Bar graph showing the average (mean ± s.e.m) threshold cycle (C T ) values for the

target genes CaV3.1 and CaV3.2, and the housekeeping genes hypoxanthine

phosphoribosyltransferase (HPRT1) and large ribosomal subunit (17S) within A7r5 cells (n=8). B, Bar graph showing relative mRNA expression of CaV3.1 (upper y-axis) and

CaV3.2 (lower y-axis), channel expression is plotted as (mean ± s.e.m) % expression of

HPRT1, for “old” A7r5 cells sub-cultured at 90-100% confluence (n=7, experiments conducted by Dr H. Duckles). C, As B except with “new”A7r5 cells sub-cultured at 70- 80% confluence (n=8, note larger axis). A7r5 cell lysates were measured in triplicate wells with the mean value used as n=1. All data were analysed using unpaired students t-tests, *p≤0.05, ****p ≤0.0001.

In order to assess the sub-cellular distribution of CaV3.1 and CaV3.2 channels,

immunofluorescence was conducted on “new” A7r5 cells. On all occasions, DAPI (4',6- diamidino-2-phenylindole) was co-applied to label cell nuclei. Initial attempts to detect the CaV3.1 and CaV3.2 antibodies within A7r5 cells using the Alexa Fluor 488 secondary

antibody (detected in green) were unsuccessful due to prominent background fluorescence, apparent upon omission of either the primary or secondary antibodies (Error! Reference source not found.). NAD(P)H is a common source of autofluorescence detected within cytosolic, mitochondrial and nuclear regions. Within bovine coronary artery smooth muscle cells, NAD(P)H-mediated autofluorescence has been observed, with peaks of excitation and emission around 340nm and 450nm respectively (Gao & Wolin, 2008). Resultantly, Alexa Fluor 555 (detected in red) was used as the secondary antibody within A7r5 cells, as no background fluorescence was observed at these wavelengths (Error! Reference source not found.). Low levels of immuno-reactivity (IR) to both CaV3.1 and CaV3.2 antibodies were detected within

some A7r5 cells. CaV3.2 channels were found to be localised around nuclear regions

(Error! Reference source not found.), whereas CaV3.1 channels appeared to have a

more generalised distribution throughout A7r5 cells (Error! Reference source not found.). Both CaV3.1 and CaV3.2 primary antibodies were found to be highly specific, as

they showed no cross-reactivity with the opposing isoform, i.e. no CaV3.2 IR was

apparent within HEK293/CaV3.1 cells and vice versa (Error! Reference source not

no 1⁰ DAPI DAPI DAPI

A

B

C

Secondary alone . 5.10.15

CaV3.1 alone . 18.10.15

CaV3.2 alone . 18.10.15

Figure 3.2. Background fluorescence is detected in “new” A7r5 cells at 488nm Control images for dual-label immunofluorescence using anti-CaV3.1 (1:100) and anti-

CaV3.2 (1:1000) primary antibodies, with Alexa Fluor-488 secondary antibody (1:1000,

left panel, green images), and DAPI to label cell nuclei (right panel, blue images). Fluorescence is observed within A7r5 cells when; A, the Alexa Fluor 488 secondary antibody is applied alone, B, when the CaV3.1 primary antibody is applied alone (i.e. no

secondary) and C, when the CaV3.2 primary antibody is applied alone (i.e. no

no 1⁰ DAPI DAPI DAPI

A

B

C

Figure 3.3. No background fluorescence is detected in “new” A7r5 cells at 555nm Control images for dual-label immunofluorescence using anti-CaV3.1 (1:100) and anti-

CaV3.2 (1:1000) primary antibodies, with Alexa Fluor-555 secondary antibody (1:500,

left panel, red images), and DAPI to label cell nuclei (right panel, blue images). No fluorescence is observed within A7r5 cells when; A, the Alexa Fluor 555 secondary antibody is applied alone, B, when the CaV3.1 primary antibody is applied alone (i.e. no

secondary) and C, when the CaV3.2 primary antibody is applied alone (i.e. no

Ca_V3.1 DAPI

A

(i)

merged

Figure 3.4. Sub-cellular distribution of CaV3.1 channels within “new” A7r5 cells Dual-label immunofluorescence using anti-CaV3.1 (1:100) with Alexa Fluor-555

secondary antibody (1:500, left panels, red images) and DAPI to label cell nuclei (centre panel, blue images), merged CaV3.1 and DAPI images (right panels). A, A7r5 cells show

low levels of diffuse CaV3.1 immuno-reactivity (IR), regions of interest (white box) are

