback that allows the rapid increase of intracellular Ca2+ concentration and muscle contraction. A molecular mechanism must exist to counter the intrinsic tendency of RyRs to remain open. One of the current hypotheses is that RyR2 close in response to the reduction of Ca2+ in the lumen of the sarcoplasmic reticulum. The sensor for this decrease in Ca2+ would be the intrareticular protein calsequestrin. David Baéz, an undergraduate student of Biochemstry from the Catholic University of Valparaíso is experimentally testing this ideas in his thesis work. Watch David working in the lab: http://ryanodine.camarades.com/

What is the Laboratory of Intracellular Ion Channels?

Intracellular Ion Channels regulate many key cellular functions by controlling the ion flux across different intracellular compartments. Our laboratory is particularly interested in ion channles normally found in membranes of the sarcoplasmic/endoplasmic reticulum. We are currently investigating how the calcium release channel (Ryanodine Receptor, RyR2) from the sarcoplasmic reticulum of cardiac myocytes is regulated by calcium content in the lumenal side of the channel.

Other questions qe want to solve is How cytochrome c normally inside the mitochondria is released to the cytosol initiating the apoptotic death of the cell. We think that Bax, a pro-apoptotic member of the Bcl-2 family, forms a pore through which cytochrome c molecules permeate out of the mitochondria.