RNA editing of the IQ domain in Ca(v)1.3 channels modulates their Ca²⁺-dependent inactivation

Abstract

Adenosine-to-inosine RNA editing is crucial for generating molecular diversity, and serves to regulate protein function through recoding of genomic information. Here, we discover editing within Ca(v)1.3 Ca²⁺ channels, renown for low-voltage Ca²⁺-influx and neuronal pacemaking. Significantly, editing occurs within the channel's IQ domain, a calmodulin-binding site mediating inhibitory Ca²⁺-feedback (CDI) on channels. The editing turns out to require RNA adenosine deaminase ADAR2, whose variable activity could underlie a spatially diverse pattern of Ca(v)1.3 editing seen across the brain. Edited Ca(v)1.3 protein is detected both in brain tissue and within the surface membrane of primary neurons. Functionally, edited Ca(v)1.3 channels exhibit strong reduction of CDI; in particular, neurons within the suprachiasmatic nucleus show diminished CDI, with higher frequencies of repetitive action-potential and calcium-spike activity, in wild-type versus ADAR2 knockout mice. Our study reveals a mechanism for fine-tuning Ca(v)1.3 channel properties in CNS, which likely impacts a broad spectrum of neurobiological functions.

Figure 1

Detecting RNA editing in the Ca_V1.3 IQ domain (A) Cartoon of Ca_V1.3 channel pore-forming α₁ subunit (α_1D), with hotspots for CaM/channel regulation shown on the amino terminus (NSCaTE (Dick et al., 2008)), and in the CI region of the carboxyl terminus (EF, EF-hand (Peterson et al., 2000); preIQ and IQ domains (Erickson et al., 2003; Pitt et al., 2001)). (B) DNA sequencing chromatogram from direct analysis of PCR of genomic DNA, yielding unique coding for IQ domain at this level. (C) Top, exemplar PCR products from RT-PCR analysis of rat thalamus. Bottom, direct sequencing results of RT-PCR from thalamus, showing distinct doublets of adenosine (A) and guanosine (G) (arrows) to either generate MQDY or IQDC from unedited IQDY. Colony screening reveals additional IQ domain editing from IQDY to IRDY. (D) Direct sequencing results for RT-PCR of cochlea, heart, DRG and pancreatic β-islets, showing identical patterns to the genomic analysis in panel B, indicating no editing.

Figure 2

RNA editing of Ca_V1.3 regulated by ADAR2 and during development (A) Schematic of ADAR2 mechanism. Transcription of pre-mRNA with formation of putative ECS duplex (top); recruitment of ADAR2 to duplex (second from top); conversion of adenosine to inosine (third from top); edited mature transcript ready for exportation to cytoplasm and translation (bottom). (B) Profile of editing in mouse lumbar and whole brain. Left column, direct DNA sequencing of mouse RT-PCR products. Right column, percent editing at three locations (I-to-M, Q-to-R and Y-to-C), as calculated by measuring electropherograms heights for adenosine versus guanosine (translucent bars), or by colony counting from colony screening analysis (filled bars). (C) No editing in ADAR2^−/−/GluR-B^R/R knockout mice. Format as in panel B. (D) Developmental profile of RNA editing in mouse and rat brains. Left panel, direct DNA sequencing of RT-PCR products from brains of different ages (n = 3 animals per age). Right panel, percent editing at three locations (I to M, Q to R and Y to C), as calculated by measuring electropherograms heights for adenosine versus guanosine (embryonic day 14, unfilled bars at zero level; postnatal day 4, translucent bars; and postnatal day 7, filled bars). (E) Equivalence of peak-height and area metrics for RNA editing, based on sequencing chromatography. Symbols represent editing percentages calculated via peak heights of sequencing chromatograms, plotted as a function of editing percentages calculated via area under sequencing chromatograms, performed for data sets in panel B. Open circles, lumbar; filled circles; whole brain; solid line, line of identity. (F) Overall frequency distribution of Ca_V1.3 IQ-domain variants, taken from mouse whole-brain via colony counting method. IRDC combination was never observed.

