Muscarinic receptor-activated cationic channels in murine ileal myocytes

Abstract

Background and purpose: There is little information about the excitatory cholinergic mechanisms of mouse small intestine although this model is important for gene knock-out studies.

Experimental approach: Using patch-clamp techniques, voltage-dependent and pharmacological properties of carbachol- or intracellular GTPgammaS-activated cationic channels in mouse ileal myocytes were investigated.

Key results: Three types of cation channels were identified in outside-out patches (17, 70 and 140 pS). The voltage-dependent behaviour of the 70 pS channel, which was also the most abundantly expressed channel (approximately 0.35 micro(-2)) was most consistent with the properties of the whole-cell muscarinic current (half-maximal activation at -72.3+/-9.3 mV, slope of -9.1+/-7.4 mV and mean open probability of 0.16+/-0.01 at -40 mV; at near maximal activation by 50 microM carbachol). Both channel conductance and open probability depended on the permeant cation in the order: Cs+ (70 pS) >Rb+ (66pS) >Na+ (47 pS) >Li+ (30 pS). External application of divalent cations, quinine, SK&F 96365 or La3+ strongly inhibited the whole-cell current. At the single channel level the nature of the inhibitory effects appeared to be very different. Either reduction of the open probability (quinine and to some extent SK&F 96365 and La3+) or of unitary current amplitude (Ca2+, Mg2+, SK&F 96365, La3+) was observed implying significant differences in the dissociation rates of the blockers.

Conclusions and implications: The muscarinic cation current of murine small intestine is very similar to that in guinea-pig myocytes and murine genetic manipulation should yield important information about muscarinic receptor transduction mechanisms.

Figure 1

Representative whole-cell cation current response to 50 μM carbachol application (a) recorded at −40 mV. Examples of the 140 pS channel activity at −80 and 80 mV in an outside-out membrane patch, formed at the maximal whole-cell response to the agonist are shown in the inset. In a membrane patch from a different cell (b) activity of 17 and 74 pS MRCC is evident; see also the all-point amplitude histogram shown in (c). The dotted lines in all figures indicate the current levels corresponding to the open (O) and closed (C) channel states. Holding potential was −40 mV. Calculated current amplitudes and open probabilities were −0.72±0.09 pA, P_o=0.03 and −2.98±0.14 pA, P_o=0.24 for the small and medium conductance channels, respectively.

Figure 2

Voltage dependence of gating of the medium conductance channel (a, b and c) determines CCh-evoked whole-cell current (d, e). I–V relation of the mean patch current evoked by 50 μM carbachol (a) was obtained as a product of NP_o and single channel amplitude at each test potential, and is U-shaped at negative potentials with the E_rev close to 0 mV (compare to e inset). The 70 pS single channel I–V relation (b) is nearly linear and typical P_o-voltage dependence (c) could be fitted by the Boltzmann relation between −120 and −40 mV as shown by the superimposed curve. Note that close to the reversal potential P_o values are significantly reduced and therefore these points have been excluded from the fit, similarly to the whole-cell conductance values positive to about −40 mV shown in panel e (see text for details). Panels on the right show examples of single channel activity at different test potentials (from top to bottom: 60 to −120 mV, with an increment of 20 mV excluding 0 and 20 mV where channel activity was undetectable). Whole-cell instantaneous (triangles) and steady-state (circles) I–V relations (d) of current evoked by 50 μM CCh measured from the superimposed traces shown in the inset, were obtained by 1.2 s duration voltage steps applied from a holding potential of −40 mV to test potentials between −120 and 80 mV with increments of 20 mV. The effect of desensitization on the I–V curve was relatively small as during the measurements the holding current was reduced by 11±6% (n=9). Whole-cell I–V relationship (e) obtained by 6 s duration negative-going voltage ramp in the presence of 50 μM carbachol (inset) and the corresponding activation curve obtained by dividing current amplitude by the driving force. Data were fitted between −120 and −40 mV by the Boltzmann relation as shown by the smooth superimposed curve. Note that the activation curve (e) is similar to the P_o-voltage dependence of the single channel (c).

Figure 3

Time course of whole-cell (a) and single channel (b) desensitization in the presence of 50 μM carbachol at −40 mV. During the first minute after patch excision multiple single channel activity was present in an outside-out patch (b). However, the number of active channels and their P_o decreased during 20 min of carbachol application (from 4 to 1 and from 0.31 to 0.04, respectively).

Figure 4

Dependence of single channel activity on the permeating cation was investigated in the same patch with CsCl-, RbCl-, NaCl- or LiCl-containing external solutions (all at 120 mM; patch pipette contained 124 mM Cs⁺). Outside-out patch was formed after cation current was fully activated by 200 μM GTPγS.

Figure 5

Effects of extracellular Ca²⁺ on the whole-cell current (a, b) and single channel activity (c, d). Ca²⁺ added to the bath solution at the indicated concentrations caused rapid whole-cell current decrease at −120, −40 and 80 mV (a). The I–V relations (b) measured with no (trace 1), 2.5 mM (trace 2) and 10 mM added Ca²⁺ (trace 3) obtained with ramp protocol. Corresponding activation curves fitted by the Boltzmann equation are shown in the inset. Single channel recordings from a representative patch (c) exposed to no, 2.5 and 10 mM added Ca²⁺. Unitary currents measured at −40 mV were reduced from 2.8±0.26 to 1.85±0.19 and 1.71±0.14 pA, respectively. The open channel I–V relations (d) at different Ca²⁺ concentrations were obtained from all-point amplitude histograms with no (squares), 2.5 mM (circles) and 10 mM added Ca²⁺ (triangles).

Figure 6

Inhibitory effects of quinine (a, b) and La³⁺ (c, d) on carbachol-induced whole-cell and single channel currents measured at −40 mV. Whole-cell (top panel) and single channel (bottom panel) currents were reversibly suppressed by the bath application of 20 μM quinine. Single channel conductance was unaltered while channel NP_o decreased from 0.18 to 0.03 (b). Bath application of 2 mM La³⁺ caused a rapid inhibition of both whole-cell (c top panel) and single channel (c bottom panel) currents. The latter were inhibited mainly via a reduction of the unitary current amplitude (top panel in d, squares and circles show the open channel I–V relationships before and after 2 mM La³⁺ application, respectively; n=3) as well as variable reduction in NP_o (bottom panel in d). No single channel activity was observed at positive potentials in the presence of La³⁺.