1. Calcium Channel Blockers
Shaojian (Stacey) Yu, Lusi Wang, Yu (Kylin) Zhang, Diana Leon Tong
PHM142 Fall 2018
Instructor: Dr. J. Henderson
Date: November 13th 2018
GOOD MORNING EVERYONE. Calcium ions are privileged, because they are the second critically important cell signaling molecules.

2. Types of Calcium Channels
Ligand-gated Calcium Channels
2. Voltage-gated Calicum Channels
Calcium channels have two subtypes; there are ligand-gated and voltage-gated calcium channels. Pharmalogoical speaking, voltage-gated calcium channels are more important, so the main focus of our presentation would be on voltage-gated calcium channels.

3. Ligand-gated Calcium Channel
Type
Ligand
Location
Function
IP3 Receptor
IP3
Endoplasmic Reticulum/Sarcoplasmic Reticulum
Release calcium from ER/SR in response to IP3
Ryandodine Receptor
DHP
Calcium-induced calcium release in myocytes
Two-pore Channel
NAADP
Endosomal/Lysosmal Membrane
NAADP-activated calcium transport across endosomal/lysosmal membrane
Cation Channel of sperm
Calcium
Sperm
Non-selective calcium-activated cation channel directing sperm in female reprodctive tract
Store-operated Channel
indirect pathway
Plasma Membrane
Provide calcium signaling to the cytoplasm
First, I will briefly touch base on ligand-gated calcium channel. There are five subtypes based on the different ligands. Their major functions are activation of calcium-dependent enzymes, gene transcription, and muscle contraction.
Striggow et al (1996)

4. Voltage-gated Calcium Channel
Type
Voltage
Associated Subunit
Location
L-type Calcium Channel
High-Voltage-Activated
α2δ, β, γ
Skeletal Muscle, Smooth Muscle, Osteoblasts, Ventricular Myocytes and Dendrites of Cortical Neurons
P-type Calcium Channel
α2δ, β, possibly γ
Purkinje Neurons
N-type Calcium Channel
α2δ, β1,3, 4, possibly γ
Throughout Brain and Peripheral Nervous System
R-Type Calcium Channel
Intermediate-Voltage-Activated
Cerebellar Granule Cells
T-Type Calcium Channel
Low-Voltage-Activated
Single α
Neurons Cells, i.e. pacemaker, bone, thalamus
Let’s move onto voltage-gated calcium channels. There are multiple types of voltage-gated calcium channels, and they can be divided into high, intermediated, and low voltage activated channels based on their voltage sensitivities. Since the most of drugs on the market right now are targeting L-type and T-type related disease, so we will pay our attention to these two types of calcium channels and their blockers.
Gerald (2017)

5. L-Type CCBs: Types and Mechanisms
Amlodipine
Both dihydropyridine and non-dihydropyridine CCBs bind to and inhibit the alpha one subunit of the L- type calcium channel with high affinity.
The general inhibition mechanism is that CCBs bind to the alpha1 subunit in different binding sites to decrease the frequency of opening in response to depolarization, and therefore reduce the calcium ions entering the channel
Zamponi et al (2015)

6. Mechanism of DHPs L-Type CCBs
The subtypes of α1 subunit include α1S (CaV1.1), α1C (CaV1.2), α1D (CaV1.3), and α1F (CaV1.4) expressed in different tissues
CaV1.3 and CaV1.4 exhibit 5-10 fold of lower sensitivity to DHPs than CaV1.2
CaV1.2 inhibits more currents in the working myocardium than in arterial muscles
there are 4 subtypes of alpha1 subunit include ….
Calcium inhibition can be induced by modification of channel gating or within the activation gates of the pore domain channel, in this case, DHP calcium blockers utilizes the 2nd mechanism of inhibition, which is labelled in red circle.
Zamponi et al (2015)

7. L-Type: DHP Selective Vasodilator
Voltaged-depedent block
Higher affinity for inactivated calcium channel conformation
Tissue-slectivity in arterial smooth muscle
At therapeutic dose, vasodilating without affecting cardiac inotropy
Dihydropyridine
ring
DHPs are voltaged-dependent blocks, and they primarily stabilize and induce inactivated channel states
provides these drugs with tissue-selectivity: inactivated channel states are favored in arterial smooth muscle due to their more depolarized resting membrane potential and long lasting depolarizations. This property also explains the potent vasodilating effect of DHPs without affecting the cardiac inotropy at therapeutic doses.
PubChem Compound Database (2005)

