Summary of Calcium Regulation inside the Cell

Transcription

1 Overview of Calcium

2 Summary of Calcium Regulation inside the Cell Plasma membrane transport a. Influx via receptor & voltage-regulated channels b. Efflux via Ca-ATPase & Na-Ca antiporter ER/SR membrane transport a. Release via Ca-activated Ca channels (Ryanodine receptors) and IP3-activated channels (IP3 receptors) b. Uptake via Ca-ATPases (SERCA), Na-Ca antiporter & Ca uniporter Mitochondrial membrane transport a. Ca-H antiporter b. Ca-Na antiporter c. Ca uniporter Berridge, M.J Nature Rev. Molec. Cellu. Biol. 4: 517

3 First Demonstration of Calcium conductance Fig. 1. A. Response of crayfish muscle fibre in 20 mme CaCl-210 mm NaCi to an outward current pulse. Intracellular electrodes for applying current and recording potential close together. B. Action potential of muscle fibre in 160 mms SrCl. Stimulating current pulse applied several mm away from position of recording. C. Action potential of muscle fibre in 160 mnm BaCJ2. Complete record included in 3 sweeps. In A and B upper horizontal line is potential recorded outside the fibre. Duration of current pulse indicated by horizontal bar. Same voltage scale for allrecords. Note sweep speed of C 1/10th that of A and B. Current seen when 210 mm NaCl replaced by glycine. Increased Ca increased the response. Citrate abolished the response. Fatt P, Ginsborg BL (1958) The production of regenerative responses in crayfish muscle fibres by the action of calcium, strontium and barium. J Physiol 140:59-60P.

4 Voltage Clamp Analysis of Squid Giant Synapse Axon Terminal Llinas R, Steinberg IZ, Walton K (1981) Presynaptic calcium currents in squid giant synapse. Biophys J 33: FIGURE 13 Sets of calcium currents recorded in 3 and 40 mm external calcium. The amplitude of these currents is significantly dependent on [Ca2"]I (note calibrations) while their time courses are similar. Note that the tail currents in 3 mm calcium are large with respect to the steady-state currents while this is not the case for 40 mm calcium. Two-electrode voltage clamp technique; currents subtracted. FIGURE 14 Plot of experimental values for r0, at 3, 10, and 40 mm [Ca2"]0 (symbols) and comparison with results obtained from numerical solutions to Eq. 17 (solid lines). Note the shift of peak rc. to the right with increased [Ca2"]0. QuickTime and a TIFF (LZW) decompressor are needed to see this picture. FIGURE 18 Action potential. Upper record, action potential reconstructed from Hodgkin-Huxley equations, including the values for gn. and gk. gc, is expressed as the ratio of current to driving forces. Lower record, current density for these three currents obtained from Hodgkin-Huxley equation and our Eq. 18.

5 Speed of transmission at vertebrate CNS synapse: Granule cell to stellate cell synapse in cerebellum Ca indicators to monitor cytoplasmic Ca Temperature dependence of AP & Ca current Temperature dependence of presynaptic Ca & post-synaptic currents Fura-2 has higher affinity & lower dissociation constant At 38ºC Ca current is high at AP peak Temperature dependence of delays: AP start to Ca current start [ ] Ca current start to EPSC start [ ] AP initiation to EPSC initiation [ ] Sabatini BL, Regehr WG (1996) Timing of neurotransmission at fast synapses in the mammalian brain. Nature 384:

6 Three Types of Ca Channel in DRG neurons T: conductance ca 8 ps; activation above -50 mv; inactivation above -40 mv; removed only at -80 mv. Not sensitive to Bay K8644. N: conductance ca 13 ps; activation above -20 mv; inactivation at -20 mv; rapid inactivation compared to L (but similar to T); not sensitive to Bay K8644. L: conductance ca 25 ps; activation at -10 mv; inactivation above -20 mv, sensitive to dihydropyridine agonist Bay K8644 Nowycky MC, Fox AP, Tsien RW (1985) Three types of neuronal calcium channel with different calcium agonist sensitivity. Nature 316:

7 Cloning of dihydropyridine receptor from skeletal muscle 1. Purified DHP binding protein (photolabelled in B lane b) 2. Further purified 170 kda peptide by HPLC [C] 3. Trypsin digested 170 kda peptide and determines a.a. sequences. 4. Designed oligos and cloned cdna by library screen. Tanabe T, Takeshima H, Mikami A, Flockerzi V, Takahashi H, Kangawa K, Kojima M, Matsuo H, Hirose T, Numa S (1987) Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature 328:

8 Structure of Muscle DHP receptor Reconstitution of Ca Current with DHP receptor cdna Control mdg-/- mdg-/- plus DHP-R cdna Mdg=muscle dysgenesis mutant Major conclusion: structurally and sequence related to voltage-dependent sodium channel Tanabe T, Takeshima H, Mikami A, Flockerzi V, Takahashi H, Kangawa K, Kojima M, Matsuo H, Hirose T, Numa S (1987) Primary structure of the receptor for calcium channel blockers from skeletal muscle. Nature 328: Sensitivity to DHP (an antagonist) Control mdg-/- plus DHP-R cdna Tanabe T, Beam KG, Powell JA, Numa S (1988) Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA. Nature 336:

9 Ca channels consist of multiple subunits α1 subunit contains ion channel pore β subunits are cytoplasmic proteins that increase surface expression of α1 subunits and affect activation and inactivation kinetics α2- and δ subunits are cleavage products of a single polypeptide that also increase surface expression of α1 subunits and affect activation and inactivation kinetics γ subunit function is poorly understood. There are multiple genes encoding each auxiliary subunit Catterall, W.A. and Few, A.P Calcium channel regulation and presynaptic plasticity. Neuron 59:882.

