Post on 17-Jan-2016
transcript
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 G• 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 subunits
McGee et al., 2004. Neuron 42:89-99
Introduction• The subunits are cytosolic components of
Ca2+ channels that are necessary for proper expression and kinetics of the subunit.
• There are two conserved regions of the 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
subunits are similar to MAGUKs; they contain a split SH3 fold that can assemble from subdomains composed to C1 (-SH3) and C3 (-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 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
Figure 6 - Kimm
utan
tw
t
Conclusions – Kim
• C terminal apoCaM tethering domains and Ca2+/CaM effector domains that regulated CDI are inseparable.
Control of ion conduction in L-type Ca2+ channels by the
concerted action of S5-6 regions
Cibulsky and Sather, 2003. Biophys J. 84: 1709-1719.
Figure 1 - Cibulsky
Fig. 2: Activation
1C: fast activation (as expected) 1S: slow activation (as expected)
1S based, No change
1C based, No change
Fig. 2: Reversal Potential
Reversal :
1C wt = 73 mV1s wt = 67.7 mV
sQuadS5-6c = 46.3 mVcQuadPs = 61.1 mVcQuadS5-6s = 63.9 mV
Fig. 2: Cd2+ Block
Fig. 3: P loop transfer from 1S to 1C
1C wt = 28.9 pS 1s wt = 16.3 pScQuadPs = 22.9 pS
Conclusion: additional parts of the channel affect unitary conductance.
Fig. 4: S5-6 transfer from 1S to 1C
1C wt = 28.9 pS
1s wt = 16.3 pS
cQuadS5-6s = 14.1 pS
Fig.5: Reciprocal transfer - 1C to 1S
1C wt = 28.9 pS
1s 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 1S and 1C 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.