Figure 1.
Amino acid sequence conservation of calmodulin binding, amino terminal (NSCaTE) and carboxyl terminal (Pre-IQ/IQ) motifs in L-type Cav1 channels.
(A) Running window of amino acid similarity of aligned Cav1.2, Cav1.3 and four representative invertebrate L-type channels (red asterisk in B). DI, DII, DIII, DIV are the location of the four major domains, each domain consisting of six transmembrane helices. (B) Multiple alignment of N-terminal sequences illustrating the conservation of NSCaTE and a downstream methionine (Met2) in L-type channels of coelomate animals. (C) C-terminal sequence alignments illustrating the nearly invariant Pre-IQ/IQ motifs in coelomate animals, with a recognizable “IQ” even in single-celled, Paramecium.
Figure 2.
Gene tree of L-type calcium channels illustrating the evolution of calmodulin binding, amino terminal (NSCaTE) and carboxyl terminal (Pre-IQ/IQ) motifs in L-type Cav1 channels.
All animal phyla including single cell protozoans (such as Paramecium) and simple multicellular organisms (placozoans, sponge) have an IQ motif. A N-terminal NSCaTE (xWxxx(IorL)xxxx) motif evolved in common invertebrate ancestors (coelomates) and retained in all major phylogenetic groups. While the Pre-IQ and IQ motifs are featured in all L-type channels of metazoans, the NSCaTE motif is missing in some Arthropod species, including many insects, suggesting that NSCaTE is not an essential feature of L-type channels. NSCaTE was lost in Cav1.1 and Cav1.4 after the speciation of L-type channels to four gene isoforms in vertebrates. Almost every NSCaTE containing L-type channel has a downstream methionine (Met2) from the start codon (Met1) which could serve as an alternative translational start site for inclusion (Met1) or exclusion (Met2) of NSCaTE in L-type channels.
Figure 3.
No difference in levels between snail LCav1 (L-type) channel mRNA transcripts containing NSCaTE (LCav1-Met1) and those lacking NSCaTE (LCav1-Met2).
A) Illustration of the location of primer sets for qPCR amplification of snail mRNA transcripts containing or lacking NSCaTE in LCav1 channels. Numbers refer to the position of the DNA sequence from LCav1-Met1 translational start site. B) Graph illustrates the lack of significant difference in mRNA expression levels (relative to HPRT control values, scaled such that the sum of all signal values across tissues are equal) in whole animals or in specific tissues of snails containing or lacking the LCav1NSCaTE sequence. Degree of correlation with adjusted R2 value = 0.95.
Figure 4.
No major differences in biophysical properties between snail LCav1 channel containing a full-length N-terminus with NSCaTE translated from upstream methionine Met1 or truncated N-terminus missing NSCaTE generated from the downstream methionine Met2.
LCav1-Met1 and LCav2-Met2 were transfected in HEK-293T cells alongside mammalian α2δ1 and β1b accessory subunits and recorded in 10 mM Barium (Ba) or Calcium (Ca) containing extracellular solution using patch clamp electrophysiology. Intracellular solution contained 9 mM EGTA. (A) Representative current traces generated from voltage steps (−40 mV to 60 mV in 10 mV steps) from a holding potential of −60 mV) illustrating the typical buffer resistant (9 mM EGTA) calcium-dependent inactivation when calcium is the charge carrier, leaving residual voltage-dependent inactivation when barium replaces calcium in the extracellular solution. (B) Normalized current-voltage relationships (n = 10), transformed and Boltzmann-fitted as activation curves in (C). (D) Steady-state availability curves (Baex: n = 10, Caex: n = 6), generated by measuring the fraction of maximal current generated after a 10 s sustained prepulse voltage from −100 to +50 mV in 10 mV steps). (E) Time of recovery from inactivation (n = 4) measured as the fraction of maximal current recovery after time delays, plotted on a log scale.
Table 1.
Summary of biophysical parameters of LCav3 channel variants containing exons 8b and 25c expressed in HEK-293T cells, with one-way analysis of variance to assess statistical significance.
Figure 5.
Full-length LCav1-Met1 channels containing NSCaTE have a buffer-sensitive form of calcium dependent inactivation not found in truncated LCav1-Met2 channels lacking NSCaTE.
