Functional characteristics of two BK_Ca channel variants differentially expressed in rat brain tissues

Abstract

cDNAs encoding large-conductance Ca 2+ -activated K + channel a-subunit (rSlo) were obtained from rat brain. From the DNA sequence of multiple rslo clones, we identified a specific sequence variation of 81 nucleotides, which is either absent from or present at the N-terminal region of a putative Ca 2+ -sensing domain of the channel. Transcripts containing such variations were detected in different ratios from several brain regions, and their functional significance was further examined. When heterologously expressed in Xenopus oocytes, both rSlo variants, named rSlo 0 and rSlo 27 , generated Ca 2+ -activated and voltage-activated K + currents characteristic of neuronal large-conductance Ca 2+ -activated K + (BK Ca ) channels. Single-channel recordings of the two channels showed almost identical permeation characteristics and steady-state gating behavior. Noticeable differences between rSlo 0 and rSlo 27 were revealed when the macroscopic currents were measured at various voltages and intracellular Ca 2+ concentrations. rSlo 27 activated was more rapidly than rSlo 0 in the presence of the same voltage stimulus, and the differences in these activation kinetics were dependent on the concentration of intracellular Ca 2+ . Despite their similar apparent affinities for Ca 2+ , rSlo 0 and rSlo 27 showed significant differences in their co-operative gating behavior. The Hill coefficient for intracellular Ca 2 was estimated to be about 3.7 for rSlo 27 regardless of the membrane voltage, and that for rSlo 0 was reduced from about 5 to 2 as the membrane voltage changed from 40 to 140 mV. As activation of BK Ca channels is involved in rapid hyperpolarization of action potentials, the differential processing of rslo transcripts, and the generation of channels with different activation kinetics and Ca 2+ cooperativity may be a mechanism for tuning the excitability of neurons in different brain regions.

Profiling the Phospho-status of the BKCa Channel α Subunit in Rat Brain Reveals Unexpected Patterns and Complexity

“…For example, expression of Strex-containing BK Ca channels is under hormonal control (46), yielding channels more sensitive to hypoxia (57), and that display a significant hyperpolarizing shift (ϳ20 mV) in V 1 ⁄2 and considerably slower rates of deactivation when compared with the insertless form (58). The Ca27 splice variant yields channels with an increased activation rate and modified Ca 2ϩ cooperativity (59). The C-DEC splice variant generates an extended C terminus resulting in BK␣ with enhanced retention of newly synthesized BK Ca channels in the endoplasmic reticulum, which finally results in decreased cell surface expression (60,61).…”

“…In rat brain, BK␣ splice variants may be differentially expressed in distinct cell types or different compartments of the same neuron. A region-specific distribution of the insertless counterpart of Ca27 splice variant has been observed in rat brain, mRNA is predominantly enriched in cerebellum, whereas Ca27 is abundant in all brain regions (59). * This work was supported, in whole or in part, by National Institutes of Health Grant NS34383 (to J. S. T.).…”

Identification and Functional Characterization of Ankyrin-Repeat Family Protein ANKRA as a Protein Interacting with BKCaChannel

“…AF135265) (Ha et al, 2000). Plasmid vector pNBC-HA for in vitro transcription and translation reaction was modified from pGEM-HE vector (Liman et al, 1992).…”

“…Complement DNA was synthesized from 5 g of total RNA by priming of 1 g of oligo(dT) 12-18 (Promega, Madison, WI) by using M-MLV reverse transcriptase (Invitrogen) at 37°C for 1 h. To test whether rANKRA and rslo transcripts are expressed at particular tissues, gene-specific primers were used to generate 390-base pair cDNA fragments corresponding to nucleotides 332-721 of rANKRA (forward, 5Ј-TAAGGCACGTCTACACACC-3Ј and reverse, 5Ј-CGTCAACTCCACAGTCTAGC-3Ј) and 783 base pairs to 2550 -3332 of rslo, respectively. The primers for rslo were described in a previous report (Ha et al, 2000). To show minor variation in RNA preparations between different tissues, ␤-actin-specific primers used as internal control for RT-PCR reaction (forward, 5Ј-ACAGCTTCACCACCACAG-3Ј and reverse, 5Ј-CG-GATGTCAACGTCACAC-3Ј).…”

“…The expression of rslo was detected in other tissues but only in low levels. Because our PCR primers were designed to flank the fifth splicing site of rslo gene, two different sizes of RT-PCR bands corresponding to rslo 0 and rslo 27 were expected (Ha et al, 2000). Although the rslo 0 band (702 base pairs) was detected widely in all tissues tested, the splicing variant of rslo 27 (783 base pairs) was observed predominantly in cerebral cortex, brain stem, and skeletal muscle.…”