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Voltage activated Ca2+ channels
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- 1What are the functions of Ca2+ channels?Voltage-activated calcium channels help regulate intracellular calcium concentration and contribute to calcium signalling.
They mediate Ca2+ entry into cells in response to depolarisation.
They control multiple cellular events:
AP generation and conduction
sensory processes,
muscle contraction
secretion of transmitters and hormones
cell differentiation and gene expression
The opening of Ca2+ channels is also modulated by a wide variety of hormones, transmitters, protein kinases and phosphatases.
Calcium channels are also the targets for various toxins and drugs.
Spontaneous mutations of Ca2+ channel subunits associated with hyperexcitable disorders - 2What is the ionic requirement for transmitter release?Calcium influx is the trigger for fast evoked transmitter release
extracellular calcium (Ca2+) is typically about 1-2 mM, intracellular calcium at rest is <0.1 mM.
An elevation in intracellular calcium concentration is an absolute requirement for transmitter release. Na+ and K+ ions not necessary for release.
Unlike other ions (Na+, K+, Cl-) it is the CHEMICAL signal carried by Ca2+ that is important, not the electrical charge of the ion. - 3Give an overview of the Ca2+ channel a subunitsPrimary structure like the a subunit of Na+ channel. Four repeat domains, each with six transmembrane segments and a membrane-associated loop between S5 and S6.
Glutamic acid residues (E) in the P regions of each domain are important for determining selectivity for calcium ions (DEKA in sodium channels)
Like Na+ channels there are auxiliary subunits. - 4Describe the subtypes of calcium channelsInitially they were divided on the basis of the voltage required to activate.
Some Ca2+ channels require a large depolarisation (e.g. to -20mV) to elicit opening - termed High-Voltage Activated (HVA) calcium channels.
Other Ca2+ channels open at much more negative potentials (e.g. -60 mV) and are termed Low-Voltage Activated (LVA) calcium channels.
HVA Ca2+ channels display variable inactivation, some hardly inactivate over a 200ms depolarising pulse, others completely inactivate in this time
LVA channels generally display rapid voltage dependent inactivation, like that observed for Na+ channels
As many HVA channels are Long-lasting they are also called L-type calcium channels
The LVA type, because they displayed fast voltage-dependent inactivation are termed Transient or T-type calcium channels
Further analyses, using voltage parameters and toxins have shown more diversification:
N-type (neuronal)
P-type (after Purkinje cell)
Q-type (found in cerebellar grangranule neurones)
R-type (so-called resistant component of neuronal current) - 5Describe the subunits of calcium channelsMolecular and cloning studies have revealed that the diversity of Ca2+ channels arises from the combination of five subunits.
The a1 subunit has the basic Na+ channel a subunit structure and there appear to be 10 (individual genes) of these.
There is a b subunit (4 separate genes identified) which is intracellularly located. Each a subunit is associated with multiple b subunits
A highly glycosylated a2 subunit, extracellularly located, is attached to the membrane through disulphide linkage to a d subunit which anchors the complex to the a1 subunit via a single transmembrane segment.
This a2d subunit occurs as 4 separate genes. i.e. a single gene encodes the a2 and d subunits
Finally there is a g subunit, a glycoprotein with four transmembrane segments, may be up to 8 genes but involvement is not entirely clear - 6Describe L-type calcium channelsAs many HVA channels are Long-lasting they are also called L-type calcium channels
L-type are found in a wide range of cells
HVA type, blocked by dihydropyridines (DHPs)
Function = E-C coupling, hormone sec^n, muscle contraction - 7Describe N-types calcium channelsN-type (neuronal) are found mostly in neurons
HVA type, blocked by conotoxin (w-CTx) and GVIA
Function = neurotransmitter (N-t) release - 8Describe P-type calcium channelsP-type (after Purkinje cell) are found mostly in neurones
HVA type, blocked by agatoxin (w-Aga) and IVA (spider)
Function = neurotransmitter release - 9Describe Q-type calcium channelsQ-type (found in cerebellar granule neurones) are mostly found in neurons
HVA type, blocked by conotoxin (w-CTx) and MVIIC
Function = neurotransmitter release - 10Describe R-type calcium channelsR-type (so-called resistant component of neuronal current) are mostly found in neurons
H/LVA type, blocked by SNX-482 (tarantula toxin)
Function = Ca action potentials and neurotransmitter release - 11Describe T-type calcium channelsThe LVA type, because they displayed fast voltage-dependent inactivation are termed Transient or T-type calcium channels
Found in a wide range of cells
Blocked by mibefradil
Function = repetitive firing - 12Describe the 3 calcium channel familiesChannel type Current type Location
CaV1.1-4 L Cardiac &
Skeletal
muscle,
neurones,
endocrine cells
CaV2.1 P/Q Nerve
terminals,
dendrites
CaV2.2 N Nerve
terminals,
dendrites
CaV2.3 R Cell
bodies, Nerve
terminals
dendrites
CaV3.1-3 T Cardiac
& smooth
muscle,
neurons - 13Describe the L-type channels mode-switchingSingle channel recordings have demonstrated that L type Ca2+ channels display multiple gating kinetics
during series of consecutive sweeps
of depolarising pulses, see clusters of
sweeps in 3 different kinetic modes
Mode 1 = normal short openings
Mode 0 = no openings
Mode 2 = long opening times - 14Describe phosphorylation regulation of the Cav1 familyL-type Ca2+ channels are modulated by hormones and neurotransmitters in muscle (skeletal, smooth and cardiac) and neurones.
