Biophysical characterization of Ca_V1.4 L-type calcium channel mutants causing congenital stationary night blindness type 2 in humans

Ca_V1.4 L-type calcium channels show unique biophysical properties such as slow inactivation due to the lack of calcium-dependent inactivation (CDI). These properties make Ca_V1.4 channels appropriate candidates for triggering persistent glutamate release at retinal photoreceptor cell synapses. Mutations in the CACNA1F gene encoding for the Ca_V1.4 α1 subunit are described in patients with X-linked congenital stationary night blindness type 2 (CSNB2). Impaired transmission between rod photoreceptor cells and second-order neurons manifests as night blindness and various other visual symptoms in the affected individuals.

The aim of this study was to investigate the functional properties of Ca_V1.4 mutants L849P and R1816stop compared to wild-type (WT) in transiently transfected tsA 201 cells (+β3,+α2δ-1) via whole-cell patch clamp technique using 15 mM Ba²⁺ and Ca²⁺ as charge carrier. For statistics, either Mann-Whitney (two groups) or Kruskal-Wallis test and Dunn’s Post hoc test (multiple comparisons) were used.

L849P was mainly characterized by a reduced current density (pA/pF: WT: −16.3 ± 1.6 (n = 38), L849P: −2.5 ± 0.3 (n = 12), p < 0.0001; Ca²⁺), only minor, not significant (p > 0.05) changes in the voltage-dependent activation properties were observed. In presence of the dihydropyridin-activator BayK8644 (5 μM) the current density was increased ~10-fold (p < 0.001). The fold-increase in current density was comparable to WT. As expected R1816stop, which lacks an intrinsic C-terminal modulator (CTM), exhibited CDI (f-value: WT: 0.11 ± 0.03 (n = 8); R1816stop: 0.63 ± 0.02 (n = 22)) and shifted the voltage-dependence of activation to more negative voltages (V0.5_act in mV: WT: 1.8 ± 0.3 (n = 74), R1816stop: −12.3 ± 0.3 (n = 23)). In presence of the Ca_V1.4-CTM; comprising the last 122 C-terminal residues WT conditions were fully restored, e.g. V0.5_act 2.2 ± 1.0 mV (n = 14) (p < 0.0001).

We assume that the reduced current density observed in mutant L849P derives from decreased channel expression, which might be explained by a folding defect of the Ca_V1.4 channel protein rather than a reduced open probability. Moreover, the fact that the functional phenotype of the R1816stop can be rescued bears a potential pharmacotherapeutic concept based to the C-terminal modulatory mechanism present in Ca_V1.4 channels.

Financial support was given by the Austrian Science Fund (FWF, grant P22526 to A.K.).

Author notes

Affiliations

1. Department of Neurophysiology and Neuropharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, 1090, Vienna, Austria

   • Klaus W Schicker
   • , Verena Burtscher
   • , Dagmar Knoflach
   • , Thomas Stockner
   •  & Alexandra Koschak

2. Department of Pharmacology and Toxicology, Institute of Pharmacy, University of Innsbruck, 6020, Innsbruck, Austria

   • Anamika Singh

Corresponding author

Correspondence to Alexandra Koschak.

Klaus W Schicker, Verena Burtscher contributed equally to this work.

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