Metabolic regulation of calcium channels

• Endothelium


  Single layer of cells lining the intima of blood vessels, influences multiple important physiological processes
• Endothelium-derived vasoconstrictors


  • Endothelin-1
  • Thromboxane A2
• Endothelium-derived vasodilators


  • Nitric oxide (NO)
  • Prostacyclin (PGI2)
  • Endothelium-derived hyperpolarizing factor (EDHF)
• Endothelin-1


  Binds to ETA receptors on vascular smooth muscle cells, activates a phospholipase C-protein kinase C driven pathway that ultimately elevates intracellular calcium levels to trigger smooth muscle contraction
• Nitric oxide (NO)


  Produced by endothelial nitric oxide synthase (eNOS), stimulates vascular smooth muscle guanylyl cyclase, which converts GTP to cyclic GMP (cGMP), activates protein kinase G to promote closure of vascular smooth muscle membrane voltage gated calcium channels and promote calcium uptake by sarcoplasmic reticulum calcium-ATPases (SERCA), reducing intracellular calcium levels and preventing calcium-induced smooth muscle contraction
• Prostacyclin (PGI2)


  Acts by activating adenylyl cyclase, causing increased conversion of ATP to cyclic AMP (cAMP), after which cAMP activates protein kinase A (PKA), which promotes sarcoplasmic reticulum calcium uptake and inhibits myosin light chain kinase via phosphorylation, leading to smooth muscle relaxation and vasodilation
• Endothelium-derived hyperpolarizing factor (EDHF)


  Non-NO, non-PGI2 inducer of vasodilation that acts on vascular beds by increasing potassium conductance through opening of endothelial and smooth muscle potassium channels, leading to cell membrane hyperpolarization and vasodilation

  
• Categories of calcium-activated potassium channels

  • Small/intermediate conductance (SK/IK)
  • Large conductance (BK)

  
• Subtypes of SK/IK channels

  • SK1 (KCa2.1)
  • SK2 (KCa2.2)
  • SK3 (KCa2.3)
  • SK4 (KCa3.1)
• SK/IK channels


  • Exhibit lower unitary conductance (5-15 pS) compared to BK channels (100-300 pS)
• KCa2.3 (SK3) and KCa3.1 (IK)

  Particularly abundant in endothelial cells
• BK channels have been definitively identified in vascular smooth muscle, but their presence in endothelial cells is more complicated</b>

  
• SK channel structure


  Tetrameric assembly, each alpha subunit consists of 6 transmembrane domains with a pore-forming p-loop between S5 and S6 domains, N and C terminal domains are cytosolic, calmodulin binding domains at C-terminus

  
• SK channel activation is dependent on binding of calcium/calmodulin complex to the C-terminus of the alpha subunits

  
• SK channels


  • Resemble voltage-gated potassium channels
  • Tetrameric assembly
  • Length of ~95 angstroms
  • Width of ~120 angstroms in the plane of the plasma membrane

  
• SK alpha subunits


  Encoded by KCNN1, KCNN2, and KCNN3 genes for SK1, SK2, and SK3 respectively
• SK alpha subunits


  • Consist of 6 hydrophobic transmembrane alpha helical domains (S1-S6)
  • Have a pore forming p-loop between S5 and S6 that governs potassium selectivity
  • Have N and C terminal domains that are cytosolic
  • Have calmodulin binding domains at the proximal C terminal end of S6

  
• SK and IK channels

  Are voltage-independent, regulated by calcium through a calmodulin-dependent mechanism

  
• SK channels are located in close proximity to intracellular calcium stores and cell membrane calcium channels

  
• SK channels will not inactivate at negative membrane potentials, facilitating a hyperpolarization response

  
• Calmodulin binding to SK channels

  • From 2 to 8 calmodulin molecules can bind to one SK channel
  • Only one calmodulin can bind to each SK subunit, resulting in a maximum of 4 bound calmodulins per tetramer

  
• Calcium binding to the N-lobes of calmodulin triggers a structural change in the bound calmodulin-SK channel-calmodulin-binding-domain conformation, leading to channel opening and potassium flow

  
• Endothelium-dependent hyperpolarization


  Involves activation of SK and IK channels in the endothelium

  
• Pharmacological and genetic studies on SK/IK channels

  1. Application of selective KCa3.1 inhibitors TRAM-34 and TRAM-39 abolish IK-mediated potassium currents and block nitric oxide and prostacyclin independent vasodilation

  2. Application of selective SK/IK channel activator NS309 increases endothelial SK/IK currents and promotes arteriolar vasodilation

  3. Deletion of IK or SK3 channel genes in mice impairs endothelial hyperpolarization and vasodilation

  4. Overexpression of SK3 in transgenic mice increases KCa currents and reduces myogenic tone

  
• EDHF


  A NO-independent, PGI2-independent vasodilator that acts through activation of SK and IK channels

  
• EDHF signaling pathway


  1. Endothelial agonists like acetylcholine, bradykinin, and substance P bind to receptors on endothelial cells, mobilize calcium release, and potentially induce EDHF synthesis

  2. Elevated intracellular calcium and EDHF stimulate calcium-activated potassium channels (SK and IK) in endothelial cells

  3. Potassium efflux through endothelial SK/IK channels causes endothelial hyperpolarization, which is conducted to vascular smooth muscle via gap junctions or directly stimulates smooth muscle hyperpolarization

  4. Smooth muscle hyperpolarization closes voltage-gated calcium channels, lowering intracellular calcium and promoting vasodilation

  
• The specific identity of EDHF remains unclear, with several potential candidates proposed
• Potassium released through SK channels can itself trigger endothelial-dependent vascular smooth muscle relaxation, so "EDHF" may simply be potassium

  
• Endothelial hyperpolarization


  Leads to closure of voltage-gated calcium channels on vascular smooth muscle cells, lowering intracellular calcium levels and inhibiting vasoconstriction, promoting vasodilation
• EDHF is a critical component of the framework for endothelial hyperpolarization
• There is uncertainty regarding the specific place of EDHF in these pathways, contributing to controversy surrounding its identity
• EDHF


  If it activates SK channels through increasing intracellular calcium, then molecules like EETs are strong candidates for EDHF because they can stimulate cell membrane TRPV4 channels that mediate calcium influx

  
• EDHF


  Potassium released through SK channels can itself trigger endothelial-dependent vascular smooth muscle relaxation, so it is possible that "EDHF" is simply potassium

  
• Calcium


  • The most important ionic regulator of SK channels
  • Co-localization of SK channels with L-type voltage gated cell membrane calcium channels like Cav1.3 and Cav1.2
  • Experimental manipulation of intracellular calcium affects SK channel trafficking

  
• Other divalent cations


  • Can bind to charged inner pore residues of the SK channel S6 transmembrane domain, which are important regulators of SK channel sensitivity and open probability

  
• Nitric oxide


  Inhibits EDHF-SK/IK channel induced vasodilation
• Activation of SK3 and IK channels

  May promote endothelial nitric oxide synthase activation

  
• The interaction between nitric oxide and EDHF is an unresolved matter, and further work needs to be done to better characterize their relationship
• Protein kinase C


  • Can modulate SK channel activity
  • Phosphorylation of SK channel-bound calmodulin at the threonine 80 residue produces an almost 5-fold reduction in SK channel calcium sensitivity, leading to channel inhibition
  • Protein phosphatase 2A dephosphorylation of SK channel-bound calmodulin reverses the inhibitory effects of protein kinase C

  
• Most of the experiments on protein kinase C regulation of SK channels have been performed on SK2 and SK3 channels, so verifying these findings for SK1 and IK channels is needed