Table 4

Comparison of the binding affinities of wild-type and TMS2 and TMS3 mutant D₄ and D₂ dopamine receptors

Receptor or Mutant	Drug Equilibrium Affinity ConstantsK_i (Fold change in K_i away from the wild-type background)^4-a
	Methylisobutylamiloride nM^4-b	Zinc μM
D₄-WT	199 ± 32	39 ± 5.8
	(1)	(1)
D₄-M107V	10 ± 2.4	18 ± 2.5
	(−20)^4-c	(−2)
D₄-TLMAM105-109IFVTL	14 ± 4	17 ± 3.9
	(−14)^4-c	(−2)
D₄-LM106-107FV	14 ± 2.1	ND
	(−14)
D₄-MA107-108VT	31 ± 8.7	ND
	(−6)
D₄-L87W	290 ± 31	20 ± 0.52
	(1.5)	(−2)
D₄-F88V	253 ± 52	11 ± 4.7
	(1)	(−4)^4-c
D₄-S91L	221 ± 27	23 ± 3.8
	(1)	(−2)
D₄-LM106-107FV+F88V	133 ± 36	8 ± 3.4
	(−1.5)	(−5)^4-c
D₄-TLMAM105-109IFVTL+F88V	105 ± 22	4.6 ± 1.1
	(−2)	(−8)^4-c
D₂-WT^4-d	270 ± 81 (8421 ± 2526)	3.2 ± 0.75 {184 ± 43}
	(1 {40})	(−12 {5})
D₂-IFVTL109-113TLMAM^4-d	>1000^4-e	17.1 ± 1.9 {131 ± 14.4}
	(>5)	(5 {1})

ND, not determined.
↵4-a The fold changes in parentheses for the D₂ wild-type dopamine receptor are relative to the D4 wild-type dopamine receptor, whereas all fold changes for mutant receptors are relative to the wild-type background from which they were derived.
↵4-b K _i values for MIA binding to the wild-type and mutant D₄ receptor were calculated with the competitive form of the Cheng-Prusoff equation because MIA inhibition of [³H]methylspiperone binding to the wild-type D₄dopamine receptor is highly cooperative.
↵4-c A significantly better fit at 95% CI with a shallow slope (n _H < 0.8). In these cases, the values reflect an apparent K _0.5 rather than a K _ivalue.
↵4-d Because both MIA and zinc binding to the D₂ subtype are characterized by negative heterotropic cooperativity, their actual affinity values lie between their IC₅₀ values andK _i values derived from the competitive form of the Cheng-Prusoff equation. The values in brachets are IC₅₀ values. Note that the K _i value for zinc at the “unoccupied” D₂ receptor expressed in Chinese hamster ovary cells was previously determined to be ∼40 μM (Schetz and Sibley, 1997).
↵4-e Only partial inhibition (∼30%) was achieved at the highest concentration.