T-Type Ca2+ Channels Boost Neurotransmission in Mammalian Cone Photoreceptors

Abstract

It is a commonly accepted view that light stimulation of mammalian photoreceptors causes a graded change in membrane potential instead of developing a spike. The presynaptic Ca²⁺ channels serve as a crucial link for the coding of membrane potential variations into neurotransmitter release. Ca_v1.4 L-type Ca²⁺ channels are expressed in photoreceptor terminals, but the complete pool of Ca²⁺ channels in cone photoreceptors appears to be more diverse. Here, we discovered, employing whole-cell patch-clamp recording from cone photoreceptor terminals in both sexes of mice, that their Ca²⁺ currents are composed of low- (T-type Ca²⁺ channels) and high- (L-type Ca²⁺ channels) voltage-activated components. Furthermore, Ca²⁺ channels exerted self-generated spike behavior in dark membrane potentials, and spikes were generated in response to light/dark transition. The application of fast and slow Ca²⁺ chelators revealed that T-type Ca²⁺ channels are located close to the release machinery. Furthermore, capacitance measurements indicated that they are involved in evoked vesicle release. Additionally, RT-PCR experiments showed the presence of Ca_v3.2 T-type Ca²⁺ channels in cone photoreceptors but not in rod photoreceptors. Altogether, we found several crucial functions of T-type Ca²⁺ channels, which increase the functional repertoire of cone photoreceptors. Namely, they extend cone photoreceptor light-responsive membrane potential range, amplify dark responses, generate spikes, increase intracellular Ca²⁺ levels, and boost synaptic transmission.SIGNIFICANCE STATEMENT Photoreceptors provide the first synapse for coding light information. The key elements in synaptic transmission are the voltage-sensitive Ca²⁺ channels. Here, we provide evidence that mouse cone photoreceptors express low-voltage-activated Ca_v3.2 T-type Ca²⁺ channels in addition to high-voltage-activated L-type Ca²⁺ channels. The presence of T-type Ca²⁺ channels in cone photoreceptors appears to extend their light-responsive membrane potential range, amplify dark response, generate spikes, increase intracellular Ca²⁺ levels, and boost synaptic transmission. By these functions, Ca_v3.2 T-type Ca²⁺ channels increase the functional repertoire of cone photoreceptors.

Keywords: Cav3.2; calcium; cone photoreceptors; exocytosis; spike.

Figure 1.

The presence of I_LVA and I_HVA in cone photoreceptors. A, Example transmission microscope image of a patch-clamp recording from a cone photoreceptor terminal in a mouse horizontal retinal slice. White arrowhead, Cone photoreceptor terminal; black arrowhead, rod photoreceptor terminal. B₁, Example whole-cell patch-clamp recording from mouse cone photoreceptor terminals. Currents were monitored in response to a voltage ramp protocol from −89 to +49 mV, with a speed of 0.1875 mV/ms. Prepulse duration was 800 ms at different V_h values (black, −89 mV; gray, −39 mV). B₂, Example trace shows the current–voltage relationship of I_Ca. Arrowheads indicated I_LVA and I_HVA components. C₁–C₃, Peak amplitudes of I_LVA: V_h = −89 mV: −12.72 ± 2.15 pA, n = 12; V_h = −39 mV: −6.90 ± 1.53 pA, n = 12; p < 0.0001, paired t test. Peak amplitudes of I_HVA: V_h = −89 mV: −19.66 ± 3.33 pA, n = 12; V_h = −39 mV: −20.19 ± 3.45 pA, n = 12; p = 0.4511, paired t test. I_Ca peak position: LVA: −42.48 ± 0.46 mV, n = 12; HVA: −20.75 ± 0.71 mV, n = 12; p < 0.0001, Mann–Whitney test.

Figure 2.

