Abstract
Voltage-gated Cav1 and Cav2 Ca2+ channels are comprised of a pore-forming α1 subunit (Cav1.1-1.4, Cav2.1-2.3) and auxiliary β (β1-4) and α2δ (α2δ-1-4) subunits. The properties of these channels vary with distinct combinations of Cav subunits and alternative splicing of the encoding transcripts. Therefore, the impact of disease-causing mutations affecting these channels may depend on the identities of Cav subunits and splice variants. Here, we analyzed the effects of a congenital stationary night blindness type 2 (CSNB2)-causing mutation, I745T (IT), in Cav1.4 channels typical of those in human retina: Cav1.4 splice variants with or without exon 47 (Cav1.4+ex47 and Cav1.4Δex47, respectively), and the auxiliary subunits, β2X13 and α2δ-4. We find that IT caused both Cav1.4 splice variants to activate at significantly more negative voltages and with slower deactivation kinetics than the corresponding WT channels. These effects of the IT mutation, along with unexpected alterations in ion selectivity, were generally larger in channels lacking exon 47. The weaker ion selectivity caused by IT led to hyperpolarizing shifts in the reversal potential and large outward currents that were evident in channels containing the auxiliary subunits β2X13 and α2δ-4 but not in those with β2A and α2δ-1. We conclude that the IT mutation stabilizes channel opening and alters ion selectivity of Cav1.4 in a manner that is strengthened by exclusion of exon 47 and inclusion of β2X13 and α2δ-4. Our results reveal complex actions of IT in modifying the properties of Cav1.4 channels, which may influence the pathological consequences of this mutation in retinal photoreceptors.
Highlights
Voltage-gated Cav[1] and Cav[2] Ca21 channels are comprised of a pore-forming a1 subunit (Cav1.1-1.4, Cav2.1-2.3) and auxiliary b (b1-4) and a2d (a2d2124) subunits
We analyzed the effects of a congenital stationary night blindness type 2 (CSNB2)-causing mutation, I745T (IT), in Cav1.4 channels typical of those in human retina: Cav1.4 splice variants with or without exon 47 (Cav1.41ex[47] and Cav1.4Dex[47], respectively), and the auxiliary subunits, b2X13 and a2d-4
The weaker ion selectivity caused by IT led to hyperpolarizing shifts in the reversal potential and large outward currents that were evident in channels containing the auxiliary subunits b2X13 and a2d-4 but not in those with b2 variants with exon 7A (b2A) and a2d-1
Summary
Exon 47 resides in the CTM of Cav1.4 (Fig. 1A); deletion of this exon, like the IT mutation, causes a large negative shift in the voltage dependence of channel activation (16, 18). Exponential fits of the rising phase of the peak currents yielded time constants for activation (tact) that were significantly longer (Table 2) and with weaker voltage dependence for Cav1.41ex47IT (v = 250.3 mV) than for Cav1.41ex[47] (v = 226.9 mV; F2,7 = 16.4, p = 0.002; Fig. 2, A and B). IT increased tdeact more than 10-fold for Cav1.4Dex[47] versus ;4fold for Cav1.41ex[47] upon repolarization to 260 mV (Table 2) These results indicate that deletion of exon 47 augments the gain-of-function effects of IT by modifying the kinetics and voltage-dependence of channel activation and deactivation. With intracellular solutions containing Na1 or K1, IT caused a negative shift in Erev and lowered PBa/Px (Fig. 8, Table 3) Taken together, these results signified a reduction in the ionic selectivity of Cav1.4Dex47IT compared with WT channels. The identity of the auxiliary b and a2d subunits critically determines the effects of IT on selectivity of Cav1.4
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