8. Clinical Pharmacology
Dihydropyridine (DHPs): primarily used in hypertension
Reduce systemic vascular resistance & arterial pressure
Amlodipine
Longer T1/2 (36 hr) than other CCBs
Slower induction of vasodilator action
Greater vascular/cardiac effect ratios without significant fluid retention
Side effects:
Headache
Edema
Reflex Tachycardia
· too much vasodilation resulting in increased oxygen demand on heart, and the heart needs to beat harder and faster to keep up with the blood supply to the heart.
Amlodipine
Wang et al. (2017)

9. Non-DHP: Phenylalkylamines
Tertiary amines, (+) charged under physiological pH
Verapamil: prototype, clinically used
Methoxyverapamil and desmethoxyverapamil: higher affinity to Ca2+ channels, used in the lab
Three interactions with the Ca2+ channel:
IVS6 conserved residues: e.g. H-bond with the hydroxyl group on Y1463 residue
IIIS6 conservered residues: Y1152, F1164, V1165
Pore region: ionic interaction with Ca2+ binding sites
Let’s move on to Non-DHP blockers. The first type is phenylalkylamines. Although the tertiary amines in the structures are positively charged under physiological pH, a small uncharged portion can still diffuse across the membrane and bind to the transmembrane domains of calcium channels to exert their inhibition effect.
Verapamil is the prototype and the only clinically used phenylalkylamine. Its methoxy and desmoethoxy derivatives have higher affinity to calcium channels, but are only used in the lab.
The binding between PAAs and the calcium channels is stabilized by three interactions, including two hydrophobic interactions with S6 segment of transmembrane domains three and four, and one ionic interaction between the charged amine and the calcium binding site in the pore region.
For example, the oxygen atoms on desmethoxyverapamil can form hydrogen bonds with the proton of the hydroxyl group on the Y1463 residue)
The positive charge on the verapamil can also interact ionically with the Ca2+ binding sites on the pore region and further contribute to binding stability.
(All PAA are said to be state-dependent, as they stabilize the inactivated calcium channels to reduce the total amount of activated channels, thereby limit calcium influx and muscle contraction)
Hockerman et al (1997)

10. Non-DHP: Benzothiazepine
Diltiazem / Cardizem
Prototype benzothiazepine
Only drug of this class being used clinically
Distinctive binding site on alpha1 subunit
bind in between III and IV transmembrane segment (previously shown)
Allosterically linked to phenylalkylamine (-) and DHP site (+)
Both cardio-depressant effect and vasodilatory effect
Negative inotropic effect: decrease contractility of the heart
Negative chronotropic effect: decrease the heart rate
Negative dromotropic effect: decrease conduction velocity within heart, esp. AV node
Treat hypertension, angina and arrhythmias
Hockerman et al (1997)
second type of the Non-DHP drug is benzothiazepine which is an intermediate class between DHP and phenylalkylamine
diltiazem or say cardizem is the prototype and the only drug of this class being used clinically
it distinctively bind to the third and the fourth transmembrane segments of the alpha1 subunit shown on the previous
this drug it has both cardio-depressant effect and vasodilatory effect
it can be used to treat hypertension, angina and arrhymias.
For hypertension, Since it is more cardiac selective. Therefore, it can reduce arterial pressure without producing the same degree of reflex tachycardia as side effect of DHP

11. Clinical use of Non-DHP
As antiarrhythmic drug (Class IV)
Decrease the electrical firing rate of the pacemaker site within the heart
Decrease the force needed by the heart to contract
Decrease the conduction velocity and prolong the repolarization esp. at AV node
block blood re-entry mechanism
As anti-Anginal drug
Decrease oxygen demand of the muscle
Dilate the arterial walls to reduce the pressure
Decrease the ventricular afterload
Lower electrical activity
lower the force generated by the heart to contract
lower the heartbeat rate
Increase oxygen supply to the heart muscle
it works on the Pacemaker sites to decrease electrical firing rate
works on the ventricular muscle to decrease the heart contractility
meanwhile it decrease the conduction velocity and prolong the repolarization phase especially at AV node
all three work together to decrease the tachycardia and bring the heart beat rate to normal
Angina or say chest pain as a symptom for insufficient heart received heart muscle receive less oxygen than usual
As an anti-angina drug, therefore, cardizem has the similar effect, working on the SA node, ventricular myocardium to decrease the working load of the heart muscle thus to decrease the oxygen demand of the heart
meanwhile, it also have the vasodilation effect on the coronary arteries to increase oxygen supply to the heart.
afterall, these two works together to ensure sufficient oxygen to the heart, alleviating the symptom
Systemic vasodilation reduces arterial pressure,
which reduces ventricular afterload (wall stress) : the resistance of the ventricles need to overcome to eject the blood from ventricles chamber
thereby decreasing oxygen demand of the heart muscle
meanwhile, diltiazem and verapamil lower the electrical activity