10 Different Ca channel classes reflect presence of different α subunits Catterall, W.A Structure and regulation of voltage-gated Calcium channels. Annu. Rev. Cell Dev. Biol. 16: 521.

11 Interactions of CaV alpha subunits Catterall, W.A. and Few, A.P Calcium channel regulation and presynaptic plasticity. Neuron 59:882

12 Transmitter-mediated Inhibition of Calcium Current in DRG Neurons Effect of a, 10-5 M 5-HT; b, 10-4 M GABA; c, d 10-4 M NA on the DRG neuron soma action potential. The two sweeps show spike before and after drug. Return to control duration shown in d with 10-sec intervals between sweeps. Calibration, 20mV and 2ms M GABA decreased rate of rise, overshoot & duration of Ca 2+ spike recorded in TTX. B, 10-4 M NA Decreased rate of rise,overshoot and duration of Ba 2+ spike Recorded in TTX. Calibration: 20 mv and 5 msec. Dunlap K, Fischbach GD (1978) Neurotransmitters decrease the calcium ocmponent of sensory neurone action potentials. Nature 276:

13 Transmitter and G protein regulation of calcium channel function Effect of Gα and Gβγ subunits on voltage dependence of activation of P/Q channels in tsa-201 cells expressingα1a,, β1b and α2δ subunits. Enhanced tonic inhibition and decreased modulation of calcium Current in sympathetic neurons mediated by Gβγ subunits Herlitze S, Garcia DE, Mackie K, Hille B, Scheuer T, Catterall WA (1996) Modulation of Ca2+ channels by G-protein beta gamma subunits. Nature 380: Hess P, Tsien RW (1984) Mechanism of ion permeation through calcium channels. Nature 309:

14 Binding of various cytoplasmic domain sequences in α1b to syntaxin Structure and binding activity of subsegments of the syntaxin binding site Sheng ZH, Rettig J, Takahashi M, Catterall WA (1994) Identification of a syntaxin-binding site on N-type calcium channels. Neuron 13:

15 Injection of N-channel α1b peptide a.a to dissociate interactions with syntaxin Increases asynchronous release Increases paired pulse facilitation Mochida, S., Few, A.P., Scheuer, T., and Catterall, W.A. (2008). Regulation of presynaptic Ca(V)2.1 channels by Ca2+ sensor proteins mediates short-term synaptic plasticity. Neuron 57,

16 Differential regulation of Ca channel by CaM and related proteins Regulation of CaV2.1 Channels by CaM and CaM-like ncas Proteins (A) Ca2+/CaM-dependent regulation of CaV2.1 channels expressed in tsa-201 cells. Currents were evoked by 5ms depolarizations from _80 to +20 mv (Ca2+) or +10 mv (Ba2+) at 100 Hz. Ca2+ currents (black) show initial Ca2+-dependent facilitation followed by Ca2+-dependent and voltage-dependent inactivation. The regulation remaining in Ba2+ currents is voltage dependent. (B) Model for sequential Ca2+/CaM-dependent facilitation and inactivation. Local rises in intracellular Ca2+ activate the two C-terminal Ca2+-binding EF-hands of CaM, which strengthens or initiates an interaction with the IQ-like motif causing facilitation. Following prolonged Ca2+ entry, global rises in intracellular Ca2+ allow CaM to become fully liganded where it interacts with both the IQ-like motif and CaM-binding domain (CBD) to produce inactivation. Adapted from Lee et al.(2003). (C) Schematic representation of CaM and two CaM-like ncas proteins, CaBP1 and VILIP-2, showing N-terminal myristoyl group (CaBP1 and VILIP-2), EF-hands,and central helical linker. Filled boxes represent Ca2+-binding EF-hands. Open boxes represent EF-hands that are inactive in binding Ca2+. (D) Averaged normalized Ca2+ currents elicited as in panel (A) in tsa-201 cells expressing CaV2.1 channels modulated by endogenous CaM (black) or overexpressed CaBP1 (blue) or VILIP-2 (green). Catterall, W.A. and Few, A.P Calcium channel regulation and presynaptic plasticity. Neuron 59:882; Mochida, S., Few, A.P., Scheuer, T. & Catterall, W,A Regulation of presynaptic Cav2.1 channels by Ca2+ sensor proteins mediates short-term synaptic plasticity. Neuron 57: 210.

17 Differential Role of two calmodulin-binding motifs in Paired-Pulse Facilitation and Depression Catterall, W.A. and Few, A.P Calcium channel regulation and presynaptic plasticity. Neuron 59:882. Mochida, S., Few, A.P., Scheuer, T. & Catterall, W,A Regulation of presynaptic Cav2.1 channels by Ca2+ sensor proteins mediates short-term synaptic plasticity. Neuron 57: 210.

18 Interactions of CaV alpha subunits are important Catterall, W.A. and Few, A.P Calcium channel regulation and presynaptic plasticity. Neuron 59:882