Abstract
Calcium channel beta subunits are essential regulatory elements of the gating properties of high voltage-activated calcium channels. Co-expression with beta(3) subunits typically accelerates inactivation, whereas co-expression with beta(4) subunits results in a slowly inactivating phenotype. Here, we have examined the molecular basis of the differential effect of these two subunits on the inactivation characteristics of Ca(v)2.2 + alpha(2)-delta(1) N-type calcium channels by creating a series of 22 chimeric beta subunits that are based on various combinations of variable and conserved regions of the parent beta subunit isoforms. Our data show that replacement of the N terminus region of beta(4) with a corresponding 14-amino acid stretch of beta(3) sequence accelerates the inactivation kinetics to levels seen with wild type beta(3). A similar kinetic speeding is observed by a concomitant substitution of the second conserved and variable regions, but not when these regions are substituted individually, suggesting that 1) the second variable and conserved regions cooperatively regulate N-type calcium channel inactivation and 2) that there are two redundant mechanisms that allow the beta(3) subunit to accelerate N-type channel inactivation. In contrast with previous reports in Ca(v)2.1 calcium channels, deletion of the C-terminal region of Ca(v)2.2 did not alter the regulation of the channel by wild type and chimeric beta subunits. Hence, the molecular underpinnings of beta subunit regulation of voltage-gated calcium channels appear to vary with calcium channel subtype.
Highlights
The influx of calcium ions through voltage-gated calcium channels triggers a range of intracellular responses, ranging from gene transcription [1] and activation of second messenger pathways [2, 3] to the release of neurotransmitters from presynaptic nerve termini [4]
We have examined the molecular basis of the differential effect of these two subunits on the inactivation characteristics of Cav2.2 ؉ ␣2-␦1 N-type calcium channels by creating a series of 22 chimeric  subunits that are based on various combinations of variable and conserved regions of the parent  subunit isoforms
Our data show that replacement of the N terminus region of 4 with a corresponding 14-amino acid stretch of 3 sequence accelerates the inactivation kinetics to levels seen with wild type 3
Summary
3/4 Chimeras—Unique restriction sites were introduced via the QuikChange site-directed mutagenesis kit (Stratagene) into the rat 3 (GenBankTM accession number M88751) and 4 (GenBankTM accession number L02315) cDNAs (both in the pMT2 expression vector) at exactly corresponding positions. The downstream antisense primers restricted the amplification region and introduced a novel 3Ј NotI restriction site immediately following the stop codon The pMT2/3 and pMT2/4 constructs were cut, and the fragments isolated from T-Easy were ligated This resulted in four new cDNA constructs: 3 and 4 without the extensive 5Ј-untranslated region but containing novel 5Ј KpnI sites and Kozak start sequences, as well as chimeric 33344 and 44433 constructs. After 12 h of incubation at 37 °C in the presence of the calcium phosphate/cDNA mixture, the cells were washed with fresh Dulbecco’s modified Eagle’s medium and allowed to recover for an additional 12 h. Forward primer sequences are shown, because the reverse mutagenesis primers are the reverse complement of the shown sequences
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