Table 1. Ca2+ affinity and Ca2+ cooperativity of wild‐type and mutant piccolo C2A‐domains in isolation (intrinsic Ca2+ affinity) and in the presence of phospholipids (apparent Ca2+ affinity)
1. Intrinsic Ca2+ affinitiesa,b
Protein domainsKdHillcoeff.N
Piccolo C2A‐domain1.52 ± 0.18 mM2.1 ± 0.24
Piccolo C2A‐domain V4688S/M4689S0.80 ± 0.05 mM2.3 ± 0.14
Piccolo C2A‐domain V4690S/V4691S0.82 ± 0.08 mM2.2 ± 0.14
Protein domains30% PS/70% PC liposomes50% PI/50% PC liposomes
  EC50 (μM)Hillcoeff.NEC50 (μM)Hillcoeff.N
Piccolo C2A‐domain164 ± 131.9 ± 0.116127 ± 161.8 ± 0.25
Piccolo C2A‐domain V4688S/M4689S14 ± 18.4 ± 2.83not determined
Piccolo C2A‐domain V4688S97 ± 51.7 ± 0.23not determined
Piccolo C2A‐domain M4689S38 ± 41.7 ± 0.33not determined
Piccolo C2A‐domain V4690S/V4691S12 ± 111.0 ± 1.03not determined
Piccolo C2A‐domain Q4692A/N4693A103 ± 161.9 ± 0.53not determined
Piccolo C2A‐domain A4694S187 ± 282.3 ± 0.83not determined
Piccolo PDZ/C2A‐domain75 ± 81.4 ± 0.5349 ± 11.2 ± 0.12
Synaptotagmin I C2A‐domain12 ± 15.9 ± 0.5310 ± 14.7 ± 0.32
  • 2+ 2. Apparent Ca affinities with phospholipid membranes

  • 50% PI/50% PC liposomes

  • Intrinsic Ca‐binding affinities were determined by titrations of purified recombinant proteins monitored with HSQC spectra as described in Figure 8.

  • Data shown are mean ± SEM from the number of experiments indicated (N).

  • Affinities were determined using Ca‐dependent binding of radiolabeled liposomes with the indicated compositions, performed in triplicate as described in Figure 6.