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. 1997 Jun 24;94(13):7006-11.
doi: 10.1073/pnas.94.13.7006.

Heat transduction in rat sensory neurons by calcium-dependent activation of a cation channel

D B Reichling  1 , J D Levine
Affiliations

Heat transduction in rat sensory neurons by calcium-dependent activation of a cation channel

D B Reichling et al. Proc Natl Acad Sci U S A. .

Abstract

The mechanism of heat transduction in vertebrate sensory neurons was investigated in vitro by using cultured dorsal root ganglion neurons from adult rat. In response to a physiologically relevant range of stimulus temperatures (23-45 degrees C), a subpopulation of small dorsal root ganglion neurons are depolarized by a cation current (heat-activated current, Iheat) that is antagonized by extracellular cesium. Heat-induced single-channel currents in cell-attached patches are evoked at a similar range of temperatures. Iheat is a calcium-dependent current activated indirectly by heat-evoked release of calcium from intracellular stores. This suggests that the channel itself is not the transducer of thermal energy. Similar to nociceptive heat sensation in vivo, Iheat is enhanced by the hyperalgesic agent prostaglandin E2 and only partially adapts during prolonged heat stimuli. To our knowledge, these data provide the first demonstration that ion channels can mediate heat transduction in mammalian sensory neurons and provide evidence that heat causes the channels to open via an increase in the intracellular second messenger calcium.

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Figures

Figure 1
Figure 1
Heat-evoked depolarizing inward current in a subpopulation of cultured DRG neurons. (A) In a neuron current-clamped at 0 pA, heating to 39°C caused a depolarization from −66 mV to −35 mV. (B) A neuron voltage-clamped at −65 mV responded to heat with an inward current (Iheat). Input resistance decreased during Iheat; the current required to deliver a −20-mV 10-msec voltage step command increased from 35 pA before heating to 47 pA during heat, indicating a decrease in input resistance of approximately 25%. (C) The magnitude of Iheat is progressively larger as the peak stimulus temperature increases. This cell was heated to peak stimulus temperatures of approximately 30, 35, 40, and 45°C. (D) Some DRG neurons exhibit no detectable response to heat, as in this neuron heated to 45°C. (E) Expression of Iheat is greatest in small-diameter neurons. The response to a 40°C stimulus was measured in small-diameter (≤28 μm) and large-diameter (≥45 μm) DRG neurons. The magnitude of Iheat increases markedly over time during the first 24 hr neurons are maintained in culture (data not shown). Therefore, to study the distribution of Iheat among DRG neurons of different sizes, recordings were performed only between 12 and 18 hr after cell plating, in a sequence that strictly alternated between small- and large-diameter DRG neurons. Data were obtained from cells isolated from six rats. To correct for different membrane areas in different-sized neurons, current density was calculated based on the whole-cell capacitance. Most small-diameter neurons exhibit Iheat of greater than 1.75 pA/pF (broken line) and most large-diameter neurons exhibit Iheat of less than 1.75 pA/pF. (F) The response to a 40°C stimulus was measured in sympathetic neurons isolated from the superior cervical ganglion. All neurons tested responded with an inward current of less than 1.75 pA/pF.
Figure 2
Figure 2
Heat activates single-channel currents in rat dorsal root ganglion neurons. Cell-attached patch recordings were performed with the electrode voltage clamped at 0 mV. No single-channel activity was observed at 22°C. Upon heating the bath, brief single-channel inward currents began to appear at approximately 29°C. At 37°C, channel openings became more frequent and longer in duration. At 39°C, openings of a second heat-activated channel became more frequent. Both heat-activated single-channel currents in this patch exhibited a similar amplitude of approximately 2 pA. If a resting potential of −60 mV is assumed, this would reflect a single-channel conductance of 33 pS. Finally, subsequent cooling of the bath to 22°C caused single-channel activity to cease.
Figure 3
Figure 3
Characteristics of the Iheat current. (A) Iheat was reduced by 67% in this DRG neuron when extracellular Na+ was substituted by choline. (B) Iheat was reduced by 37% in another DRG neuron when Ca2+ was removed from the extracellular solution. (C) At 10 mM, extracellular Cs+ reduced Iheat in this neuron by 85%. Iheat recovered nearly to control levels within 2 min after removal of Cs+. (D) La3+ (100 μM) does not reduce Iheat. (E) A plot of leak-subtracted Iheat during a voltage ramp shows outward rectification at membrane potentials more negative than approximately −40 mV. (Inset) Protocol in which a voltage ramp is applied at the peak of the response to a 38°C stimulus (ramp 1), and leak current is measured by the same ramp applied after recovery from the stimulus (ramp 2).
Figure 4
Figure 4
Iheat is activated by calcium released from intracellular stores. (A) Loading cells with the calcium chelator BAPTA reduces Iheat. Iheat was evoked in a neuron by heating to 40°C (Control), then the neuron was exposed to a bath containing 100 μM BAPTA-AM for 5 min at room temperature, BAPTA-AM was washed out of the bath, and the stimulus was reapplied (BAPTA). In this neuron, BAPTA reduced the peak Iheat by 78%. The control response of this neuron also illustrates that in some neurons, Iheat does not return to baseline (broken line) immediately upon cessation of the heat stimulus. (B) Heat stimulates [Ca2+]i increases in the absence of extracellular Ca2+. Heating to 40°C caused [Ca2+]i to increase by approximately 200 nM. Removal of Ca2+ from the extracellular bath completely blocked Ca2+ entry evoked by K+ depolarization but the heat-evoked increase in [Ca2+]i persisted.
Figure 5
Figure 5
Iheat exhibits properties consistent with heat transduction in vivo. (A) Magnitude of Iheat adapts to maintained stimuli. In a neuron heated to approximately 43°C for about 2 min, Iheat reached a peak during the first minute and then decayed to a plateau level of about 50% of peak for the remainder of the stimulus. (B) PGE2 enhances Iheat. PGE2 (10 nM) was perfused through the recording chamber for 10 min. Within 1 min after the beginning of wash-out of PGE2, the 43°C stimulus was reapplied. In this neuron, the peak amplitude of Iheat after exposure to PGE2 was 192% of the control value obtained before PGE2.
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