Convergent evolution of sodium ion selectivity in metazoan neuronal signaling

Abstract

Ion selectivity of metazoan voltage-gated Na(+) channels is critical for neuronal signaling and has long been attributed to a ring of four conserved amino acids that constitute the ion selectivity filter (SF) at the channel pore. Yet, in addition to channels with a preference for Ca(2+) ions, the expression and characterization of Na(+) channel homologs from the sea anemone Nematostella vectensis, a member of the early-branching metazoan phylum Cnidaria, revealed a sodium-selective channel bearing a noncanonical SF. Mutagenesis and physiological assays suggest that pore elements additional to the SF determine the preference for Na(+) in this channel. Phylogenetic analysis assigns the Nematostella Na(+)-selective channel to a channel group unique to Cnidaria, which diverged >540 million years ago from Ca(2+)-conducting Na(+) channel homologs. The identification of Cnidarian Na(+)-selective ion channels distinct from the channels of bilaterian animals indicates that selectivity for Na(+) in neuronal signaling emerged independently in these two animal lineages.

Graphical abstract

Figure 1

Current Recordings from NvNa_v2.1, NvNa_v2.2, and NvNa_v2.1^DEKA Channels Expressed in Xenopus Oocytes Oocytes were clamped at −80 mV holding potential, and currents were elicited by 200 ms depolarizations from −75 mV to 50 mV. (A) Ca²⁺-activated Cl⁻ currents recorded in ND96 bath solution from an oocyte expressing NvNa_v2.1. (B–E) NvNa_v2.1 currents recorded in bath solution with Ba²⁺ substituting for Ca²⁺, and in addition with choline substituting for Na⁺ (C) and also without Ba²⁺ as control (see inset). See Figure S1 for further characterization of NvNa_v2.1. (D) Current-voltage relations of NvNa_v2.1 (circles: E_rev = 16.2 ± 0.8 mV; n = 14) and with choline substituting for Na⁺ (triangles: E_rev = 13.3 ± 0.9 mV; n = 7). (E) Inward currents elicited by 200 ms depolarizing pulse to −30 mV in the presence of increasing concentrations of lidocaine. The inhibitory effect of lidocaine was removable by washes with bath solution (gray). (F) Outward and tail currents elicited by 1 s depolarizations from −75 mV to 50 mV, measured for an oocyte expressing NvNa_v2.2 in ND96 bath solution. (G and H) Currents decreased in the presence of 5 mM lidocaine (G) and were eliminated when Ca²⁺ was substituted with Ba²⁺ ions in the bath solution (H). (I and J) NvNa_v2.1^DEKA currents in ND96 bath solution (I) and with choline substituting for Na⁺ (J). (K) Current-voltage relations of NvNa_v2.1^DEKA in ND96 bath solution (E_rev = 16.7 ± 1.1 mV; n = 14). Each point represents the mean ± SEM of n cells. See also Figure S1.

Figure 2

Current-Voltage Relations for NvNa_v2.1 Mutants in Na⁺ and K⁺ Single Ion Solutions and Relative Permeabilities Currents were elicited for 200 ms from −95 up to 70 mV from a holding potential of −100 mV. (A–D) Current-voltage relations of representative oocytes expressing NvNa_v2.1 (A), NvNa_v2.1^DEKA (B), NvNa_v2.1^DKEA (C), or NvNa_v2.1^{NvNav2.5(p−loops)} (D). Circles: Na⁺ single ion solution; squares: K⁺ single ion solution. The relative ion permeability was calculated from the difference in reversal potential between K⁺ and Na⁺ single ion solutions of identical concentrations (see Experimental Procedures). The values provided are the mean ± SEM of n cells. The protonation state of each SF residue is indicated in parentheses, with x designating an uncharged residue. See also Figure S2.

Figure 3

Sequence Alignment of NvNa_v2 Channels and Current Recordings from NvNa_v2.1^DKEA and NvNa_v2.1^{NvNav2.5(p−loops)} Channels Expressed in Xenopus Oocytes (A) Alignment of the pore-loop regions of the five N. vectensis (Nv) channels (see also Figure S3 for spatiotemporal expression of Na_v2 cnidarian channels), as well as a channel from the medusae P. penicillatus (Pp) and C. capillata (Cc) and the mammalian brain channel Na_v1.2. Substitutions of NvNa_v2.1^{NvNav2.5(p−loops)} are underlined and substitutions unique to the Na_v2.5 channel subfamily are in yellow boxes. For current recordings the oocytes were clamped at −80 mV holding potential and currents were elicited by 200 or 500 ms depolarizing voltage pulses from −75 mV to either 50 or 70 mV. (B) NvNa_v2.1^DKEA in ND96 bath solution. (C) NvNa_v2.1^DKEA with choline substituting for Na⁺ in the bath solution. (D) NvNa_v2.1^DKEA with Ca²⁺ in the bath solution chelated by EGTA. (E and F) NvNa_v2.1^{NvNav2.5(p−loops)} in ND96 bath solution (E) and with choline substituting for Na⁺ (F). (G) Current-voltage relations of NvNa_v2.1^{NvNav2.5(p−loops)} (E_rev = 46.5 ± 1.2 mV; n = 19). Each point represents mean ± SEM of n cells (see Figure S2 for analysis of the SF in NvNa_v2.1^{NvNav2.5(p−loops)}).

