Increased facilitation in early post-natal and adult DRG neurons. An increase in Ba2+ current facilitation following a depolarizing pre-pulse to 80 mV is consistent with constitutive coupling of GPCRs with voltage-dependent Ca2+ channels (VDCCs) through Gβγ subunits. Facilitation was quantified by a two-step protocol comparing the current amplitude with (P2) or without a pre-pulse (P1), the P2/P1 ratio. A. Early post-natal DRG neurons i. Whole cell patch clamp recordings for all experiments were conducted in small DRGs (highlighted by the arrow, the scale-bar represents 10 μM), the population of cells most likely to express the μ receptor. ii. Exemplar recordings from β-arr2+/+ and β-arr2-/- cells, in which GTP-γ-S was included in the intracellular recording solution, demonstrate the relative increase in current following a depolarizing pre-pulse to 80 mV (P2). Horizontal and vertical scale-bars represent 20 ms and 0.4 nA respectively. iii. The P2/P1 ratio reveals increased facilitation in βarr2-/- but not β-arr2+/+ early post-natal neurons. This difference was enhanced in the presence of GTP-γ-S and absent in neurons lacking both β-arr2 and the μ receptor (μβarr2-/-). B. Adult DRG neurons. i. Similar to the early post-natal DRGs, recordings were made in small DRG neurons from adult mice (highlighted by the arrow, the scale-bar represents 10 μM). ii. Exemplar recordings from adult DRG neurons in which GTP-γ-S was included in the intracellular recording solution, similarly demonstrate an increased current after the depolarizing pre-pulse (P2) in βarr2-/- neurons. Horizontal and vertical scale-bars represent 20 ms and 1.0 nA, respectively. iii. Recordings from adult DRG neurons show a similar effect as early post-natal DRGs in which the P2/P1 ratio was enhanced in β-arr2-/- vs β-arr2+/+ neurons. This effect was enhanced by GTP-γ-S. ***p < 0.0001 vs β-arr2+/+., * p < 0.05 vs β-arr2+/+, #p < 0.05 vs untreated β-arr2-/- recordings.
Lam et al. Molecular Pain 2011 7:24 doi:10.1186/1744-8069-7-24