Involvement of Regions in Domain I in the Opioid Receptor Sensitivity of a 1 B Ca 2 1 Channels

The structural basis of Ca channel inhibition by G proteins has received considerable attention recently, and multiple regions on Ca channels that interact with G protein subunits have been identified. We have demonstrated previously that a region extending from the N terminus to the I/II loop of the Ca channel is involved in determining the differences between a1B and a1E Ca channels with respect to inhibition by G proteins. Here we explore this region of the channel in greater detail in an effort to further define the regions involved in determining inhibition. Chimeric Ca channels constructed from a1B and a1E Ca channels revealed that the N terminus, the I/II loop, and domain I all play an important role in determining inhibition. We identified a 70-amino acid fragment from domain I that mediates the effects of domain I, and a 50-amino acid fragment from the I/II loop that mediates the effects of the I/II loop. When these regions from a1B were exchanged into a1E, inhibition identical with that of a1B was observed. The differences between a1B and a1E in the identified region of domain I involve residues that are predicted to be almost exclusively extracellular. Mutations to some of the high-affinity G protein binding regions of a1B (a interaction domain, CC14, and a C-terminal Ga binding site) caused relatively little change in inhibition, which suggests that these sites are not necessary individually for G proteinmediated inhibition and may help to explain the small effects of exchanging these regions in isolation. The inhibition of Ca influx through presynaptic Ca channels by G proteins is an important mechanism for the modulation of synaptic transmission (Wu and Saggau, 1997; Miller, 1998). The Ca channels involved in presynaptic Ca entry are usually of the N, P/Q, and R-type (Dunlap et al., 1995; Reid et al., 1998; Wu et al., 1998). Cloned N-type (a1B) Ca channels have generally been found to be more sensitive to inhibition by G proteins than are P/Q(a1A) or R(a1E) type channels (Bourinet et al., 1996; Toth et al., 1996; Yassin et al., 1996; Zhang et al., 1996; Simen and Miller, 1998; but see Meza and Adams, 1998). These differences in sensitivity to inhibition by G proteins may therefore represent one mechanism mediating the efficacy of synaptic depression by presynaptic receptors. The structural basis of G protein binding to Ca channels has been the subject of a number of studies. Gbg subunits have been shown to interact with two distinct regions in the I/II loop of nondihydropyridine (DHP)-sensitive Ca channels, and one site also interacts with Ca b subunits (De Waard et al., 1997; Herlitze et al., 1997; Qin et al., 1997; Zamponi et al., 1997; Hamid et al., 1999). It has also been shown that the C terminus of Ca channels can bind Gbg (Qin et al., 1997) as well as Ga (Furukawa et al., 1998a, 1998b) subunits. Despite a clear role for the I/II loop region in Gbg binding, this region does not seem to be primarily responsible for the differences in G protein sensitivity between the non-DHP sensitive Ca channels (Page et al., 1997; Zhang et al., 1996). Similar to the findings of Zhang et al. (1996) and Stephens et al. (1998), we have previously demonstrated that the insertion of domain I from the human a1B Ca channel into the human a1E channel yields a construct with significantly increased sensitivity to G proteins (Simen and Miller, 1998) and that the addition of the I/II loop and the N terminus increased inhibition further. The I/II loop region alone did not affect modulation. The involvement of the N terminus has recently been further defined (Page et al., 1998; Canti et al., 1999). Although these studies suggest an important role of the N-terminal portion of the channel in determining sensitivity to modulation, it remains unclear what portions of this region are involved and how large their individual contributions are to the overall level of modulation of Ca channels. We therefore examined the effects of transfer of regions between a1B (highly modulated) and a1E (minimally modulated) to determine sequences responsible for the differences in G protein sensitivity between the two channels. Our findThis work was supported by National Institute of Health Grants DA02121, MH40165, NS33826, DK44840, and NS21442. A.A. S. was supported by Grants HD07009 and DA02575. ABBREVIATIONS: DHP, dihydropyridine; norBNI, nor-binaltorphimine; AID, a interaction domain; ND1, N-terminal domain I region; GID, G protein interaction domain; kOR, k-opioid receptor. 0026-895X/00/051064-11$3.00/0 MOLECULAR PHARMACOLOGY Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics MOL 57:1064–1074, 2000 /12985/822097 1064 at A PE T Jornals on M ay 0, 2017 m oharm . aspeurnals.org D ow nladed from ings suggest that the N terminus alone contributes substantially to inhibition, a region extending from the S1/S2 loop to the S3/S4 loop of domain I is involved, and that a fragment of the I/II loop spanning the AID region and a downstream Gbg binding region is also involved in mediating inhibition. The identification of the region of domain I involved is of particular interest because the residues involved are almost entirely extracellular and are therefore unlikely to directly interact with G proteins. Materials and Methods Chimeric Ca channel a1 subunit constructs were created using methods described previously (Simen and Miller, 1998). Final constructs were confirmed by a combination of restriction analysis and DNA sequencing. The native human a1B construct consisted of residues 1 to 2340 of GenBank 2284339 (gift from Dr. R. Harpold, SIBIA Neurosciences, San Diego, CA). The native human a1E construct consisted of residues 1 to 2271 of GenBank 21082919 (gift from Dr. R. Harpold, SIBIA Neurosciences). The negative chimeras were named by appending the amino acids from a1E that were transferred into a1B to the letter “E,” using the numbering system shown in Fig. 5. For example, the construct E120–132 consisted of a1B with amino acids 120 to 132 replaced with the corresponding amino acids from a1E. The positive chimeras were named in a similar fashion, by appending the residues from a1B that were transferred into a1E to the letter “B. ” For example, the construct B1–93 consisted of residues 1 to 93 from a1B in the a1E background. The construct D2037–2087 was created by replacing the cDNA coding for residues 2037 to 2087 of a1B with a HindIII restriction site, coding for the amino acids RL. The construct D1875–2339 was created by deleting the nucleotides coding for amino acids 1875 to 2339 of a1B. The construct BQ1 was created from a1B by site-directed mutagenesis to make the mutations Q383A, E386A, and R387A. The construct BQ2 was created from a1B by site-directed mutagenesis to make the mutations Q383A, Q384A, I385A, E386A, and R387A. tsA-201 cell culture, transfections, solutions for electrophysiological recording, and drug solutions were made as described previously (Simen and Miller, 1998). Cells were transfected with Ca a2/d, Ca b1b, and wildtype or recombinant Ca a1 subunits, along with the mouse k-opioid receptor and CD8a. The mouse k-opioid receptor was a gift from Dr. Graeme Bell (Howard Hughes Medical Institute, University of Chicago, Chicago, IL). Currents were elicited by a dual-pulse protocol consisting of two 50-ms depolarizations (pulse 1 and pulse 2) to test potentials varying from 240 to 140 mV from a holding potential of 290 mV, separated by 800 ms at the holding potential, with a 30-ms, 90-mV depolarization (“prepulse”) ending 5 ms before the second pulse (Fig. 1A), at an acquisition rate of 10 kHz and filtered with a four-pole Bessel filter at 2 kHz. To facilitate comparison of the different recombinant channel constructs, cumulative integrals (CI) were computed on the current voltage data from each construct, and were defined as