LCav1-Met1 and LCav2-Met2 were transfected in HEK-293T cells alongside mammalian α2δ1 and β2a accessory subunits and recorded using patch clamp electrophysiology. (A) Overlapping sample traces illustrating the ultra-fast calcium-dependent inactivation (red trace) of LCav1-Met1 channel currents in low (0.5 mM) EGTA buffering conditions. (B) Graph of fraction of peak current size at 300 ms time point of inactivation decay (R300). * represents a statistically significantly smaller R300 value (p<0.001, ANOVA) for LCav1-Met1 calcium currents (red trace), reflecting the buffer-sensitive calcium dependent inactivation uniquely in LCav1 channels with a full N-terminus, containing NSCaTE.
Figure 6.
Snail NSCaTELCav1 and mammalian NSCaTECav1.2 peptides assume an alpha-helix upon binding calmodulin (CaM).
(A) 17 mer NSCaTE and 21 mer IQ peptide sequences are used in circular dichroism (Figures 6) and Gel Shift Mobility Assays (Figures 7). (B,C) Differential spectra of NSCaTE peptide indicate a helical transformation with snail and mammalian NSCaTE upon addition of the helix stabilizing agent, trifluoroethanol (TFE: dashed line; no TFE: solid line, Figure 6B) or upon addition to CaM (solid line = 1∶1 CaM:peptide, dashed line = 1∶2 CaM:peptide, Figure 6C). Each trace is obtained by subtracting the 10 µM CaM-alone trace from the corresponding NSCaTE+CaM spectrum. Y axis units are mean residue ellipticity or (θ), (difference in spectra corrected for total protein/peptide concentration).
Figure 7.
Gel Mobility Shift Assays illustrate that snail NSCaTE of LCav1 and mammalian NSCaTE of Cav1.2 can displace calcium-calmodulin (Ca2+-CaM) prebound to either snail IQ or mammalian IQ motifs.
Gel shift mobility assays of CaM and individual peptides (A) or CaM pre-bound to an IQ peptide competing with increasing amounts of NSCaTE (B). Each lane contains 300pmol wild-type Ca2+-CaM; first lane is a CaM-only control for reference (Positions #2,#3,#5). (A) Each subsequent lane has increasing ratios of C-terminal (IQ) and/or N-terminal NSCaTE peptides at the ratios indicated. The changing conformation of CaM by IQ peptide causes a mobility shift of the CaM band (from Positions #2 → #1, top panels). Neither snail NSCaTELCav1 or mammalian NSCaTECav1.2 changed CaM mobility alone, at any ratio (Position #3, bottom panels). (B) First lane is again Ca2+-CaM-only control (Position #5). Second lane is Ca2+-CaM with 1.5×IQ peptide alone control (for the maximum shift reference, Position #4). In subsequent lanes, increasing the amount of added NSCaTE peptide eventually reversed the slow mobility of CaM-IQ peptide back to the faster mobility of CaM without IQ peptide (Position #4 → #5), but not at a 100% effectiveness.
Figure 8.
Isothermal Calorimetry (ITC) analysis indicates a 2∶1 stoichiometry of NSCaTE:CaM in the absence of IQ, and a 1∶1 NSCaTE:CaM stoichiometry when CaM is first pre-bound to IQ.
(A) Representative raw sample data for several CaM-peptide titrations. (B) Summary Table of ITC data. NSCaTE or IQ peptides were titrated into Ca2+-CaM alone or Ca2+-CaM pre-bound to a competing peptide at a 1∶1 ratio. All binding reactions exhibited negative enthalpy under the experimental conditions used (exothermic, ΔH <0). Despite the lack of mobility shift of Ca2+-CaM (Figure 7), NSCaTE does bind Ca2+-CaM alone. LCav1 and Cav1.2 IQ peptides have a higher affinity (Kd = 80 nM and 130 nM, respectively) for Ca2+-CaM than do either of the NSCaTE peptides (Kd = 0.83 µM and 3.24 µM for Cav1.2 and LCav1 NSCaTEs). Both mammalian NSCaTECav1.2 and snail NSCaTELCav1 is able to bind to Ca2+-CaM pre-bound to IQ motifs, and both IQ peptides are able to bind to CaM when it is first bound to NSCaTE, which is consistent with the possibility of a NSCaTE-CaM-IQ complex in vivo. N values indicate that NSCaTELCav1 and NSCaTECav1.2 can bind to Ca2+-CaM in a 2∶1 stoichiometry, consistent with NSCaTE possibly binding simultaneously to both the N- and C-lobes of CaM, as previously reported [30].