Figure 3

Modulation of Ca²⁺-dependent inactivation by IQ domain editing (A) Wildtype (IQDY) channel gating properties. Top panel, exemplar traces of currents evoked from holding potential of −90 mV to test potential of −10 mV, with Ba²⁺ as charge carrier (black), Ca²⁺ as charge carrier (red). Throughout, vertical scale bar pertains to Ca²⁺ current, and Ba²⁺ currents are scaled down ~3× to facilitate visual assessment of kinetic decay. Second panel, averaged inactivation profiles shown by r₅₀, the fraction of peak currents remaining after 50-ms depolarization to indicated voltages (V). VDI characterized by Ba²⁺ data; CDI was quantified by a f-value that is the difference of r₅₀-values between Ca²⁺ and Ba²⁺ profiles at −10 mV. Symbols represent the average of n = 10 cells. Third panel, semi-log plot of fractional recovery from Ca²⁺-dependent inactivation. Symbols are averages of 9–11 cells. Fourth panel, Ba²⁺ tail-activation curves averaged from ~15 cells. Bottom panel, peak Ba²⁺ current versus voltage relation, averaged from 10–13 cells. Currents were normalized to maximum values before averaging. For second through bottom panels, s.e.m. bars are shown when larger than symbol size. (B) Gating profile for MQDY channel variant. Format as in panel A. Slowed CDI onset and enhanced CDI recovery apparent in top three panels, with wildtype profiles reproduced as dashed gray curves, and shaded region emphasizing important differences. Activation gating unchanged, as shown in bottom two panels. (C) Gating profile for IRDY variant, showing lesser effects on CDI. Format as in panel B. (D) Gating profile for MRDY variant, showing strongest effects on CDI. Format as in panel B.

Figure 4

Comparison of SCN rhythmicity in wild-type and ADAR2 knockout mice. (A₁) Analysis of RNA editing of Ca_V1.3 IQ domain in mouse SCN. Format as in Figure 2. (A₂) Timecourse of normalized current decay in voltage-clamped SCN neurons in acute slice preparations. Overall format as in Figure 2A, top. Blue trace averaged from 7 wildtype neurons, with 10 mM Ba²⁺ as charge carrier. Cyan trace averaged from 6 knockout neurons, with 10 mM Ba²⁺ as charge carrier. Black trace averaged from 7 wildtype neurons, with 10 mM Ca²⁺. Red trace averaged from 6 knockout neurons, with 10 mM Ca²⁺. Ca²⁺ traces shown for 0-mV depolarizing voltage step. Ba²⁺ trace shown for −10-mV step, to account for ~10-mV surface-charge shift. (A₃) Analysis of inactivation in SCN neurons, using r₁₀₀ metric defined as the fraction of peak current remaining after 100-ms depolarization. Ba relation averaged from 13 wildtype or knockout neurons. Ca WT relation averaged from 7 wildtype neurons. Ca KO relation averaged from 5–6 knockout neurons. Red shading highlights effects of editing on CDI, with P<0.05 difference between wildtype and knockout relations denoted by *. (B) Na spikes in wildtype (WT, top black) and knockout mouse (KO, bottom red) SCN neurons. (C) Overlays of time-aligned Na spikes from preparation in panel B, confirming diminished depolarization prior to Na spikes in KO mice. Solid lines represent graphically the mean depolarization rate averaged over multiple spikes. Symbols plot mean depolarization rate (±SEM) preceding Na spikes in exemplar wild-type versus KO mouse presented in panel B. (D) Average frequency of action potential (Na spikes) in wild-type (n = 6) versus KO (n = 10) mouse SCN neurons. Upper scale tick, 1 Hz. Asterisk denotes significance at P < 0.05 level. (E) Ca spike activity (in 1 µM TTX) recorded in wildtype (top, black) versus knockout mouse SCN neurons (bottom, red). In the knockout, Ca spike frequency is decreased, and troughs are depolarized. Red trace at extreme bottom shows abolition of Ca spikes by 10 µM nimodipine. (F) Time-aligned, averaged Ca spikes confirm that KO mouse neurons manifest a depolarization of minimal troughs between spikes. Data averaged from n = 6 WT and n = 7 KO mouse SCN slices. Bars, standard error of mean. (G) Decreased average frequency of Ca spikes in wild-type versus KO mouse SCN slices analyzed in panel F (p < 0.01). Format as in panel D. (H) Exemplar Ca spikes from wild-type mouse SCN slice, illustrating effect of Bay K 8644 to increase spike frequency and deepen troughs between spikes. (I) Time-aligned, averaged Ca spikes confirm that Bay K 8644 deepens troughs between Ca spikes. Averaged from n = 11 mouse SCN slices. Format as in panel F. (J) Increased average frequency of Ca spikes in wild-type SCN slices exposed to 1–10 µM Bay K 8644 (P < 0.05), averaged from same slices as analyzed in panel I. Format as in panel D.

