Hair cells use active zones with different voltage dependence of Ca2+ influx to decompose sounds into complementary neural codes.

For sounds of a given frequency, spiral ganglion neurons (SGNs) with different thresholds and dynamic ranges collectively encode the wide range of audible sound pressures. Heterogeneity of synapses between inner hair cells (IHCs) and SGNs is an attractive candidate mechanism for generating complementary neural codes covering the entire dynamic range. Here, we quantified active zone (AZ) properties as a function of AZ position within mouse IHCs by combining patch clamp and imaging of presynaptic Ca(2+) influx and by immunohistochemistry. We report substantial AZ heterogeneity whereby the voltage of half-maximal activation of Ca(2+) influx ranged over ∼20 mV. Ca(2+) influx at AZs facing away from the ganglion activated at weaker depolarizations. Estimates of AZ size and Ca(2+) channel number were correlated and larger when AZs faced the ganglion. Disruption of the deafness gene GIPC3 in mice shifted the activation of presynaptic Ca(2+) influx to more hyperpolarized potentials and increased the spontaneous SGN discharge. Moreover, Gipc3 disruption enhanced Ca(2+) influx and exocytosis in IHCs, reversed the spatial gradient of maximal Ca(2+) influx in IHCs, and increased the maximal firing rate of SGNs at sound onset. We propose that IHCs diversify Ca(2+) channel properties among AZs and thereby contribute to decomposing auditory information into complementary representations in SGNs.

Cell-type-specific tuning of Cav1.3 Ca(2+)-channels by a C-terminal automodulatory domain.

Cav1.3 L-type Ca(2+)-channel function is regulated by a C-terminal automodulatory domain (CTM). It affects channel binding of calmodulin and thereby tunes channel activity by interfering with Ca(2+)- and voltage-dependent gating. Alternative splicing generates short C-terminal channel variants lacking the CTM resulting in enhanced Ca(2+)-dependent inactivation and stronger voltage-sensitivity upon heterologous expression. However, the role of this modulatory domain for channel function in its native environment is unkown. To determine its functional significance in vivo, we interrupted the CTM with a hemagglutinin tag in mutant mice (Cav1.3DCRD(HA/HA)). Using these mice we provide biochemical evidence for the existence of long (CTM-containing) and short (CTM-deficient) Cav1.3 α1-subunits in brain. The long (HA-labeled) Cav1.3 isoform was present in all ribbon synapses of cochlear inner hair cells. CTM-elimination impaired Ca(2+)-dependent inactivation of Ca(2+)-currents in hair cells but increased it in chromaffin cells, resulting in hyperpolarized resting potentials and reduced pacemaking. CTM disruption did not affect hearing thresholds. We show that the modulatory function of the CTM is affected by its native environment in different cells and thus occurs in a cell-type specific manner in vivo. It stabilizes gating properties of Cav1.3 channels required for normal electrical excitability.

Structural determinants of CaV1.3 L-type calcium channel gating.

A C-terminal modulatory domain (CTM) tightly regulates the biophysical properties of Ca(v)1.3 L-type Ca(2+) channels, in particular the voltage dependence of activation (V(0.5)) and Ca(2+) dependent inactivation (CDI). A functional CTM is present in the long C-terminus of human and mouse Ca(v)1.3 (Ca(v)1.3(L)), but not in a rat long cDNA clone isolated from superior cervical ganglia neurons (rCa(v)1.3(scg)). We therefore addressed the question if this represents a species-difference and compared the biophysical properties of rCa(v)1.3(scg) with a rat cDNA isolated from rat pancreas (rCa(v)1.3(L)). When expressed in tsA-201 cells under identical experimental conditions rCa(v)1.3(L) exhibited Ca(2+) current properties indistinguishable from human and mouse Ca(v)1.3(L), compatible with the presence of a functional CTM. In contrast, rCa(v)1.3(scg) showed gating properties similar to human short splice variants lacking a CTM. rCa(v)1.3(scg) differs from rCa(v)1.3(L) at three single amino acid (aa) positions, one alternative spliced exon (exon31), and a N-terminal polymethionine stretch with two additional lysines. Two aa (S244, A2075) in rCa(v)1.3(scg) explained most of the functional differences to rCa(v)1.3(L). Their mutation to the corresponding residues in rCa(v)1.3(L) (G244, V2075) revealed that both contributed to the more negative V 0.5, but caused opposite effects on CDI. A2075 (located within a region forming the CTM) additionally permitted higher channel open probability. The cooperative action in the double-mutant restored gating properties similar to rCa(v)1.3(L). We found no evidence for transcripts containing one of the single rCa(v)1.3(scg) mutations in rat superior cervical ganglion preparations. However, the rCa(v)1.3(scg) variant provided interesting insight into the structural machinery involved in Ca(v)1.3 gating.

Functional properties of a newly identified C-terminal splice variant of Cav1.3 L-type Ca2+ channels.

An intramolecular interaction between a distal (DCRD) and a proximal regulatory domain (PCRD) within the C terminus of long Ca(v)1.3 L-type Ca(2+) channels (Ca(v)1.3(L)) is a major determinant of their voltage- and Ca(2+)-dependent gating kinetics. Removal of these regulatory domains by alternative splicing generates Ca(v)1.3(42A) channels that activate at a more negative voltage range and exhibit more pronounced Ca(2+)-dependent inactivation. Here we describe the discovery of a novel short splice variant (Ca(v)1.3(43S)) that is expressed at high levels in the brain but not in the heart. It lacks the DCRD but, in contrast to Ca(v)1.3(42A), still contains PCRD. When expressed together with α2δ1 and ß3 subunits in tsA-201 cells, Ca(v)1.3(43S) also activated at more negative voltages like Ca(v)1.3(42A) but Ca(2+)-dependent inactivation was less pronounced. Single channel recordings revealed much higher channel open probabilities for both short splice variants as compared with Ca(v)1.3(L). The presence of the proximal C terminus in Ca(v)1.3(43S) channels preserved their modulation by distal C terminus-containing Ca(v)1.3- and Ca(v)1.2-derived C-terminal peptides. Removal of the C-terminal modulation by alternative splicing also induced a faster decay of Ca(2+) influx during electrical activities mimicking trains of neuronal action potentials. Our findings extend the spectrum of functionally diverse Ca(v)1.3 L-type channels produced by tissue-specific alternative splicing. This diversity may help to fine tune Ca(2+) channel signaling and, in the case of short variants lacking a functional C-terminal modulation, prevent excessive Ca(2+) accumulation during burst firing in neurons. This may be especially important in neurons that are affected by Ca(2+)-induced neurodegenerative processes.