Calcium-activated potassium channels in rat visceral sensory afferents

BRAIN RESEARCH ELSEVIER

Brain Research 639 (1994) 333-336

Short Communication

Calcium-activated potassium channels in rat visceral sensory afferents Meredith Hay *, Diana L. Kunze Department of Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA (Accepted 16 December 1993)

Abstract

The purpose of the present study was to describe, at the single-channel level, the activity of a calcium-sensitive potassium channel in rat visceral-sensory neurons which has been suggested to be involved in sensory neuron excitability. Single-channel recordings in the inside-out configuration identified a 220 pS conductance calcium-activated potassium channel (KCa). From a - 2 0 mV holding potential, increasing [Ca2+] i from 0.01 /zM to 1.0/zM increased the open probability of this channel 92% (from 0.12 to 0.23). However, from a + 20 mV holding potential, increasing [Ca 2÷ ]i from 0.01 to 1.0/xM increased the open probability by 326% (from 0.15 to 0.64). In addition, this large conductance KCa channel was blocked by TEA (1.0 ~M) and charybdotoxin (40/zM) when applied to the external surface. These results are the first to characterize a large conductance KCa channel in the sensory afferent neurons of the rat nodose ganglia and should further expand the understanding of the ionic currents involved in the regulation of sensory afferent neuronal activity. Key words: Channel; Calcium; Nodose; Dissociated; Charybdotoxin

Calcium dependent after-potentials have been described in the neurons of the nodose ganglia [5,8,18], and it has been suggested by others that these channels may contribute to the resting membrane potential in nodose neurons and regulate neuronal excitability. Thus, the purpose of the present study was to identify and characterize the KCa channels present in visceral afferent neurons of the nodose ganglia. Nodose neurons were isolated and cultured as previously described [11]. Patch clamp recordings using the inside-out and outside-out configurations [4] were performed at room temperature on nodose neurons 1-2 days in culture. Single-channel recordings were made using a List EPC-7 patch clamp amplifier, filtered at 3 kHz, and digitized on line at 10 kHz using pCLAMP software. The digitized records were analyzed using T R A N S I T analysis program [14]. In all figures, inward currents represent the movement of positive ions from the ex-

* Corresponding author. Present address: Department of Physiology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7756, USA. Fax: (1) (210) 567-4410. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved

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ternal surface to the internal surface of the membrane and are depicted as downward deflections. The closed state of the channel is indicated by the letter C. Pipette solution consisted of (in raM): 145 KAsp, 10 HEPES, 2.2 E G T A and sufficient CaC12 to yield a final concentration of 0.01/zM. Symmetrical potassium bath solution consisted of (in mM): 5.0 N-methyl-oglucamine, 145 KAsp, 10 glucose, 10 HEPES, 2.2 EGTA, and sufficient CaCI z to yield either 0.01/xM or 1.0 /zM final calcium concentration [2]. Different calcium concentrations were applied directly to the patch via a multibarrel gravity-fed perfusion system. Potassium channels which were activated by increases in [Ca2+] i were observed in 55% (15/27) of the patches studied. The majority of membrane patches contained 2 - 3 active K ÷ channels with channel openings generally occurring in approximately 100 ms bursts typical of KCa channel activity recorded in other preparations [10,13]. Fig. 1A illustrates the activity of the large conductance K ÷ channel from a single patch at different membrane potentials in an inside-out patch in the presence of 1.0/zM [Ca2+]i. These traces were chosen to illustrate discrete open and closed events and current amplitudes but not open probabilities. However, burst duration and frequency did appear to

M. ttav D.L. Kunze / Brain Re,warch 03 ~) (1994) 333 3.'th

334

increase with increased membrane depolarization. As seen in Fig. 1B, in symmetrical [K+]~ bath solution, the reversal potential obtained was - 1.0 _+ 1 mV (n = 10). After substituting 145 mM [KAsp]+ with 140 mM [NaAsp] i + 5.0 mM [KAsp] i, the reversal potential of the single channel current was a projected 82 mV (n = 4). Under these conditions, the calculated K + equilibrium potential was 84.2 mV, indicating that the primary current-carrying species was K +. Fig. 1C illustrates the effects of increased [Ca 2 +]~ on an inside-out nodose neuron patch containing 2 large conductance channels. At a + 20 mV holding potential in symmetrical 145 mM KAsp, increasing [Ca2+]i from 0.01 /xM to 1.0 /xM markedly increased the activity of the large conductance channel. This effect was reversible when [Ca2+]i was returned to 0.01/xM. Importantly, KCa activity was present at 0.01 /xM [Ca2+]~ suggesting a relatively high sensitivity of this channel to