Pharmacological separation of I_LVA from I_HVA. A₁, Example current–voltage relationship of ramp-evoked I_Ca in mouse cone photoreceptors in the presence and absence of 100 μm NiCl₂. Arrowheads indicate I_LVA and I_HVA component (LVA, HVA). A₂, Peak I_LVA amplitude: control: −11.03 ± 1.80 pA, n = 5; nickel (Ni²⁺): −6.22 ± 1.13 pA, n = 5; p = 0.0030, paired t test. Peak I_HVA amplitude: control: −37.45 ± 5.25 pA, n = 5; nickel (Ni²⁺): −11.01 ± 1.52 pA, n = 5; p = 0.0022, paired t test. B₁, Example current–voltage relationship of evoked I_Ca in the presence and absence of 1 μm mibefradil (Mibe). B₂, Peak I_LVA amplitude: control: −11.15 ± 0.94 pA, n = 6; mibefradil: −9.81 ± 0.93 pA, n = 6; p = 0.0641, paired t test. Peak I_HVA amplitude: control: −37.04 ± 3.48 pA, n = 6; mibefradil: −35.06 ± 3.27 pA, n = 6; p = 0.1712, paired t test. C₁, Example current–voltage relationship of evoked I_Ca in the presence and absence of 5 μm Z944. C₂, Peak I_LVA amplitude: control: −12.51 ± 1.58 pA, n = 7; Z944: −6.05 ± 0.82 pA, n = 7; p = 0.0003, paired t test. Peak I_HVA amplitude: control: −36.61 ± 4.06 pA, n = 7; Z944: −49.25 ± 6.04 pA, n = 7; p = 0.0371, paired t test. D₁, Current response to a voltage ramp protocol from −89 to +61 mV, with a speed of 0.1875 mV/ms in the presence (gray) and absence (black) of 3 mm CsCl. Prepulse duration was 800 ms. D₂, Example trace shows current–voltage relationship of I_Ca during 3 mm CsCl application. D₃, Peak I_LVA amplitude: control: −15.72 ± 0.87 pA, n = 7; CsCl: −15.71 ± 1.25 pA, n = 7; p = 0.9786, paired t test. Peak I_HVA amplitude: control: −29.42 ± 3.71 pA, n = 7; CsCl: −32.09 ± 4.05 pA, n = 7; p = 0.0933, paired t test. E₁, Example current–voltage relationship of evoked I_Ca in the presence and absence of 10 μm nifedipine (Nife). E₂, Peak I_HVA amplitude: control: −37.45 ± 5.25 pA, n = 5; nifedipine: −19.73 ± 6.42 pA, n = 5; p = 0.0022, paired t test. Peak I_LVA amplitude: control: −10.83 ± 0.87 pA, n = 5; nifedipine: −10.86 ± 1.00 pA, n = 5; p = 0.9082, paired t test. F₁, Example current–voltage relationship of evoked I_Ca in the presence and absence of 1 μm TTX. F₂, Peak I_LVA amplitude: control: −12.80 ± 0.26 pA, n = 5; TTX: −12.92 ± 0.47 pA, n = 5; p = 0.7628, paired t test. Peak I_HVA amplitude: control: −34.51 ± 0.81 pA, n = 5; TTX: −33.61 ± 1.02 pA, n = 5; p = 0.2789, paired t test. G₁, Example current–voltage relationship of evoked I_Ca in the presence and absence of 10 μm ω-conotoxin GVIA (Cono). G₂, Peak I_LVA amplitude: control: −12.05 ± 1.77 pA, n = 5; Cono: −11.81 ± 2.06 pA, n = 5; p = 0.7978, paired t test. Peak I_HVA amplitude: control: −31.37 ± 2.92 pA, n = 5; Cono: −31.03 ± 3.60 pA, n = 5, p = 0.7205; paired t test. H₁, Example current–voltage relationship of evoked I_Ca in the presence and absence of (3 mm CsCl, 10 μm nifedipine, 5 μm Z944 and 100 μm nickel). H₂, Peak I_LVA amplitude (LVA), −10.72 ± 1.53 pA; peak I_HVA amplitude (HVA), −40.16 ± 5.78 pA; peak I_Ca amplitude in the presence of L-type and T-type Ca²⁺ channel blockers (Mix), −3.0 ± 0.33 pA. LVA versus mix, p = 0.005; HVA versus mix, p = 0.0028; n =5, unpaired t test.

Figure 3.

