Consideraciones éticas

Extracellular and internal store Ca2+ _{are primary sources of signal Ca}2+ 21_{. These} two components of the signal event are introduced into the cytosol through different

storage organelles22_{. Various stimuli, including changes in membrane potential and} ligand binding, activate the opening of these channels spurring cellular changes induced by the accumulation of cytosolic Ca2+21_{. In the following subsections, we will discuss} the channels responsible for mobilizing Ca2+ _{into the cytosol.}

1.2.2.1 Voltage-gated Ca2+ _channels

Voltage-gated Ca2+ _{channels (VGCCs or VOCs) allow Ca}2+ _{into the cell in}

response to voltage changes across the plasma membrane39_{. The diverse Ca}2+

activation currents found in cells can be divided into low voltage currents, with electrical potentials of -60 to -70 mV that are rapidly disabled, and high energy currents, with depolarizing potentials greater than -40 mV and slower inactivation39_{. The high voltage} currents are further categorized based on physical properties and antagonists of the current. L-type currents are large conductance currents and are sensitive to 1,4- dihydropyridine. N-type currents are neuronal and can be inhibited by ω-conotoxin GVIA peptide from cone snail. P/Q-type currents were first isolated in Purkinje neurons and are blocked by ω-agatoxin IVA from spider venom. R-type currents are residual or resistant currents and are inhibited by SNX-482. Low voltage currents are designated T-type currents, or transient currents, and show insensitivity to Ca2+ _{blockers. L-type} and T-type currents can be detected in many cell types such as cardiac, skeletal, and neuronal; however, the remaining types dominate in neurons40,39_.

Structurally, VGCCs are complexes composed of multiple subunits including the

transmembrane α2 and δ disulfide associated subunit, the cytoplasmic β subunit, the

transmembrane γ subunit, and the chief transmembrane Cavα1 subunit that forms the

containing six transmembrane segments (S1-S6). The loop that connects S5 and S6 forms the channel pore. In each domain, S1-S4 forms the gating machinery pairing activity with depolarization. S5 and S6 form the inner lining of the pore. Channel deactivation, regulation, and subunit binding occur at the cytosolic N- and C-terminals and loops I-II, II-III, and III-IV that link the domains of the Cavα140. Ten isoforms of the Cavα1 subunit exist. Due to its importance for channel function, the Cavα1 subunit is used to divide VGCCs into three families: Cav1, Cav2, and Cav339. Cav1.1-Cav1.4 produce L-type currents and are distributed in cardiac, skeletal muscle, endocrine, neuronal, and retinal cells. Cav2.1-Cav2.3 generate P/Q, N, and R-type currents, respectively, and are distributed in nerve terminals and dendrites. Cav3.1-Cav3.3 produce T-type currents and are expressed in cardiac muscle, skeletal muscle, and neurons42_{. The β subunit has four isoforms, and its association with the Ca}

vα1 leads to

increased channel expression and trafficking which increases the Ca2+ _{current. The α2δ} subunit, like the β subunit, increases channel trafficking to the plasma membrane. The γ subunit aides in the modification of the biophysical properties of the Cavα1 subunit39,40.

1.2.2.2 Transient receptor potential channels

Transient receptor potential channels (TRP) are similar in function to VGCCs in that they are voltage sensitive, but only weakly. TRPs are non-selective ion channels

that allow the influx of Ca2+ _{and sodium ions across the plasma membrane into the}

cytosol leading to depolarization43_{. TRPs and their isoforms are divided into the} following six subfamilies based on sequence similarities: TRPC, TRPML, TRPV, TRPM, TRPA, and TRPP. The modeled channel structure includes six transmembrane domains with the ion pore region between transmembrane segments five and six.

TRPs can integrate multiple stimuli that induce activity and signal amplification44_{. These}

activators include ligands such as 2-APB, DAG, Ca2+_{, and Mg}2+_{, receptors such as}

GPCRs, and changes in temperature. Because TRPs are considered signal integrators and amplifiers, they function mainly as cellular sensors and aide in growth cone

guidance43_.

