Organelle membrane derived patches: reshaping classical methods for new targets

Abstract

Intracellular ion channels are involved in multiple signaling processes, including such crucial ones as regulation of cellular motility and fate. With 95% of the cellular membrane belonging to intracellular organelles, it is hard to overestimate the importance of intracellular ion channels. Multiple studies have been performed on these channels over the years, however, a unified approach allowing not only to characterize their activity but also to study their regulation by partner proteins, analogous to the patch clamp "golden standard", is lacking. Here, we present a universal approach that combines the extraction of intracellular membrane fractions with the preparation of patchable substrates that allows to characterize these channels in endogenous protein environment and to study their regulation by partner proteins. We validate this method by characterizing activity of multiple intracellular ion channels localized to different organelles and by providing detailed electrophysiological characterization of the regulation of IP₃R activity by endogenous Bcl-2. Thus, after synthesis and reshaping of the well-established approaches, organelle membrane derived patch clamp provides the means to assess ion channels from arbitrary cellular membranes at the single channel level.

Figure 1

Principal diagram of the approach. The diagram illustrates fraction isolation and preparation of patchable substrates. Top (fractionation): a flowchart presenting example avenues leading to preparation of various types of membrane fraction extracts. Middle (mixture preparation): an illustration of the process of the preparation of the fraction extract/lipid mixture suitable for - bottom (functional characterization) – containing an illustration of the giant uni-lamellar vesicle (GUV) preparation and patch-clamping.

Figure 2

Bichemical characterization of the isolated fractions and localization of the IP₃R protein. (a) A principal diagram of gradient centrifugations yielding crude and better isolated fractions following single and double-gradient centrifugation, showing fraction numbers. (b,c) Western blot characterization of organelle-specific markers in the isolated fractions following single- (b) and double- (c) gradient isolation of subcellular membrane fractions. ER membranes are reported by the detection of calnexin (upper panels), while Golgi apparatus and mitochondria are characterized by the detection of golgin-97 and voltage-dependent anion channel 1 (VDAC1) (middle and lower panels), respectively. Note that, following the double-gradient isolation, Fractions 8, 9 and 10 did not contain sufficient amounts of protein and were thus not characterized. Traces below Western blot images indicate presence (b) or absence (c) of VDAC-like activity in the patches made of GUVs produced from isolated fractions. (d) Western blot characterization of the IP3R presence in the isolated fractions following single- (left) and double- (right) gradient isolation protocol. Note the enrichment of IP3R presence in purer ER and MAM sub-fractions following double-gradient isolation. Splices of the double-gradient gel, where two non-adjacent parts of the same gel with the same exposure time have been emerged together, are indicated with vertical lines, between fractions 7 and 11. The original uncut gel can be seen in Fig. S1. Experiments were reproduced 4 times independently.

Figure 3

Characterization of IP₃R activity in whole-membrane extracts from HEK293 and in ER fractions from LNCaP cells. (a) Typical basal (left) and stimulated by 10 μM IP3 (right) activity at the series of applied potentials indicated on the left. C, O1 and O2 on the right indicate closed, open states and double openings respectively. (b) Typical IV relationship. Note the bend indicating smaller single-channel conductance near zero, characteristic of IP₃R activity in the presence of Mg²⁺. (c) Sample IP₃R activity showing basal region, followed by increased activity in response to application of 10 μM IP3 and, finally, reduction of activity upon washout. (d) Average NPopen in basal, IP3 stimulated and wash regions is summarized in a barplot (mean ± s.e.m.; **denotes significant difference with p < 0.01; n = 7). See also Fig. S2.

Figure 4

Isolation and single-channel activity in the lysosomal fraction. (a) Diagram illustrating the distribution of HEK293 lysate material following the procedure of the lysosome isolation kit protocol and the numbering of the isolated fractions. Lysosomal fraction is positioned in the top-most region of the gradient, directly below the narrow white band of light material that did not enter the Iodixanol gradient. The top edge of the gradient (17%) is indicated by a dotted line and another dotted line below indicates the split of wide lysosomal band into Fractions 2 and 3. (b) Distribution of molecular markers in the isolated fractions. (c–e) Patch-clamp characterization of TPC2 activity in lysosomal fraction, showing sample IV activity (c), IV curve under symmetric 150 mM KCl conditions, yielding representative full-open state conductance of 207 ± 16 pS (d) and [Ca²⁺] dependence of TPC2 activity (e).

Figure 5

Isolated ER fractions retain interaction of endogenous proteins: activity of IP₃R compared in control and Bcl-2 expressing WEHI7.2 cells. (a) Recording of IP₃R gating in control and Bcl-2 expressing cells shows a significant decrease in its activity in the presence of Bcl-2, as summarized in the barplot in (b) (mean ± s.e.m.; ***Denotes significant difference with p < 0.001; n = 11 and 12). (c) Sample IP₃R activity in control WEHI7.2 cells can be inhibited by addition of a diffusible fragment of Bcl-2, BH4 (upper panel), while inhibition of IR3P activity in the Bcl-2 expressing cells could be reversed by a diffusible Bcl-2 inhibitor, pep2 (lower panel); n = 7 and 8 correspondingly.