A good example of this is the enhancement of Ca2+ current in the heart by catecholamines, which underlies their positive inotropic action
b-adrenergic agonists increase cardiac action potential amplitude, and muscle contractility and rate
Single channel recordings show 2 actions of b1-adrenergic stimulation - 15What regulates phosphorylation of the channels?Adrenergic stimulation increases probability of opening (more mode 2 behaviour) and the apparent number of functional Ca2+ channels
These actions can be mimicked by an increase in cAMP in the cell
Shown that cAMP-dependent protein kinase A (PKA) phosphorylates a subunit on serine 1928 of CaV1.2 to mediate an increase in Ca2+ current - but this increase is much less than observed for native channels.
Presence of a b-subunit (b2a) considerably enhanced b-adrenergic agonist activation of CaV1.2 (by phosphorylation of serine 478/479)
PKA phosphorylates native b subunits in cardiac tissue.
This is an area of intense study as other protein kinases, activated by hormones/transmitters likely modulate Ca2+ channel activity - 16What regulates the Cav2 family?The CaV2 family is regulated by G-proteins
GPCRs couple to Cav channels - N, P/Q
e.g. a2 adrenoceptors
m and d opioid receptors
GABAB
Adenosine A1 receptors
G-protein coupling inhibits calcium current - local membrane action
Responsible for a decrease in synaptic transmission as calcium entry at nerve terminal reduced
All G-proteins show this behaviour (Gs, Gi, Go, Gq)
A common feature responsible - Gbg dimer mimics agonist actions - tonic inhibition of calcium current. Gbg dimer in membrane patch was sufficient to inhibit calcium current
A depolarizing prepulse reduces/abolishes the inhibitory effect
Integration of multiple signalling pathways regulates CaV channel activity - 17What effects can Ca2+ knock outs have?die at birth of asphyxiation, die before birth as unable to contract cardiac muscle, Deaf, cardiac arrhythmias, blind
- 18What drugs target L-type (Cav1.x) Ca2+ channels?The major classes of drugs that act on L-type Ca2+ channels are:
Dihydropyridines (DHPs) e.g. nifedipine
Phenylalkylamines e.g. verapamil
Benzothiazepines e.g. diltiazem
Used therapeutically to treat hypertension, cardiac arrhythmias and ischaemic heart disease. - 19How do drugs targeting L-type (Cav1.x) Ca2+ channels work?These drugs act to block Ca2+ influx and they do so by more than one mechanism, drugs may bind to the same site but have converse effects
The dihydropyridines act as allosteric modulators, in that they alter the gating behaviour of L-type Ca2+ channels
DHP antagonists act to stabilise mode 0 behaviour. Agonists are possible and BAYK8644 increases Ca2+ current by stabilising mode 2 behaviour - 20How are calcium channels blocked?Mutation studies indicate that the DHPs bind to specific sites associated with S5 & S6 segments of the a subunit (particularly in domains III & IV) although there is a contribution from IS6 segment
Major contribution to DHP site from Y1485, M1486 & I1493 of IVS6
The phenylalkylamines (e.g. verapamil) block L-type Ca2+ channels in a use -dependent manner from the intracellular side of the membrane - similar to the action of LAs on Na+ channels; I.e. open channel block
It is thought that the PAAs bind to the inner end of the transmembrane pore (sites in S6 in domains III & IV - some overlap with the DHPs)
The benzothiazepines (e.g diltiazem appear to act extracellularly - by binding to residues in S5 - S6 linker of domain IV - 21How are Ca channels targeted for pain?Ziconotide a peptide toxin that interacts with N-type calcium channel (chronic neuropathic pain)
Gabapentin/pregabalin - originally synthesized as GABA- mimetics (for treatment of epilepsy)- used to treat chronic pain through their interaction at the 2 subunit - 22What is Hypokalemic periodic paralysis (type1)?Cav1.x channelopathy
Cav1.1 expressed specifically in skeletal muscle: mutations (S4 regions) result in reduced calcium current and muscle weakness - 23What is Timothy Syndrome?Cav1.x channelopathy, a rare childhood multi-organ disorder - cardiac defects, immune deficiency, cognitive abnormalities etc. Generally sporadic. Due to mutations in Cav1.2 causing loss of channel inactivation and so enhanced calcium entry leading to severe cardiac dysregulation.
- 24What is night blindness?Cav1.x channelopathy, One form of this condition is caused by decreased transmitter released from retinal photoreceptor terminals. Multiple mutations (> 60) associated with loss of function of CaV1.4 (L-type calcium channel)
- 25Describe the migraine inherited Ca2+ channelopathyOne form (Familial Hemiplegic Migraine - FHM) a rare hereditary disorder resulting in transient migraine attacks is associated with mutations of the CaV2.1 gene (P/Q channel a subunit) causing increased channel activity and transmitter release.
- 26Describe the Ataxia: Episodic Ataxia Type 2 inherited Ca2+ channelopathypatients have recurrent attacks of motor (cerebellar) dysfunction. Also associated with disruption of CaV2.1 gene (>20 known mutations) preventing formation of normal functional channels and so loss of calcium current
- 27Describe the epilepsy inherited Ca2+ channelopathyRecently it has been suggested that mutations in Ca2+ channel auxiliary subunits may be associated with certain forms of epilepsy. The b4, g2 & 2 subunits have mutations that alter P/Q channel function
- 28Describe the autism spectrum disorder inherited Ca2+ channelopathymutations in Cav3.2 (T-type) associated with this disorder (patients also suffer from epilepsy) - all reduce channel activity
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- Biomembranes Karteikarten am Voltage activated Ca2+ channels, erstellt von Megan MacLeod am 06/06/2019.
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- Megan MacLeod · Jun 2019
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