1.2.2.3 Ryanodine receptor and the inositol 1,4,5-triphosphate receptor

The Ca2+ _{released from the ER/SR makes up the bulk of the Ca}2+ _{signal. The}

inositol 1,4,5-triphosphate receptor (IP3R) and the ryanodine receptor (RyR) are the

main Ca2+ _{release channels on the ER/SR membrane. Ca}2+ _{activates both receptors}

through a process called Ca2+_{-induced Ca}2+ _{release (CICR), allowing more Ca}2+ _to discharge from the ER/SR to propagate the Ca2+ _signal25_.

The RyR exists in three isoforms in mammals: RyR1, RyR2, and RyR3. RyR1 and RyR2 are the predominant isoforms found in skeletal and cardiac muscle,

respectively, while RyR3 is dominant in the thalamus, hippocampus, corpus striatum,

and smooth muscle45,46_{. High resolution cryo-electron microscopy structures of the RyR}

show it exists as a homotetramer with a total mass over 2 MDa making it the largest ion channel. The large cytosolic portion of the RyR has a mushroom-like shape with the

rest of the channel embedded in the SR membrane47,48_{. This large cytosolic portion of}

the RyR is where many proteins bind to regulate its function such as FKBP12 (RyR1),

FKBP12.6 (RyR2), calmodulin, CaMKII, S100A proteins, and DHPRs49_{. Ca}2+ _activates

and inhibits RyR1 activity. When Ca2+ _{concentration is ~1µM, the receptor is active,}

and it is deactivated when the Ca2+ _{concentration reaches 1 mM}50_{. Myoplasmic free}

physiological concentration of magnesium is lowered from 1 mmol/L to 0.05 mmol/L, the receptor opens resulting in a steep decrease in SR Ca2+_{. Cytosolic ATP also triggers} the Ca2+ _{release channel activity}51_{. Caffeine exerts a stimulatory effect on RyRs by} enhancing their affinity for Ca2+ _{without disrupting magnesium binding}52_{. 4-chloro-m-} cresol (4-CmC) is a potent agonist of the RyR, having a 10 fold higher sensitivity than

caffeine for inducing SR Ca2+ _{release with both regularly employed to study RyR-}

mediated Ca2+ _{release in healthy and diseased cells}53_{. RyR1 is highly localized in the} terminal cisternae region of the SR membrane facing the t tubules in skeletal muscle cells. The DHPRs, situated on the t tubule membrane, interact directly with RyR1 to

activate Ca2+ _{release when membrane depolarization occurs}54_{. Residues 1,635-2,636}

of RyR1 were shown to interact with the DHPR to mediate Ca2+ _{release and E-C}

coupling55_{. Later, it was discovered that residues 1-1,680 at the N-terminal portion of}

RyR1 facilitate E-C coupling56_{. RyR1 also enriches the activity of the DHPR through a}

backward current57_{. RyR1 and RyR2 are also controlled luminally through interactions}

with calsequestrin, junctin, and triadin58_{. The process linking depolarization to muscle} contraction, E-C coupling, the specialized junction it occurs in, and the associated proteins will be discussed more in section 1.4.1.

The inositol-1,4,5-triphosphate receptor (IP3R) is the major Ca2+ release channel on the ER membrane of non-excitable cells59_{. Its presence in excitable cells servers to} amplify the Ca2+ _{signal generated from depolarization}60_{. The IP3R is a member of a} vast ion channel superfamily61_{. Like RyR, the IP}

3R functions as a tetramer having a large cytosolic domain resembling a mushroom and six transmembrane segments with the Ca2+ _{binding portion homologous to that of the RyR}62_{. Three isoforms of the IP}

exist with IP3R1 being well studied63. IP3 is generated as a second messenger from the

breakdown of phosphoinositol-4,5-bisphosphate (PIP2) by phospholipase C (PLC)

through activation of GPCRs59_{. Channel opening is activated by both Ca}2+ _{and IP3} where IP3 increases the response of the channel to Ca2+ giving it a bell-shaped

response curve similar to RyR5_{. Channel opening allows the release of Ca}2+ _{from the} ER and other internal stores expressing the receptor59_{. Several molecules interact} indirectly or directly with the IP3R triggering its activation. ATP increases IP3-mediated Ca2+ _{release through the IP3R at concentrations of 100 µM}64_{. ATP binds to the}

purinergic receptor P2YR, a GPCR, triggering production of IP3 that binds to the IP3R to release Ca2+ _{from the ER}65,66_.