Physiological properties of I_LVA and I_HVA. A, Representative examples of I_LVA in cone photoreceptors evoked by 50 ms voltage steps to different potentials (V_h = −62, −38, −32, −20, −8, and 52 mV) in the presence of 8 μm CNQX, 3 mm CsCl, and 10 μm nifedipine. B, Representative examples of I_HVA evoked by 50 ms voltage steps to different potentials (V_h = −62, −38, −32, −20, −8, and 52 mV) in the presence of 8 μm CNQX, 3 mm CsCl, and 5 μm Z944. C₁, Current–voltage relationship of I_LVA and I_HVA currents evoked by voltage steps from −77 to 43 mV by 6 mV steps. C₂, Voltage dependence of steady-state activation of I_LVA and I_HVA (g/g_max). Data points were fitted with the Boltzmann function (dashed lines). C₃, Mean V₅₀: I_LVA, −44.17 ± 1.98 mV; I_HVA, −34.38 ± 1.78 mV; n = 6; p = 0.0043, unpaired t test. Mean activation slope factor (slope): I_LVA, −4.77 ± 0.70; I_HVA, 4.99 ± 0.83; n = 6; p = 0.8417, unpaired t test. D₁, Typical maximal I_LVA in cone photoreceptors evoked by a voltage step from −69 to 41 mV (50 ms). The dashed red line indicates I_LVA decay fitted with an exponential function. D₂, Corresponding time to I_LVA peak, 13.18 ± 0.81 ms. Decay, tau of single exponential function: 28.82 ± 2.38 ms. E₁, Typical maximal I_HVA in cone photoreceptors evoked by a voltage step from −69 to −17 mV (50 ms). E₂, Corresponding fast inactivation ratio, 0.85 ± 0.03. F, Example traces showing the steady-state inactivation of I_LVA. A 300-ms-long conditioning pulse over a series of V_h, from −100 to −20 mV (20 mV increments), was applied before the test pulse (−39 mV, 50 ms). G, Example traces showing the steady-state inactivation of I_HVA. The 300-ms-long conditioning pulse over a series of V_h values from −100 to −20 mV (20 mV increments) was applied before the test pulse (−19 mV, 50 ms). H₁, Steady-state inactivation of I_LVA and I_HVA were calculated by normalizing the test pulse-evoked current amplitude to the maximum current amplitude and were plotted over the prepulse V_m. Data points were fitted with Boltzmann function and illustrated as solid black (I_HVA) and gray (I_LVA) lines. Dashed lines are the Boltzmann fits of the I_LVA and I_HVA activation kinetics from C₂. H₂, Mean V₅₀: I_LVA, −54.82 ± 0.81 mV; I_HVA, −40.11 ± 2.76 mV; n = 8 and n = 5; p < 0.0001, unpaired t test. Mean inactivation slope factor (slope): I_LVA, 5.06 ± 0.49; I_HVA, 5.45 ± 0.87; n = 8 and n = 5; p = 0.6773, unpaired t test.

Figure 4.

Ca²⁺ spikes present at cone photoreceptors. A₁, Example of cone photoreceptor V_m change to current injections (6, 1, −4, −9, −14, and −24 pA) in current-clamp mode using K⁺-gluconate-based intracellular pipette solution. Arrowhead labels the initial fast rise of the V_m (spike) at the beginning of the stimulus. A₂, Example trace of cone photoreceptor V_m change to the same level of current injections in the presence of 100 μm nickel (Ni²⁺). A₃, V_m amplitude versus stimulus intensity plot. Significance was tested with a paired t test among each stimulus intensity (−22 pA, p = 0.7346; −18 pA, p = 0.1548; −14 pA, p = 0.2918; −10 pA, p = 0.3324; −6 pA, p = 0.0389; −2 pA, p < 0.0001; 2 pA, p < 0.0001; 6 pA, p = 0.2151; n = 6). B₁, B₂, Example traces of cone photoreceptor V_m change to current pulses (0, −5, −10, −15, −20, and −25 pA), in the absence (control, B₁) and presence (gray, B₂) of 5 μm Z944. B₃, V_m amplitude versus stimulus intensity plot. Significance was tested with a paired t test in each stimulus intensity (−25 pA, p = 0.9846; −20 pA, p = 0.9583; −15 pA, p = 0.9467; −10 pA, p = 0.1408; −5 pA, p = 0.3731; 0 pA, p = 0.0485; 5 pA, p = 0.0498; 10 pA, p = 0.2916; n = 7). C₁, C₂, Example traces of cone photoreceptor V_m change to current pulses (2, −4, −10, −16, and −22 pA), in the absence (control; C₁) and presence (Nife; gray; C₂) of 10 μm nifedipine. C₃, V_m amplitude versus stimulus intensity plot. Significance was tested with a paired t test in each stimulus intensity (−35 pA, p = 0.1143; −30 pA, p = 0.1352; −25 pA, p = 0.1362; −20 pA, p = 0.9151; −15 pA, p = 0.5775; −10 pA, p = 0.0150; −5 pA, p = 0.0199; 0 pA, p = 0.050; n = 6). D₁, D₂, Example traces of cone photoreceptor V_m change to current pulses (0, −8, −12, −16, and −24 pA), in the absence (control; D₁) and presence (TTX; gray; D₂) of 1 μm tetrodotoxin. D₃, V_m amplitude versus stimulus intensity plot. Significance was tested with paired t test in each stimulus intensity (−25 pA, p = 0.296; −20 pA, p = 0.1322; −15 pA, p = 0.2485; −10 pA, p = 0.1641; −5 pA, p = 0.6813; 0 pA, p = 0.1703; 5 pA, p = 0.3681; n = 4). E₁, E₂, Example traces of cone photoreceptor V_m change to current pulses (2, −4, −10, −13, and −19 pA), in the absence (control; E₁) and presence (Cs⁺; gray; E₂) of 1 μm CsCl. E₃, V_m amplitude versus stimulus intensity plot. Significance was tested with paired t test in each stimulus intensity (−25 pA, p = 0.2575; −20 pA, p = 0.2498; −15 pA, p = 0.2172; −10 pA, p = 0.1939; −5 pA, p = 0.1366; 0 pA, p = 0.0712; 5 pA, p = 0.0512; n = 5).