12. Side effects for benzothiazepine and phenylalkylamine
Excessive bradycardia
Impared conduction
Depressed contractility
Uncommon side effect
Heart failure
especially can NOT co-administer with beta-blocker drug
As a drug, non-DHP also has some common side effects such as excessive bradycardia, impared conduction and depressed contractility.
One thing more importantly, these drug can not be prescribed to patients who also take beta blocker. Since both drugs exhibit the similar effect, co-administration will lead to unexpected heart failure

13. T-type Ca2+ Channel Blocker
Cav3.2 expression: primary sensory afferent neurons, spinal cord
Mediate pain and priprioceptive stimuli
Increased expression in neuropathic pain
Cav3.2-selective T-type Ca2+ channel blockers are state-dependent: selectively treat neuronal hyperactivity
Epilepsy
Chronic pain
e.g. ABT-639, TTA-A2, Ethosuximide
Last but not least, we’ll briefly introduce T-type calcium channel blockers. There are three subtypes of T-type calcium channels, and the subtype Cav3.2 is selectively targeted by pharmacological agents.
These channels are expressed on the membrane of sensory afferent neurons, and they mediate the sensation of pain. The expression of Cav3.2 is often upregulated in patients with neuropathic pain. Since T-type CCBs are also state-dependent, they are more effective in treating conditions related to neuronal hyperactivity, for example epilepsy and chronic pain.
Examples of T-type blockers include ABT-639, TTA-A2 and an epilepsy drug Ethosuximide. Most T-type blockers are still under development, and hopefully will become the next generation of analgesic drugs.
In neuropathic pain rats models, the expression is upregulated, which might be related to post-translational modulations such as glycosylation on the extracellular loops and deubiquitination on the intracellular domains. Both modulations increase the stability of the channel in the membrane thereby increasing channel expression on the membrane
Snutch, Zamponi (2018)

14. Summary
Calcium channels are divided into ligand-gated and voltage-gated calcium channels. Voltage-gated calcium channels are the major target in pharmacology.
Main types of voltage-gated calcium channels are L-type and T-type calcium channels, and most drugs on the market are L-type and T-type calcium blockers.
Calcium channel blockers binds and inhibits alpha 1 subunit of the calcium channel, to decrease the frequency of opening in response to depolarization, thus reducing the Ca2+ from entering the channel
L-type drug
DHP: acts selectively on vascular smooth muscle (for hypertension)
Amlodipine: reduce systemic vascular resistance & arterial pressure. long half-life (36hr); causes reflex tachycardia
Non-DHP: target cardiac and vascular smooth muscles
Phenylalkylamine
Benzothiazepine (diltiazem/cardizem)
exhibit (-) inotropic, (-) chronotropic, (-) dromotropic effect
treat angina, hypertension and arrythmias
T-type Drug: selectively target Cav3.2, treat conditions related to neuronal hyperactivity (e.g. epilepsy, chronic pain). Example of drugs: Ethosuximide.

15. Reference
National Center for Biotechnology Information. PubChem Compound Database; CID=104822, (accessed Nov. 13, 2018).
Opie L.H., Pharmacological differences between calcium antagonists. European Heart Journal. 1997; 18(supplement A): A71-A79
Snutch T.P., Zamponi, G. W. (2018) Recent advances in the development of T‐type calcium channel blockers for pain intervention. Brit J Pharmacol, 175: 2375–2383. doi: /bph
Striggow F., Ehrlich B.E. Ligand-gated calcium channels inside and out. Curr Opin Cell Biol. August 1996; 8(4):
Striessnig J., Ortner N. J., Pinggera A.. Pharmacology of L-type Calcium Channels: Novel Drugs for Old Targets? Curr Mol Pharmacol. August 2015; 8 (2): Doi: /
Wang L. A., Ladecola C., Wang G.. New generations of dihydropyridines for treatment of hypertension. J Geriatr Cardiol. January 2017; 14: doi: /j.issn
Zamponi G. W., Striessnig J., Koschak A., Dolphin A. C., Sibley D. R.. The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential. Pharmacological Reviews. October 2015; 67(4): DOI:
Zamponi G.W. A Crash Course in Calcium Channels. ACS Chem Neurosci. December ; 8(12): Doi: /acschemneuro.7b Epub 2017 Nov 13.
Hockerman, G. H., Peterson, B. Z., Johnson, a.,Barry D., & Catterall, W. A. (1997). Molecular Determinants of Drug Binding and Actin on L-type Calcium Channels. Annu Rev Pharmacol, 37(1), doi: /annurev.pharmtox