A mouse model to study the C-terminal regulation of Ca_V1.3 L-type calcium channels

Background

Ca_V1.3 voltage-gated L-type Ca²⁺ channels (LTCCs) play an important role for hearing, cardiac pacemaking and neuronal excitability. The C-terminus of Ca_V1.3 tightly controls channel gating by an intramolecular protein interaction involving two putative α-helices (termed PCRD, DCRD), which form a C-terminal modulatory mechanism (CTM) only in full-length Ca_V1.3 variants. In short (Ca_V1.3_42A and Ca_V1.3_43S) Ca_V1.3 α1 subunit splice variants CTM is absent which leads to profound changes in channel gating: activation occurs at more negative voltages and Ca²⁺-dependent inactivation (CDI) is faster.

Methods

We quantified Ca_V1.3 splice variants by qPCR analysis and transcript scanning, using different mouse tissues. To assess the physiological relevance of CTM, we generated a mutant mouse strain in which CTM function is disrupted by an HA-tag (Ca_V1.3-DCRD^HA/HA mice). We used anti-HA antibodies to detect the expression of the HA-tagged full length channel by Western blot analysis.

Results

The short variants Ca_V1.3_42A (highest relative abundance in substantia nigra (SN) and ventral tegmental area (VTA)) and Ca_V1.3_43S are both less abundant in mouse brain indicating that the full length form Ca_V1.3_L comprises the most abundant form (about 50% of all transcripts). In mouse heart short transcripts are rare and Ca_V1.3_L represents about 90% of all known transcripts. Ca_V1.3-DCRD^HA/HA mice contain a homozygous interruption of the CTM by disrupting the DCRD helix with an HA-tag. We show that this induces "short" gating properties in this mutant full-length variant. Homozygous mice are viable and display no gross anatomical and functional abnormalities. Expression of the HA-tagged full-length channel could be detected in mouse whole brain membrane preparations. Heterozygous mice show no overt differences in locomotor activity during daytime.

Conclusions

We have successfully generated a mouse model which will enable us to study the physiological role of CTM function in vivo. It mimics the (permanent) pharmacological inhibition of CTM function and will thus allow predictions about its potential as a new drug target. Furthermore, the HA-tagged α1 subunit will provide a tool to specifically determine the expression of Ca_V1.3_L channels with anti-HA antibodies in mouse tissues.