Figure 5.

Spontaneous spike activity at dark V_m. A₁–C₂, Example V_m changes in control condition and during 100 μm nickel (Ni²⁺), 10 μm nifedipine (Nife), and 5 μm Z944 application. C₃, Peak V_m of spontaneous spike events: control, −13.3 ± 2.50 mV; Z944, −19.45 ± 3.13 mV; n = 7; p = 0.0337, paired t test. Spontaneous spike frequency: control, 1.45 ± 0.38 Hz; Z944, 1.1 ± 0.32 Hz; n = 7; p = 0.2462, paired t test. Half-width: control, 38.0 ± 2.50 ms; Z944, 50.68 ± 3.13 ms; p = 0.0145, paired t test; n = 7. D, Summary of spike parameters generated in control condition. Frequency, 1.47 ± 0.23; peak V_m, −13.56 ± 1.72 mV; baseline V_m, −40. 57 ± 0.50 mV; half-width, 31.96 ± 1.72 ms. E₁, Example of a spontaneous spike in cone photoreceptors. E₂, Time derivative of the V_m of the trace in E₁ plotted against V_m. E₃, Zoomed in region of the rising phase from E₂. Dashed gray line, Exponential fit to estimate the average of the trace; straight gray lines, 7.5% of the maximal rise rate reveals the V_m threshold of the spike. E₄, V_m at 0.22 dV/dt: −36.17 ± 0.29 mV; n = 14; maximal V_m rise rate: 3.17 ± 0.24 mV/ms; n = 14. F₁, Example V_m changes at near body temperature (33°C). F₂, Frequency of spikes, 1.05 ± 0.17 Hz; peak V_m, −10.65 ± 2.05 mV; baseline, −35.52 ± 1.1 mV; half-width, 27.77 ± 4.3 ms. Room temperature versus 33°C: frequency, p = 0.2610; peak V_m, p = 0.3458; baseline, p = 0.0002; half-width, p = 0.4844, unpaired t test; n = 12 and n = 5, respectively. G₁, Example trace showing V_m changes evoked by full-field light flash (300 ms, ∼130 W/cm² irradiance) in dark-adapted cone photoreceptor. G₂, V_m before light stimulation (Dark V_m), −39.65 ± 0.99 mV; light response evoked V_m hyperpolarization (V_min), −53.96 ± 2.46 mV; offset of the light response (V_max), −3.61 ± 4.08 mV.

Figure 6.

Spikes cause intracellular Ca²⁺ rise in cone photoreceptor terminals. A, Example of simultaneous current-clamp recording (from −13 to −4 pA, 5 s) and Ca²⁺ imaging with 100 μm Fluo-4 in the cone photoreceptor terminal. Pictures in the top panel were taken from the patched cone photoreceptor terminal at the time points labeled with arrows (t₁–t₃). B, Example of simultaneous current-clamp recording and Ca²⁺ imaging during stronger stimulation (from −13 to −2 pA, 5 s). Arrows indicate Ca²⁺ rise because of the individual spike events. C, Linear relationships between the timing of spike and Ca²⁺ signal peak (p < 0.0001, R² = 0.9992). D, Relative Ca²⁺ change during spikes (ΔF/F₀), 1.08 ± 0.33; Δt between spike peak amplitude and Ca²⁺ signal peak (Δt), 30.58 ± 8.36 ms.

Figure 7.

