Voltage-Gated Calcium Channels

Daniel Blackman, Zhihui Zhou, Thomas Arnold

Calcium Ion Channel Family

• Cav1 = initiate contraction, secretion, and regulation of gene expression, integration of synaptic input in neurons, and synaptic transmission at ribbon synapses of specialized sensory cells

• Cav2 = synaptic transmission of fast synapses

• Cav3 = important for repetitive or rhythmic firing of Aps (cardiac, thalamic)

Physiology of Voltage-Gated Ca2+ Channels

Image taken from Caterall 2011

Image taken from Caterall 2011

Image taken from Caterall 2011

Images taken from Caterall 2011

Images taken from Caterall 2011

Cav1 channel

• Excitation-contraction coupling• Excitation-transcription coupling• Excitation-secretion coupling

Excitation-contraction coupling

http://www.studyblue.com/notes/note/n/chapter-14-cardiovascular-physiology/deck/9845939

http://pharmaceuticalintelligence.com/2013/09/08/the-centrality-of-ca2-signaling-and-cytoskeleton-involving-calmodulin-kinases-and-ryanodine-receptors-in-cardiac-failure-arterial-smooth-muscle-post-ischemic-arrhythmia-similarities-and-differen/

Regulation of excitation-contraction coupling

• PKA phosphorylation and its anchoring via a kinase anchoring protein (APAK).

• An autoinhibited Ca2+ channel complex with noncovalently bound distal carboxyl-terminus.

• Ca2+/ calmodulin-dependent inactivation

Image taken from Caterall 2011

Excitation-transcription coupling• Calmodulin binds to the proximal caboxy-

terminal domain, the Ca2+ /calmodulin complex moves to the nucleus

• The distal carboxy-terminal domain is regulated by Ca2+ in neurons.

Image taken from Caterall 2011

Excitation-secretion coupling

• Initialization of the secretion of hormones from endocrine cells and release of neurotransmitters.

• The distal carboxy-terminal domain plays an autoregulatory role in some Cav 1 channel, such as Cav1.3, Cav1.4.

Cav2 Channels

http://physrev.physiology.org/content/90/4/1461

Image taken from Caterall 2011

Cav2 specific information

Initiate fast release of glutamate, GABA, and acetylcholine

SNARE proteins G Protein subunits are responsible for modulation Additional binding proteins

Cav3 Channels

• Molecular structure:• Negative potential activation (fast inactivation)• Similar to Cav1 and 2 by 25%

• Functional• Present in rhythmic structures:

• SA node (pacemaker), relay neurons of thalamus (sleep), adrenal cortex (aldosterone)• Mutations can cause absence epilepsy (sleep-like state)

• Regulation• Dopamine & NTMs• Angiotensin II

Conlcusion

• Ca2+ channel complexes – effector and regulator• Four cases effectors enhance Cav1 & Cav2• Skeletal muscle• SNARE proteins• Ca2+/CaM-dependent protein kinase II• RIM

• Common Theme: “Effector Checkpoint”

Point of interest

LOF for Nav1.7 causes anosmia Cav2.2 is involved with the first synapse of the

olfactory system Cacna1b LOF mutation causes an absence of Cav2.2

channels Effects of Lacking Cav2.2 on Olfactory Sensory

Neurons (OSN) in the Main Olfactory Bulb (MOB) and on Vomeronasal Sensory Neurons (VSN) in the Accessory Olfactory Bulb (AOB)

Summary

N-type Cav Channels are main contributors to presynaptic release

MOB and AOB respond differently to Cav2.2 mutation

Presence of unknown Cav channel type in MOB Lack of Cav2.2 does not cause anosmia Mutation causes hyperaggressive behavior

Question?

• Ca2+ and Na+ own nearly identical diameters (2A)• The extracellular concentration of Na+ is 70-fold higher than Ca2+

• The conductance of Na+ is more than 500-fold lower than Ca2+ via Cav channel

How the Cav channel keeps the high selectivity of Ca2+ ?

Selectivity filter

NavAb: 175TLESWSM181, outward sodium current

CavAb: 175TLDDWSD181 , inward calcium current

No significant alteration in backbone structure between NavAb and CavAb------the selectivity is mainly determined by the side chains.

Three Ca2+ - binding sites:

• Site 1: the carboxyl groups of D178.

• Site 2: four carboxylate oxygen atoms from D177 and four backbone carbonyl oxygen atoms from L176.

• Site 3: a plane of four carbonyls from T175The bound Ca2+ ion is continuously

stabilized in a fully hydrated state through the pore.

D178 VS S178:

• Site 1

• Over 100-fold change in PCa:PNa.

• D178: forms the first hydrated Ca2+ - binding site

• S178: blocks the conduction of Ca2+ by directly binding Ca2+ and displacing the hydration shell

D177 VS E177:

• Site 2

• 5.5-fold change in PCa:PNa.

• D177: interacts with Ca2+

• E177: swings away from the selectivity filter

D181 VS N181 VS M181:

• Site 1

• 4- to 5-fold change in PCa:PNa.

• D181 an N181: constrains the side-chain of the D178 ring by forming a hydrogen bond.

• M181: unconstrains the side-chain of the D178 and results in a blocking Ca2+ tightly bound at Site1.

Binding forces:

Site 2> Site 1 > Site 3Ca2+ can’t occupy adjacent sites

simultaneously due to electrostatic repulsive interactions.

High extracellular concentration of Ca2+ and weak binding of Ca2+ to Site 3 generate a unidirectional flux of Ca2+ .

Direct Recording and molecular identity of the

calcium channel of primary cilia

Daniel Blackman, Zhihui Zhou, Thomas Arnold

Primary Cilia

• Specialized compartments• Calcium signaling • Hedgehog

pathways

• Human retina pigmented epithelium cells tagged with GFP

Image taken from DeCaen et al

Image taken from DeCaen et al

Image taken from DeCaen et al

Polycystin proteins (PC/PKD)

• Identified in polycystic kidney disease• Form ion channels at high densities in multiple cell types• Two structural classes (PKD1s and PKD2s)

• Hypothesis: PKD1L1-PKD2L1 heteromultermerize to form calcium-permeant ciliary channels

Image taken from DeCaen et al

Conclusion

• No Ca2+ current with just PKD1L1 (current observed with PKD2L1)

• Only with both PKD1L1 and PKD2L1 was current observed matching human