Figure 4

Phylogeny of Voltage-Gated Sodium Channels A maximum-likelihood tree was constructed using the LG (+F +G +I) model. The bootstrap support out of 100 is indicated at the branches. A Bayesian analysis using the WAG model resulted in identical topology. Posterior probabilities of 1.0 are indicated by a red asterisk, and those of 0.95 < X < 1.0 are indicated by a blue asterisk. All sequences are from cloned cDNA unless otherwise mentioned. Accession numbers and species names are available in Table S1. Animal clades are indicated by colors.

Figure S1

Current Recordings from NvNa_v2.1 Expressed in Xenopus Oocytes, Related to Figure 1 Oocytes were clamped at −100 or −80 mV holding potential in ND96 bath solution, and currents were elicited by depolarizing pulses from −75 mV to 50 mV for 200 ms. (A) Current-voltage relations of calcium-activated chloride channels from a representative oocyte, which were activated by Ca²⁺ influx through NvNa_v2.1 (E_rev = −15.9 mV). (B) Ca²⁺-activated Cl⁻ currents recorded from an oocyte expressing NvNa_v2.1. Addition of 5 mM lidocaine reduced the Ca²⁺ inward current through NvNa_v2.1, which decreased the chloride currents. (C) Current-voltage relations of NvNa_v2.1 expressed in oocytes injected with BAPTA prior to the measurement (E_rev = 21.6 ± 2.2 mV; n = 7). Each point represents mean ± SEM of n cells. Injection of BAPTA to oocytes eliminated the Ca²⁺-activated Cl⁻ currents (see inset). (D) NvNa_v2.1 inward current elicited by 200 ms depolarizing pulse to −30 mV with BaCl₂ substituting for CaCl₂ in the ND96 bath solution. The current was not affected by 100 μM TTX (red).

Figure S2

Currents Mediated by Channel Mutants NvNa_v2.1^{NvNav2.5(p−loops DEEA)} and NvNa_v2.1^{NvNav2.5(p−loops DEKA)} Expressed in Oocytes, Related to Figure 3 (A) Currents elicited by 500 ms depolarizing pulses from −75 to 50 mV mediated by NvNa_v2.1^{NvNav2.5(p−loops DEEA)} in ND96 bath solution. Note the large tail currents upon returning to the −80 mV holding potential. (B) Inward currents elicited by 200 ms depolarizing pulses from −75 to 50 mV mediated by NvNa_v2.1^{NvNav2.5(p−loops DEKA)} in ND96 bath solution. Note the absence of tail currents (see inset). The E_rev value obtained for the current-voltage relations is 19.1 ± 1.6 (n = 6). Each point represents mean ± SEM of n cells. (C) Currents elicited by 200 ms depolarizing pulses from −90 to 50 mV mediated by NvNa_v2.1^{NvNav2.5(p−loops DEKA)} in K⁺ single ion bath solution.

Figure S3

Spatiotemporal Expression of Cnidarian Na_vs, Related to Figure 3 (A) Developmental time series of expression for the five Na_v2 channel genes from N. vectensis. The data were generated from qPCR for five developmental stages. Expression is presented in molecules per μl cDNA. Bars represent mean + SE of n = 3 replicates. (B–F) In situ hybridization with probes for NvNa_v2.1 (B), NvNa_v2.2 (C), NvNa_v2.3 (D), NvNa_v2.4 (E), and NvNa_v2.5 (F) revealed the spatial expression pattern of the different channel subtypes in 5-day-old N. vectensis larvae. A similar assay on C. hemisphaerica revealed the spatial expression patterns of two channel homologs. (G–J) Probes for ChNa_v2.1 (G) and ChNa_v2.5 (H) in the gastrozoid developmental stage, and for ChNa_v2.1 (I) and ChNa_v2.5 (J) in the medusa stage. The oral end is indicated by an asterisk.