TABLE 2

NE and NX uptake and affinity at human NET and mutants

Data are means ± S.E.M. of three or four independent experiments separately analyzed in Prism 5. Bmax and Vmax are per 20 μg of total cellular protein and ∼50,000 cells, respectively.

Mutation Position[3H]NX Kd[3H]NE KmNE IC50aNX IC50a[3H]NX Bmax[3H]NE Vmax
nMμMnM%
hNET4.1 ± 0.51.03 ± 0.0259 ± 3.43.4 ± 0.7100100
D75ALoop1a-1bLow NET expression (∼3% of hNET)
L76ATMH1bNo detectable NET expression
W80ATMH1b0.8 ± 0.3*N.D.R.32 ± 1.7N.D.64 ± 7.2N.D.R.
R81ATMH1bN.D.R.N.D.R.N.D.N.D.N.D.N.D.
R81DTMH1bN.D.R.N.D.R.N.D.N.D.N.D.N.D.
R81QTMH1b1.98 ± 0.6N.D.R.97 ± 18.2N.D.21 ± 5*N.D.R.
Y84ATMH1b19.6 ± 2.5*0.57 ± 0.678 ± 7.638 ± 10*112.7 ± 16.281 ± 16
L160ATMH32.7 ± 0.51.14 ± 0.189 ± 6.86.7 ± 2.545 ± 792.6 ± 28.7
F164ATMH33.9 ± 1.230.2 ± 0.01*70 ± 7.46.6 ± 229 ± 0.9*28.5 ± 11*
W308ATMH6aN.D.R.N.D.R.N.D.N.D.N.D.N.D.
Q314ATMH6a0.91 ± 0.1*N.D.R.43 ± 17.4N.D.69.1 ± 9.65N.D.R.
F316ATMH6a9.3 ± 0.41.20 ± 0.2647 ± 17N.D.95.2 ± 13.44 ± 0.02*
F317ATMH6aN.D.R.N.D.R.N.D.N.D.N.D.N.D.
F388AEL4N.D.R.N.D.R.N.D.N.D.N.D.N.D.
S419ATMH8Misfolded NET
Y467ATMH10N.D.R.N.D.R.N.D.N.D.N.D.N.D.
Y467FTMH102.56 ± 0.20.61 ± 0.13211 ± 30*N.D.16.7 ± 4.9*10.9 ± 4.9*
D473ATMH100.95 ± 0.3*N.D.R.371 ± 82*N.D.95 ± 17.6N.D.R.
D473RTMH100.98 ± 0.4*N.D.R.93 ± 8.8N.D.35.6 ± 5.8N.D.R.
R81D/D473RTMH1b/100.87 ± 0.3*N.D.R.115 ± 12.2N.D.26.6 ± 2.9*N.D.R.
R81Q/Q314RTMH1b/6aN.D.R.N.D.R.N.D.N.D.N.D.N.D.
R81A/Q314ATMH1b/6aN.D.R.N.D.R.N.D.N.D.N.D.N.D.

  • N.D.R., no detectable response for either specific NX binding up to 200 nM [3H]NX or specific NE uptake up to 5 μM [3H)]NE (three to five separate experiments); N.D., not determined.

  • ↵* Statistically significant compared with hNET wild type (one-way analysis of variance, P < 0.05).

  • ↵a NE IC50 determined from displacement of [3H]NX, and NX IC50 determined from inhibition of [3H]NE uptake.