Figure 5

In silico simulations of experimental changes in SCN activity via altered Ca_V1.3 CDI (A) Ca_V1.3 gating mechanism and operational profiles utilized in simulations of spontaneous SCN activity. Left, gating mechanism, with first-order activation (rate constants a_r and b_r have customized voltage dependence), and with slow-CaM CDI formulation appropriate for C-lobe-dominant inactivation of these channels. Middle, simulated CDI profiles during voltage-step protocols, illustrating appropriate behavior for wild-type condition (WT, denoting mixture of mainly MQ and IQ channels) and ADAR2 knockout condition (KO, denoting pure IQ population). Compare to Figure 3 for appropriateness of simulated CDI behavior. Right, simulated WT (with mixture of MQ and IQ channels) and WT plus Bay K 8644 (slowed CDI and augmented peak current of mixture of MQ and IQ channels). See Supplementary Information, section 6, for detailed computational methods and setup. (B) Na spikes seen with WT and KO Ca_V1.3 profiles in panel A (middle). Note the lower frequency in the ADAR2 KO setting, quantified in panel D below. (C) Decreased depolarization rate prior to Na spikes in KO. Format as in Figure 4C. (D) Decreased frequency of simulated Na spikes in KO configuration. Upper scale tick, 1 Hz. (E) Ca spikes seen with WT and KO Ca_V1.3 profiles in panel A (middle). Na currents eliminated to produce Ca spikes. Note the lower frequency and depolarization of troughs between spikes in the ADAR2 KO setting; these trends are quantified further in panels F and G. (F) Expanded view of Ca spikes, confirming trough depolarization in KO configuration. (G) Bar graph summary of drop in Ca spike frequency in KO configuration. Format as in D. (H) Bay K 8644 increases Ca spike frequency, and hyperpolarizes troughs between spikes. Ca_V1.3 profile as in panel A (right column). Trends shown at higher resolution in panels I and J. (I) Hyperpolarization of troughs between Ca spikes, upon addition of Bay K 8644. (J) Increased Ca spike frequency with Bay K 8644. Format as in panel D.

Figure 6

Schematic overview of RNA editing effects on Ca_V1.3 Ca²⁺-dependent inactivation (CDI) and calcium load in neurons. Channel schematic as defined in Figure 1A. Increased RNA editing (right scenario) favors channels with MQ and other edited versions of the IQ domain, decreasing overall CDI and presumably increasing cellular Ca²⁺ load (intense yellow-green coloration). Decreased editing (left scenario) favors default channels with IQ version of IQ domain, increasing overall CDI and potentially decreasing cellular Ca²⁺ load (weak yellow-green coloration). Actual neurons reside on a continuum between these two extremes, as represented by ramp schematics at bottom.