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Identification of Ca(2+)-activated K+ channel splice variants and their distribution in the turtle cochlea.

ABSTRACT: Turtle auditory-hair cells are frequency-tuned by the activity of calcium-activated potassium (KCa) channels, a cell's characteristic frequency being determined by the KCa channel density and kinetics which both vary systematically along the cochlea. As a first step towards identifying the source of KCa channel variation, we have isolated, by reverse-transcription polymerase chain reaction on dissociated hair cells, the main cDNAs homologous to the slo gene which encodes the channel's alpha-subunit. A total of six alternatively spliced variants were identified, the smallest of which is 94% identical to a mouse Slo sequence. Variation occurs by insertion of exons at only two splice sites, two of these exons encoding novel 31- and 61-amino acid sequences. As we were unable to detect splicing at other potential sites, we infer that the six variants correspond to naturally occurring combinations. The spatial distribution of the variants, defined by isolating hair cells from different regions of the cochlea, indicated that some isoforms were non-uniformly distributed. Those containing large inserts in the first splice site were notably absent from the highest-frequency region. We suggest that alternative splicing of the slo gene may contribute to variation in KCa channel properties.

SUBMITTER: Jones EM

PROVIDER: S-EPMC1689023 | biostudies-other | 1998 Apr

REPOSITORIES: biostudies-other

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Identification of Ca(2+)-activated K+ channel splice variants and their distribution in the turtle cochlea.

Jones E M EM Laus C C Fettiplace R R

Proceedings. Biological sciences 19980401 1397

Turtle auditory-hair cells are frequency-tuned by the activity of calcium-activated potassium (KCa) channels, a cell's characteristic frequency being determined by the KCa channel density and kinetics which both vary systematically along the cochlea. As a first step towards identifying the source of KCa channel variation, we have isolated, by reverse-transcription polymerase chain reaction on dissociated hair cells, the main cDNAs homologous to the slo gene which encodes the channel's alpha-subu ...[more]

PMID: 9608728

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Project description:The large conductance Ca(2+)-activated K(+) or BK channel has a role in sensory/neuronal excitation, intracellular signaling, and metabolism. In the non-mammalian cochlea, the onset of BK during development correlates with increased hearing sensitivity and underlies frequency tuning in non-mammals, whereas its role is less clear in mammalian hearing. To gain insights into BK function in mammals, coimmunoprecipitation and two-dimensional PAGE, combined with mass spectrometry, were used to reveal 174 putative BKAPs from cytoplasmic and membrane/cytoskeletal fractions of mouse cochlea. Eleven BKAPs were verified using reciprocal coimmunoprecipitation, including annexin, apolipoprotein, calmodulin, hippocalcin, and myelin P0, among others. These proteins were immunocolocalized with BK in sensory and neuronal cells. A bioinformatics approach was used to mine databases to reveal binary partners and the resultant protein network, as well as to determine previous ion channel affiliations, subcellular localization, and cellular processes. The search for binary partners using the IntAct molecular interaction database produced a putative global network of 160 nodes connected with 188 edges that contained 12 major hubs. Additional mining of databases revealed that more than 50% of primary BKAPs had prior affiliations with K(+) and Ca(2+) channels. Although a majority of BKAPs are found in either the cytoplasm or membrane and contribute to cellular processes that primarily involve metabolism (30.5%) and trafficking/scaffolding (23.6%), at least 20% are mitochondrial-related. Among the BKAPs are chaperonins such as calreticulin, GRP78, and HSP60 that, when reduced with siRNAs, alter BKalpha expression in CHO cells. Studies of BKalpha in mitochondria revealed compartmentalization in sensory cells, whereas heterologous expression of a BK-DEC splice variant cloned from cochlea revealed a BK mitochondrial candidate. The studies described herein provide insights into BK-related functions that include not only cell excitation, but also cell signaling and apoptosis, and involve proteins concerned with Ca(2+) regulation, structure, and hearing loss.

