Transcript Document

Voltage-gated Ca2+ Channels
(VGCCs)
For review, see:
Catterall, 2000. Annu. Rev. Cell Dev. Biol. 16: 521-555.
Voltage-gated calcium channels
Calcium is “THE ION” because of its physiological role in nearly
every cellular process, including:
• Gene regulation
• Signal transduction
• Neurotransmitter release
• Hormone secretion
• Ca2+-dependent action potentials
• Fertilization
• Cell death (apoptosis)
• Modulation of ion channel activity
• Excitation-contraction coupling (muscle)
• and on and on and on…
Voltage-gated calcium channels
These channels are regulated by:
• Phosphorylation (cAMP-dependent protein kinase)
• G proteins (uncommon modulation by Gbg
• Calcium and Ca2+/CaM
• Intracellular effector proteins (such as the RyR,
SNARE proteins)
Voltage-gated calcium channels
Figure 3 - Catterall
Calcium channel function
regulated by the SH3-GK
module in b subunits
McGee et al., 2004. Neuron 42:89-99
Introduction
• The b subunits are cytosolic components of
Ca2+ channels that are necessary for proper
expression and kinetics of the a subunit.
• There are two conserved regions of the b
subunit:
– C1
– C2
– Three variable regions (V1-3) flank C1/C2 and
are targets for postranslational modification
(e.g., phosphorylation/palmitoylation)
Figure 1 McGee
Figure 2 McGee
Figure 3 McGee
Figure 4 McGee
Table 1 - McGee
Figure 5 McGee
Figure 6 McGee
Figure 7 - McGee
Conclusions – McGee
b subunits are similar to MAGUKs; they
contain a split SH3 fold that can assemble
from subdomains composed to C1 (b-SH3)
and C3 (b-GK) regions in either an intra- or
intermolecular fashion.
Identification of the components
controlling inactivation of
voltage-gated Ca2+ channels
Kim et al., 2004. Neuron 41: 745-754.
Introduction
• Ca2+ entry is limited by Ca2+-dependent
inactivation (CDI).
• CDI depends on constitutively bound calmodulin
(CaM).
• apoCaM = calmodulin lacking bound calcium
Question: How do CaM and the channel form a
calcium-sensing apparatus???
Figure 1 - Kim
• IQ motif. In C terminus of pore-forming a subunit. Acts as
a Ca2+/CaM effector site.
• EF hand, classically thought of as a Ca2+ binding site.
• 110 amino acids in between IQ and EF:
• Peptide A = 1588-1609; can bind CaM in absence of Ca2+
• Peptide C = binds CaM with k1/2 for Ca2+ < 90 nM
Introduction
Question: do calcium-dependent inactivation
(CDI) and voltage-dependent inactivation (VDI)
utilize the same machinery, a cytoplasmic I-II
linker, to form a blocking peptide?
Figure 2 - Kim
Figure 3 - Kim
Figure 4 - Kim
Black line = IBa
Gray line = ICa
WT = - - - Mutant =
Figure 4 - Kim
Black line = IBa
Gray line = ICa
WT = - - - Mutant =
ApoCaM tethering is not
necessary nor sufficient for
producing accerated VDI.
Figure 5 - Kim
mutant
wt
Figure 6 - Kim
Conclusions – Kim
• C terminal apoCaM tethering domains and
Ca2+/CaM effector domains that regulated
CDI are inseparable.
Control of ion conduction in
2+
L-type Ca channels by the
concerted action of S5-6 regions
Cibulsky and Sather, 2003. Biophys J. 84: 1709-1719.
Figure 1 Cibulsky
Fig. 2: Activation
a1S based,
No change
a1C based,
No change
a1C: fast activation (as expected) a1S: slow activation (as expected)
Fig. 2: Reversal Potential
Reversal Y:
a1C wt = 73 mV
a1s wt = 67.7 mV
sQuadS5-6c = 46.3 mV
cQuadPs = 61.1 mV
cQuadS5-6s = 63.9 mV
Fig. 2: Cd2+ Block
Fig. 3: P loop transfer from a1S to a1C
a1C wt = 28.9 pS
a1s wt = 16.3 pS
cQuadPs = 22.9 pS
Conclusion: additional parts of the
channel affect unitary conductance.
Fig. 4: S5-6 transfer from a1S to a1C
a1C wt = 28.9 pS
a1s wt = 16.3 pS
cQuadS5-6s = 14.1 pS
Fig.5: Reciprocal transfer - a1C to a1S
a1C wt = 28.9 pS
a1s wt = 16.3 pS
sQuadS5-6c = 30.0 pS
Fig. 6
Conclusions - Cibulsky
• S5-6 region contains the structural features
that are responsible for the difference in
unitary conductance between a1S and a1C
L-type Ca2+ channels.
• The pore region alone does not confer all
properties of unitary conductance.
• Reciprocal swap indicates that no other
regions account for the characteristic ion
transport rates of the two types of channels.