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[Ca2+]i. The voltage dependence of the large conductance KCa channel activity is illustrated in Fig. I D From a +20 mV holding potential in symmetrical 145 mM KAsp, increasing [Ca2+]~ from 0.01 > M to 1.0 # M resulted in an averaged 326_+56'7{ increase in the ()pen probability (from 0.15 to 0.64) (n = 7). However, from a - 2 0 mV holding potential, increasing [Ca"+], from 0.01 to 1.0 >M resulted in only a 92 + 14c7c increase (from 0.12 to 0.23). These results show thal the large conductance KCa channels found in nodose neurons are sensilive to increases in [Ca-'+]i and this effect is voltage dependent. Tetraethylammonium (TEA) and charybdotoxin (ChTx) are known to block large conductance KCa channels in a number of different preparations [1,6,9, 13]. Fig. 2A is an example of an outside-out patch at +40 mV holding potential with 1.0 # M [Ca2+]+. A 1.0 mM concentration of TEA resulted in a flickering

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335

M. Hay, D.L. Kunze / Brain Research 639 (1994) 333-336

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block of the large conductance KCa channel when applied to the external surface of the membrane (n = 8/8). When applied to the internal surface, 1.0 mM TEA was ineffective at blocking the large conductance KCa channel (n = 2). In addition, external application ChTx (40 nM) also inhibited the large conductance channel in 4 / 4 outside-out patches tested. Fig. 2B illustrates the effect of 40 nM ChTx on a single patch with multiple KCa channels. Following application of ChTx (40 nM) long closings were observed between bursts of activity [9]. The present study is the first to provide single channel evidence for K ÷ channels in visceral sensory neurons that are activated by increases in [Ca2+]i . In symmetrical 145 mM KAsp, the conductance of this channel is approximately 220 pS and the activity of this KCa channel in nodose neurons is similar to that reported in other mammalian neurons [7,8,10,13]. The large conductance channels showed a markedly greater

sensitivity t o [Ca2+]i at more depolarized potentials and were selective for K + and were relatively impermeable to Na +. Additionally, the large conductance KCa channels in these afferent neurons were blocked by low concentrations of TEA and by ChTx when applied to the exterior surface. Importantly, the large conductance KCa channel in visceral afferent neurons is active at 0.01 /xM [ C a 2 + ] i. This rather high Ca 2÷ sensitivity may be important for the KCa channels hypothesized role in the regulation of neuronal excitability and tonic activation of these channels at rest may contribute to the maintenance of the nodose neuron resting membrane potential. [1] Blatz, A.L. and Magleby, K.L., Calcium-activated potassium channels, Trends Neurosci., 10 (1987) 463-467. [2] Fabiato, A., Computer program for calculating total from specific free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands, Methods Enzymol., 157 (1988) 378-417.

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M. Hay, D.L. Kunze / Brain Research 639 (1994) .333-336

[3] Fowler, J.C., Greene, R. and Wienreich, D., Two calcium-sensitive spike after-hyperpolarizations in visceral sensory neurons of the rabbit, J. Physiol., 365 (1985) 59-67. [4] Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high-resolution current recordings from cells and cell-free membrane patches, P)qugers Arch., 391 (1981) 85-100. [5] Higashi, H., Morita, K. and North, R.A., Calcium dependent after-potentials in visceral afferent neurons of the rabbit, J. Physiol., 355 (1984) 479-492. [6] Lancaster, R.A., Nicoll, R.A. and Perkel, D.J., Calcium activates two types of potassium channels in rat hippocampal neurons in culture, J. Neurosci., 11 (1991) 23-30. [7] Lang, D.G. and Ritcbie, A.K., Large and small conductance calcium-activated potassium channels in the GH3 anterior pituitary cell line, Pflugers Arch., 410 (1987) 614-622. [8] Latorre, R., Oberhauser, A., Labacara, P. and Alvarez, O., Varieties of calcium-activated potassium channels. Annu. Reu. Physiol., 51 (1989) 385-399.

[9] Mackinnon, R. and Miller, C., Mechanism ~f charybdotoxm block of the high-conductance, calcium-actiwtted potassium channel, Z Gen. Physiol., 91 (1988) 335--349. [10] Marty, A., The physiological role of calcium-dependent channels, Trends Neurosci., 12 (1989) 420-429. [11] Mendelowitz, D. and Kunze, D.L., Characterization of calcium currents in aortic baroreceptors, J. NeurophysioL, 59 (1992) 1798-1813. [12] Morita, K. and Katayama, Y., Calcium-dependent slow outward current in visceral primary afferent neurons of the rabbit, Pflugers Arch., 414 (1989) 171-177. [13] Smart, T.G., Single calcium-activated potassium chanpels recorded from cultured rat sympathetic neurons, Z PhysioL, 389 (1987) 337-360. [14] VanDongen, A.M.J., TRANSIT: a new algorithem for analyzing single ion channel data containing multiple conductance levels, Biophys. ,L, 61 (1993) A256.