Project description:Prevailing models postulate that high Ca(2+) selectivity of Ca(2+) release-activated Ca(2+) (CRAC) channels arises from tight Ca(2+) binding to a high affinity site within the pore, thereby blocking monovalent ion flux. Here, we examined the contribution of high affinity Ca(2+) binding for Ca(2+) selectivity in recombinant Orai3 channels, which function as highly Ca(2+)-selective channels when gated by the endoplasmic reticulum Ca(2+) sensor STIM1 or as poorly Ca(2+)-selective channels when activated by the small molecule 2-aminoethoxydiphenyl borate (2-APB). Extracellular Ca(2+) blocked Na(+) currents in both gating modes with a similar inhibition constant (Ki; ~25 µM). Thus, equilibrium binding as set by the Ki of Ca(2+) blockade cannot explain the differing Ca(2+) selectivity of the two gating modes. Unlike STIM1-gated channels, Ca(2+) blockade in 2-APB-gated channels depended on the extracellular Na(+) concentration and exhibited an anomalously steep voltage dependence, consistent with enhanced Na(+) pore occupancy. Moreover, the second-order rate constants of Ca(2+) blockade were eightfold faster in 2-APB-gated channels than in STIM1-gated channels. A four-barrier, three-binding site Eyring model indicated that lowering the entry and exit energy barriers for Ca(2+) and Na(+) to simulate the faster rate constants of 2-APB-gated channels qualitatively reproduces their low Ca(2+) selectivity, suggesting that ion entry and exit rates strongly affect Ca(2+) selectivity. Noise analysis indicated that the unitary Na(+) conductance of 2-APB-gated channels is fourfold larger than that of STIM1-gated channels, but both modes of gating show a high open probability (Po; ~0.7). The increase in current noise during channel activation was consistent with stepwise recruitment of closed channels to a high Po state in both cases, suggesting that the underlying gating mechanisms are operationally similar in the two gating modes. These results suggest that both high affinity Ca(2+) binding and kinetic factors contribute to high Ca(2+) selectivity in CRAC channels.

Project description:Previous studies report functional differences in large conductance Ca2+ activated-K+ channels (BKCa) of smooth muscle cells (VSMC) from rat cerebral and cremaster muscle resistance arteries. The present studies aimed to determine if this complexity in BKCa activity may, in part, be due to splice variants in the pore-forming ?-subunit. BKCa variants in the intracellular C terminus of the ?-subunit, and their relative expression to total ?-subunit, were examined by qPCR. Sequencing of RT-PCR products showed two ?-subunit variants, ZERO and STREX, to be identical in cremaster and cerebral arteries. Levels of STREX mRNA expression were, however, significantly higher in cremaster VSMCs (28.9±4.2% of total ?-BKCa) compared with cerebral vessels (16.5±0.9%). Further, a low level of BKCa SS4 ?-subunit variant was seen in cerebral arteries, while undetectable in cremaster arteries. Protein biotinylation assays, in expression systems and arterial preparations, were used to determine whether differences in splice variant mRNA expression affect surface membrane/cytosolic location of the channel. In AD-293 and CHO-K1 cells, rat STREX was more likely to be located at the plasma membrane compared to ZERO, although the great majority of channel protein was in the membrane in both cases. Co-expression of ?1-BKCa subunit with STREX or ZERO did not influence the dominant membrane expression of ?-BKCa subunits, whereas in the absence of ?-BKCa, a significant proportion of ?1-subunit remained cytosolic. Biotinylation assays of cremaster and cerebral arteries showed that differences in STREX/ZERO expression do not alter membrane/cytosolic distribution of the channel under basal conditions. These data, however, revealed that the amount of ?-BKCa in cerebral arteries is approximately 20X higher than in cremaster vessels. Thus, the data support the major functional differences in BKCa activity in cremaster, as compared to cerebral VSMCs, being related to total ?-BKCa expression, regardless of differences in splice variant expression.

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Identification of Ca(2+)-activated K+ channel splice variants and their distribution in the turtle cochlea.

Publications

Identification of Ca(2+)-activated K+ channel splice variants and their distribution in the turtle cochlea.

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