Identification of CaV channel types expressed in muscle afferent neurons

Abstract

Cardiovascular adjustments to exercise are partially mediated by group III/IV (small to medium) muscle afferents comprising the exercise pressor reflex (EPR). However, this reflex can be inappropriately activated in disease states (e.g., peripheral vascular disease), leading to increased risk of myocardial infarction. Here we investigate the voltage-dependent calcium (CaV) channels expressed in small to medium muscle afferent neurons as a first step toward determining their potential role in controlling the EPR. Using specific blockers and 5 mM Ba(2+) as the charge carrier, we found the major calcium channel types to be CaV2.2 (N-type) > CaV2.1 (P/Q-type) > CaV1.2 (L-type). Surprisingly, the CaV2.3 channel (R-type) blocker SNX482 was without effect. However, R-type currents are more prominent when recorded in Ca(2+) (Liang and Elmslie 2001). We reexamined the channel types using 10 mM Ca(2+) as the charge carrier, but results were similar to those in Ba(2+). SNX482 was without effect even though ∼27% of the current was blocker insensitive. Using multiple methods, we demonstrate that CaV2.3 channels are functionally expressed in muscle afferent neurons. Finally, ATP is an important modulator of the EPR, and we examined the effect on CaV currents. ATP reduced CaV current primarily via G protein βγ-mediated inhibition of CaV2.2 channels. We conclude that small to medium muscle afferent neurons primarily express CaV2.2 > CaV2.1 ≥ CaV2.3 > CaV1.2 channels. As with chronic pain, CaV2.2 channel blockers may be useful in controlling inappropriate activation of the EPR.

Keywords: CaV2.1; CaV2.2; CaV2.3; dorsal root ganglia neurons; exercise pressor reflex.

Fig. 1.

Voltage-dependent calcium (Ca_V) 2.2 channels generate the dominant Ca_V current in muscle afferent neurons. Ca_V current in 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) labeled muscle afferent neurons was measured using 5 mM Ba²⁺ external solution. A: example currents showing the effect of 0.3 μM SNX482 (SNX), 0.2 μM ω-agatoxin IVa (AgaIVa), 3 μM nifedipine (Nif), and 10 μM ω-conotoxin GVIA (GVIA). The voltage protocol is shown below the current traces. Cntl, control. B: comparison of mean ± SD calcium current block in small (S; 20–30 μm) vs. medium (M; 30–40 μm) muscle afferent neurons by the indicated blockers. The blocker concentrations are the same as indicated for A. C–E: the distribution of percentage block vs. neuron diameter for 3 μM Nif (C), 0.2 μM AgaIVa (D), and 10 μM GVIA (E). Note the different y-axis scale for GVIA. The dashed line indicates average block.

Fig. 2.

Voltage-independent block by Ca_V channel blockers. A: current-voltage relationships from a muscle afferent neuron in the presence of either 3 μM Nif, 0.2 μM AgaIVa, or 10 μM GVIA. Ca_V currents were measured in 5 mM Ba²⁺ at the end of 25-ms voltage steps to the indicated voltage. B: the mean inhibition (±SD) for each Ca_V channel blocker is shown over voltages ranging from −30 to 30 mV. The currents were measured as described for A. There was no statistical difference between the block at −30 mV vs. that at 30 mV for each blocker (n = 5 for each blocker).

Fig. 3.

Little or no Q-current in muscle afferent neurons. Ca_V current from muscle afferent neurons was recorded in 5 mM Ba²⁺ external solution. A: example currents from a muscle afferent neuron in Cntl, 10 μM GVIA, 0.2 μM AgaIVa, and 1 μM AgaIVa. B: the mean (±SD) percentage block by GVIA and the two concentrations of AgaIVa tested on the same neurons. The number of cells tested is indicated in the GVIA bar. *Ca_V current was significantly blocked.

Fig. 4.

Ca_V current block in external Ca²⁺ is similar to that in Ba²⁺. A: an example time course for Ca_V current block in 10 mM external Ca²⁺. This experiment utilized cumulative blocker application to better determine the size of the resistant current (Resistant). The blocker concentrations are indicated and were the same as those used when recording in external Ba²⁺. B: the average (±SD) percentage block by each Ca_V channel blocker. *Ca_V current was significantly blocked. The number of cells tested is indicated in each bar.

Fig. 5.

Immunocytochemistry shows Ca_V2.3 channels expressed in muscle afferent neurons. A: the upper four images show sensory neurons stained with both voltage-gated sodium (Na_V) 1.8 and Ca_V2.3 antibodies. The bright-field (left) image shows the three neurons, and the DiI panel (right) shows the one labeled muscle afferent neuron in this field. All three neurons were positive for both Na_V1.8 and Ca_V2.3. The white bar in the bright-field image indicates 50 μm. The lower four images show Na_V1.8 and Ca_V2.3 controls along with bright-field (left) image and the DiI image showing one labeled muscle afferent neuron. B: there is no correlation between Ca_V2.3 label intensity and muscle afferent neuron size. The threshold line was determined from measurement of Cntl muscle afferent neurons (e.g., lower images in A). The units for the Y-axis are arbitrary fluorescence units. C: comparison of Ca_V2.3 label intensity vs. that of Na_V1.8 in muscle afferent neurons. The threshold lines were determined from Cntl muscle afferent neurons. Only one Ca_V2.3-positive muscle afferent neuron was negative for Na_V1.8.

Fig. 6.

Roscovitine (Rosc)-induced slowed deactivation reveals functional Ca_V2 channels in the presence of GVIA and AgaIVa. All currents were recorded in 5 mM Ba²⁺. A: a muscle afferent neuron shows the effect of Rosc on Ca_V2 currents (no toxins present). Rosc (100 μM) slowed deactivation (black trace) compared with Cntl and recovery (Recov; gray traces). B: current traces from the same muscle afferent neuron as shown in A recorded in 10 μM GVIA and 0.2 μM AgaIVa. Rosc (100 μM) (black trace) slowed deactivation compared with Cntl (Toxin) and Recov (gray traces). C: a single exponential equation was fit to the deactivating currents at −40 mV to determine the deactivation τ in presence of toxin (GVIA and AgaIVa) and toxin + 100 μM Rosc. The average deactivation τ (±SD) is shown. *Significant slowing of deactivation induced by Rosc. The number of muscle afferent neurons tested is indicated in the middle bar.

Fig. 7.

ATP inhibits Ca_V2.2 channels in muscle afferent neurons. All currents were recorded in 10 mM Ca²⁺. A: the inhibition of Ca_V current induced by 10 μM ATP (black trace) compared with Cntl (gray trace) recorded from a muscle afferent neuron. The inhibition is transiently reversed by strong depolarization (+80 mV), which can be seen by comparing the prepulse (before the +80-mV step) and postpulse (following the +80-mV step) currents. B: there was no clear differences in ATP (10 μM) induced inhibition in small (<30 μm) vs. medium (30–40 μm) vs. large (>40 μm) muscle afferent neurons. The percent inhibition of prepulse current measured from 13 muscle afferent neurons is plotted vs. neuron diameter. C: the ATP (10 μM) inhibition is blocked by preapplication of 10 μM GVIA. This time course shows the inhibition induced by ATP prior to GVIA application and little or no ATP response in the presence of GVIA. The prepulse (solid circle) and postpulse (open circle) current amplitudes are plotted. D: the average (±SD) inhibition induced by 10 μM ATP is shown before (ATP) and during (GVIA + ATP) application of 10 μM GVIA. *ATP response in GVIA is significantly different from that in Cntl. The number of muscle afferent neurons tested is indicated.