1. Voltage-Gated Calcium Channels Daniel Blackman, Zhihui Zhou, Thomas Arnold

2. 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)

3. Physiology of Voltage-Gated Ca2+ Channels Image taken from Caterall 2011

6. Images taken from Caterall 2011

8. Ca v 1 channel Excitation-contraction coupling Excitation-transcription coupling Excitation-secretion coupling

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

10. 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/

11. Regulation of excitation-contraction coupling PKA phosphorylation and its anchoring via a kinase anchoring protein (APAK). An autoinhibited Ca 2+ channel complex with noncovalently bound distal carboxyl- terminus. Ca 2+ / calmodulin-dependent inactivation Image taken from Caterall 2011

12. Excitation-transcription coupling Calmodulin binds to the proximal caboxy- terminal domain, the Ca 2+ /calmodulin complex moves to the nucleus The distal carboxy-terminal domain is regulated by Ca 2+ in neurons. Image taken from Caterall 2011

13. 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 Ca v 1 channel, such as Ca v 1.3, Ca v 1.4.

14. Cav2 Channels http://physrev.physiology.org/content/90/4/1461

15. Image taken from Caterall 2011

16. Cav2 specific information Initiate fast release of glutamate, GABA, and acetylcholine SNARE proteins G Protein subunits are responsible for modulation Additional binding proteins

17. Ca v 3 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

18. 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”

20. 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)

31. 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

33. Question? Ca 2+ and Na + own nearly identical diameters (2A) The extracellular concentration of Na + is 70-fold higher than Ca 2+ The conductance of Na + is more than 500-fold lower than Ca 2+ via Ca v channel How the Ca v channel keeps the high selectivity of Ca 2+ ?

34. Selectivity filter Na v Ab: 175 TLESWSM 181, outward sodium current Ca v Ab: 175 TLDDWSD 181, inward calcium current

35. No significant alteration in backbone structure between Na v Ab and Ca v Ab------the selectivity is mainly determined by the side chains.

36.  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 T175  The bound Ca2+ ion is continuously stabilized in a fully hydrated state through the pore.

37.  D178 VS S178: Site 1 Over 100-fold change in P Ca :P Na. D178: forms the first hydrated Ca 2+ - binding site S178: blocks the conduction of Ca 2+ by directly binding Ca 2+ and displacing the hydration shell

38.  D177 VS E177: Site 2 5.5-fold change in P Ca :P Na. D177: interacts with Ca 2+ E177: swings away from the selectivity filter

39.  D181 VS N181 VS M181: Site 1 4- to 5-fold change in P Ca :P Na. 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 Ca 2+ tightly bound at Site1.

40.  Binding forces: Site 2> Site 1 > Site 3  Ca 2+ can’t occupy adjacent sites simultaneously due to electrostatic repulsive interactions.  High extracellular concentration of Ca 2+ and weak binding of Ca 2+ to Site 3 generate a unidirectional flux of Ca 2+.

41. Direct Recording and molecular identity of the calcium channel of primary cilia Daniel Blackman, Zhihui Zhou, Thomas Arnold

42. Primary Cilia Specialized compartments Calcium signaling Hedgehog pathways Human retina pigmented epithelium cells tagged with GFP Image taken from DeCaen et al

45. 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

46. Image taken from DeCaen et al

47. Conclusion No Ca2+ current with just PKD1L1 (current observed with PKD2L1) Only with both PKD1L1 and PKD2L1 was current observed matching human