LVA Ca²⁺ channels boost synaptic vesicle release. A₁, Black, Example traces showing I_Ca and C_m induced by a voltage step (from −69 to −19 mV, 25 ms) at cone photoreceptors. Stim, Stimulus; G_m, membrane conductance; G_s, series conductance; gray, I_Ca and C_m induced by a voltage step (from −39 to −19 mV, 25 ms). Labeling similar to black traces. A₂, QI_Ca: V_h = −69 mV: 1.35 ± 0.19 pC; V_h = −39 mV: 0.83 ± 0.06 pC; n = 7; p = 0.0003, paired t test; C_m: V_h = −69 mV: 75.62 ± 4.69 fF; V_h = −39 mV: 45.02 ± 2.90 fC; n = 7; p = 0.0003, paired t test. B₁, Black, Example traces of I_Ca and C_m jump induced by voltage stimulus (from −69 to −19 mV, 25 ms), Patch pipette contained 5 mm EGTA. Gray, Same as black but in the presence of 10 μm nifedipine. B₂, QI_Ca: control, 1.16 ± 0.19 pC; Nife, 0.59 ± 0.09 pC; n = 6; p = 0.0047, paired t test; C_m: control, 90.99 ± 10.73 fF; Nife, 76.23 ± 9.52 fF; n = 6; p = 0.0007, paired t test. C₁, Black, Example traces of I_Ca and C_m change induced by a voltage step (from −69 to −19 mV, 25 ms). Pipette contained 10 mm BAPTA. Gray, Same as black but in the presence of 10 μm nifedipine. C₂, QI_Ca: control: 1.28 ± 0.10 pC, n = 5; Nife: 0.74 ± 0.04 pC, n = 5; p = 0.0021, paired t test; C_m: control: 35.16 ± 5.18 fF, n = 5; Nife: 18.45 ± 2.23 fF, n = 5; p = 0.0063, paired t test. D₁, Black, Example traces of I_Ca and C_m jump induced by a voltage step (from −69 to −39 mV, 25 ms). Pipette contained 5 mm EGTA. Gray, Same as black but with 10 mm BAPTA-containing intracellular solution. D₂, QI_Ca: 5 mm EGTA, 0.36 ± 0.04 pC; 10 mm BAPTA, 0.34 ± 0.03 pC; n = 5 and n = 4; p = 0.7302, unpaired t test; C_m: 5 mm EGTA, 83.68 ± 13.76 fF; 10 mm BAPTA, 16.61 ± 3.99 fF; n = 5 and n = 4; p = 0.0159, unpaired t test. E, Paired comparison of (−19 mV, 25 ms; strong) and mild (−39 mV, 25 ms; mild) stimulus evoked QI_Ca with 5 mm EGTA containing intracellular solution. Mild, QI_Ca = 0.36 ± 0.04 pC; strong, QI_Ca = 1.09 ± 0.1 pC; n = 5; p = 0.0005, paired t test. Mild, ΔC_m = 83.68 ± 13.76 fF; strong, ΔC_m = 95.24 + 12.42 fF; n = 5; p = 0.0021, paired t test. F, Paired comparison of strong (−19 mV, 25 ms) and mild (−39 mV, 25 ms) stimulus-evoked QI_Ca with 10 mm BAPTA containing intracellular solution. Mild: QI_Ca = 0.33 ± 0.03 pC; strong: QI_Ca = 1.12 ± 0.04 pC; n = 4; p = 0.0005, paired t test; mild: ΔC_m = 16.61 ± 3.99 fF; p = 0.0005; strong: ΔC_m = 55.97 ± 9.44 fF; n = 4; p = 0.01, paired t test.

Figure 8.

Analysis of T-type Ca²⁺ channels in sorted photoreceptors. A₁, FACS strategy for isolating rod photoreceptors by FSC/SSC. A₂, FACS strategy for isolating cone photoreceptors by GFP fluorescence of Rac3-eGFP mice. B₁, Nested RT-PCR analysis of sorted cone and rod photoreceptor samples with LVA-specific primer pairs for Ca_v3.1, Ca_v3.2, and Ca_v3.3. A distinct band is present for the cone photoreceptor sample using Ca_v3.2 primers, whereas no bands can be observed for the sorted rod photoreceptor population. B₂, Conventional RT-PCR with primers for Rho, Opn1sw, and Actb on sorted rod and cone photoreceptors. Strong bands are visible for Opn1sw and Rho in cone and rod photoreceptor samples, respectively, as well as for Actb. C₁, C₂, RT-PCR analysis of whole-retina and negative control samples, in which reverse transcriptase was omitted during cDNA synthesis (retina, –RT). For whole retina, the following expected bands are present: Ca_v3.1 (271 bp), Ca_v3.2 (305 bp), Ca_v3.3 (258 bp), Rho (81 bp), Opn1sw (175 bp), and Actb (196 bp). No bands are visible for retina –RT samples.