Continuously tunable Ca(2+) regulation of RNA-edited CaV1.3 channels

Abstract

CaV1.3 ion channels are dominant Ca(2+) portals into pacemaking neurons, residing at the epicenter of brain rhythmicity and neurodegeneration. Negative Ca(2+) feedback regulation of CaV1.3 channels (CDI) is therefore critical for Ca(2+) homeostasis. Intriguingly, nearly half the CaV1.3 transcripts in the brain are RNA edited to reduce CDI and influence oscillatory activity. It is then mechanistically remarkable that this editing occurs precisely within an IQ domain, whose interaction with Ca(2+)-bound calmodulin (Ca(2+)/CaM) is believed to induce CDI. Here, we sought the mechanism underlying the altered CDI of edited channels. Unexpectedly, editing failed to attenuate Ca(2+)/CaM binding. Instead, editing weakened the prebinding of Ca(2+)-free CaM (apoCaM) to channels, which proves essential for CDI. Thus, editing might render CDI continuously tunable by fluctuations in ambient CaM, a prominent effect we substantiate in substantia nigral neurons. This adjustability of Ca(2+) regulation by CaM now looms as a key element of CNS Ca(2+) homeostasis.

Figure 1. Functional effects of RNA editing of Ca_V1.3 channels, hypothesized to occur as perturbation of Ca²⁺/CaM complexed alone with channel IQ domain

(A) Schematic of main pore-forming α_1D subunit of Ca_V1.3 channel. Shown are cytoplasmic amino (N) and carboxy (C) termini, containing main elements implicated in CDI. CI, Ca²⁺ inactivation region spanning proximal channel carboxy tail (~160 aa). CI contains elements involved in CaM regulation. IQ domain (IQ), terminal CI segment (~30 aa) believed preeminent in binding CaM. Dual vestigial EF-hand region (EF) spanning proximal ~100 aa of CI. NSCaTE element on channel N terminus of Ca_V1.2 and Ca_V1.3 channels, proposed as N-lobe Ca²⁺/CaM effector site (Dick et al., 2008; Tadross et al., 2008). (B) Popular hypothesis about how CDI arises from CaM interactions with elements described in panel A. In this view, Ca²⁺/CaM binding to the IQ element alone (right) triggers CDI. ApoCaM may prebind to the IQ element in a different way (left), positioning CaM as a ‘resident’ Ca²⁺ sensor. (C) Homology model of Ca²⁺/CaM complexed with Ca_V1.3 IQ domain (dark blue helix, carboxy-terminal end to right). Ca²⁺ ions, yellow balls. (D) Exemplar recombinant Ca_V1.3 whole-cell currents expressed in HEK293 cells. Leftmost subpanel pertains to prototypic channels with IQDY version of IQ domain. 0.2 nA scale bar pertains to Ca²⁺ current (red) throughout. Black Ba²⁺ current scaled down ~3× to facilitate comparison of decay kinetics, here and throughout. CDI metric, defined at right. Other subpanels pertain to various RNA edited variants, demonstrating a spectrum of reduced CDI strengths. Parentheses contain percent of corresponding transcripts across mouse brain. (E) FRET 2-hybrid assay for interaction of Ca_V1.3 IQ domain and Ca²⁺/CaM. Left, cartoon of relevant FRET pair. Right, 3³-FRET binding curve plots FRET efficiency (E_A, from YFP standpoint) versus free concentration of CFP-CaM_WT (D_free). Each symbol, mean ± SEM of ~19 cells. Smooth curve, E_A = E_A,max· D_free/(K_d,EFF+ D_free), where E_A,max equals plateau value and K_d,EFF is given by x-intercept of vertical dotted line. Green bar calibrates D_free units to nM. (F) Proposed strategy for quantitative confirmation of RNA editing mechanism involving Ca²⁺/CaM complex with IQ element, as portrayed in panel C. CDI metric of particular construct is to be plotted versus corresponding effective association constant K_a,EFF, deduced from data as in panel E. Symbols and fit portray hypothetical outcome: green symbol, prototypic IQ species; black symbols, RNA edited variants; smooth fit, Langmuir function as described in main text.

Figure 2. Ca²⁺/CaM effector role of IQ domain to explain functional effects of RNA editing?

