Characterization of CACNA1F mutations found in patients with a congenital form of night blindness

KW Schicker1, V Burtscher1, D Knoflach1, A Singh2, A Koschak1,2. 1Medical University Vienna, Department of Neurophysiology and -pharmacology, Austria, 2University of Innsbruck, Pharmacology and Toxicology, Institute of Pharmacy, Austria

CACNA1F encodes the alpha-subunit of Cav1.4 L-type calcium channels. Cav1.4 is mainly expressed in retinal sensory cells, such as photoreceptors and bipolar cells. Functionally they are characterized by a rather negative activation threshold and slow voltage-dependent inactivation accompanied by complete absence of calcium-dependent inactivation (CDI) making them suitable for tonic activity and continuous neurotransmitter release. In humans mutations in CACNA1F are known to cause X-linked congential stationary night blindness type-2 (CSNB2). Typical symptoms are moderate low visual acuity, myopia, nystagmus and variable levels of night-blindness. So far more than 50 mutations have been reported in the CACNA1F gene. However, most of these are not characterized functionally yet. Here we present an electrophysiological and biochemical characterization of two mutants heterologously expressed in tsA-201 cells together with beta3 and alpha2delta-1 accessory subunits. In whole-cell patch-clamp experiments, using 15mM calcium as charge carrier, a distal C-terminal truncation led to a hyperpolarizing shift in voltage-dependence of activation (V50 (mV): wildtype 1.7±0.6, n=74; mutant -11.9±0.6, n=23) and inactivation (V50 (mV): wildtype -18.9±1.8, n=22, mutant -28.9±2.3, n=14). The truncated channels elicited CDI which is explained by the loss of an intrinsic C-terminal modulator in this mutant. Co-expression of a distal C-terminal peptide however restored wildtype channel activity. A point mutation in the II-III linker, led to vastly diminished current density (n=10). This effect is either caused by a reduction in plasma membrane expression or by a change in single channel properties because Western blot analysis showed similar amount total protein compared to wildtype. A depolarizing shift in the voltage-dependence of activation due to a decrease in the slope was also observed. These data add to the accepted view that a reduction in calcium influx as well as a defect in calcium-dependent modulatory processes in Cav1.4 can cause visual defects in humans. Support: FWF-P22528 (AK).