(A)Population data for CDI metric of different RNA editing variants (left cluster in blue), and of various point-alanine substitutions (right cluster, rose and gray). Metric for prototypic IQDY species shown in green. Rose bars, strongest CDI reduction by mutations. Dashed-gray bars, mutations without appreciable CDI effects that were nonetheless chosen at random for subsequent Ca²⁺/CaM binding analysis. Bars show mean ± SEM, derived from 4–6 cells each for editing, and ~6 cells each for the alanine scan. (B)Exemplar current traces corresponding to indicated point-alanine substitutions. Format as in Figure 1D. (C) FRET 2-hybrid interaction curves for Ca²⁺/CaM versus IQ domain of point-alanine substitutions. As reference, green curve reproduces fit for prototypic IQDY species (from Figure 1E). Black data and fits correspond to whole-cell currents directly above in panel B. Each symbol bins data from ~3, 4, 7, and 8 cells (left to right). (D) FRET 2-hybrid interaction curves for Ca²⁺/CaM versus IQ domain of RNA editing variants. Format as in panel C.Each symbol bins data from ~7, 6, 9, and 6 cells (left to right). (E) CDI plotted as a function of K_a,EFF deviates from Langmuir function (Figure 1F), arguing against CDI reduction arising from diminished Ca²⁺/CaM with solitary IQ element acting as effector site. Horizontal bars, standard deviation of K_a,EFF deduced from Jacobian error analysis (Johnson, 1980).

Figure 3. ApoCaM prebinding role of IQ domain explains effects of RNA editing

(A) Channels require preassociation with apoCaM (configuration A) to undergo CDI. Channels without apoCaM (configuration E) cannot undergo CDI. RNA editing and alanine scanning might perturb association constant K_a for apoCaM preassociation, thereby reducing CDI by populating configuration E. (B) Population data for CDI metric under strong overexpression of CaM_WT (CDI_CaMhi). Different RNA editing variants (left cluster in blue). Various point-alanine substitutions (right cluster, rose and gray). Metric for prototypic IQDY species shown in green. Rose bars, strongest CDI reduction by mutations in Figure 2A. Gray bars, mutations without appreciable CDI effects in Figure 2A that were nonetheless chosen for CaM overexpression studies. Dashed-gray bars, subset of gray-bar constructs chosen at random for additional FRET analysis of apoCaM binding below. Bars show mean ± SEM derived from ~5 cells each. (C) Exemplar current traces for selected constructs during CaM overexpression. Leftmost subpanel, prototypic IQDY species. Other subpanels, RNA-editing variants and point-alanine-substitution constructs, as labeled. Format as in Figure 1D. (D) FRET assays of apoCaM binding to entire Ca_V1.3 CI region (cartoon at left). FRET 2-hybrid interaction curves. Green data and fit within MQDY subpanel display properties for IQDY construct (symbols bin ~10 cells each). Black data, interaction data and fit corresponding to RNA editing variants and alanine-substituted constructs, as labeled. Symbols average ~4, 4, 6, and 5 cells each (left to right). (E) Plotting the ratio of CDI/CDI_CaMhi (from Figures 2A, 3B) as function of K_a,EFF decorates Langmuir curve (black line), arguing for apoCaM preassociation function of the IQ domain. Wild-type data in green. (F) Homology model of C-lobe of apoCaM (cyan) complexed with Ca_V1.3 IQ domain (blue). Left, complex with prototypic IQ domain showing hotspots in red. Right, model with MQDY variant, showing potential steric clash of M[0] with apoCaM.

Figure 4. Tuning of CDI by CaM in substantia nigral neurons (SNc)

(A)Refocusing new mechanistic scheme to emphasize how changes in ambient apoCaM levels (ΔapoCaM) may continuously tune strength of CDI. (B) Confocal image of SNc neuron expressing GFP under tyrosine hydroxylase promoter. (C) Whole-cell currents from SNc neurons, averaged from n = 7 (left) and n = 6 (right)cells. Format as in Figure 1D. Left, with endogenous levels of CaM. Right, after strongly increasing CaM levels via pipet dialysis of recombinant CaM_WT. (D) Bar graph summary of data at left. (E) CDI-CaM response curves for various RNA editing species, deduced from FRET 2-hybrid data in Figure 3D. Black curves, RNA editing species in splice variants lacking a competitive inhibitor ICDI module. Blue curves, parallel behavior of RNA editing species in splice variants containing an ICDI module (Extended Discussion). (F) Aggregate CDI-CaM response curve (black), averaged over the curves of the entire population of RNA-edited species. Weighting factors specified by transcript and splice prevalence in substantia nigra. Gray and blue zones and curves reproduce response characteristics of individual RNA editing variants. Dashed lines, CDI response and CaM estimates for SNc neurons before and after CaM supplementation, taken from panel D. (G) Conceptual scheme of Ca²⁺ homeostasis, incorporating continuous CaM tuning of CDI strength as projected in panel F. Gray outline, generic pacemaking neuron with depolarizing Ca_V1.3 and hyperpolarizing SK channels. Ca²⁺ negative feedback gain on Ca_V1.3 opening is continuosly adjustable by CaM (as in panel F), in the manner of a rheostat-controlled gain element.