Ca_V3.2 DAPI

A

(i)

merged

Figure 3.5. Sub-cellular distribution of CaV3.2 channels within “new” A7r5 cells Dual-label immunofluorescence using anti-CaV3.2 (1:1000) with Alexa Fluor-555

secondary antibody (1:500, left panels, red images) and DAPI to label cell nuclei (centre panels, detected in blue), merged CaV3.1 and DAPI images (right panels). A, A7r5 cells

show low levels of CaV3.2 immuno-reactivity (IR) localised within and around cell

nuclei, regions of interest (white box) are magnified in (i). All scale bars 50µm.

Due to a wide variety of factors such as post-translational modification and dynamic membrane trafficking, mRNA expression does not necessarily equate to functional ion channel expression. For example, increased T-type Ca2+ channel current-density observed in response to chronic insulin stimulation within rat pituitary derived GH3

cells was shown to be due to increases in dynamic endosomal recycling and trafficking of CaV3.1 to the surface membrane and not alterations in mRNA levels or promoter

activity (Toledo et al., 2012). We therefore employed patch-clamp electrophysiology to assess functional T-type and L-type Ca2+ channel expression within A7r5 cells.

As illustrated by Figure 3.2, both the T- and L-type SS protocols evoked Ca2+ currents within some A7r5 cells. Whilst the SS protocols were designed to maximally activate T- or L-type Ca2+ channels it was possible that these voltage protocols were not entirely subtype selective (Chapter 2). As such, quotations marks are used when describing currents evoked using the SS protocols in A7r5 cells. The distribution of Ca2+ currents was highly heterogeneous; some A7r5 cells expressed either “T-type” or “L-type” Ca2+ currents in isolation, some cells expressed both types of current and many cells had no detectable CaV current. As approximate values, 75% of cells expressed both “T-” and

“L-type” Ca2+ currents, 10% expressed “L-type” Ca2+ currents alone, 5% expressed “T- type” Ca2+ currents in isolation and 5% showed no detectable CaV current. It would

have been advantageous to provide a more accurate quantification, however, on many occasions recordings did not last long enough to run both single-step protocols. In addition, to ensure effective voltage-clamp, only small A7r5 cells were recorded from which would have introduced a selection bias and decreased the validity of any quantification. Regarding current-voltage (I-V) relationships, cells exhibiting predominantly “L-type” Ca2+ currents showed maximal activation at +20mV. By contrast, cells exhibiting predominantly “T-type” Ca2+ currents displayed maximal activation at -20mV and cells with mixed populations showed maximal activation at 0mV.

A(i) -80mV -20mV -50mV +20mV iii ii _“T-type” “L-type” B(i) ii iii C(i) ii

Figure 3.2. T-type and L-type Ca2+ channel currents are heterogeneously expressed within “new” A7r5 cells

Whole-cell patch-clamp recordings from 3 representative “new” A7r5 cells using Ca2+ (15mM), as the charge carrier. A, Cell with both T- and L-type Ca2+ currents. B, Cell with predominant “L-type” Ca2+ current. C, Cell with predominant “T-type” Ca2+ current. (i), Current-voltage relationships with red line showing voltage producing maximal current, (ii), “T-type" Ca2+ current traces evoked from single step(SS)-depolarisations to -20mV from a holding potential of -80mV, (iii), “L-type” Ca2+ current traces evoked from SS-depolarisations to +20mV from a holding potential of -50mV. All scale bars 50pA (vertical), 50ms (horizontal). Due to the potential activation of L-type mediated Ca2+ current using the T-type SS protocol and vice versa, “T-type” and “L-type” currents are labelled with quotation marks.

For single step (SS) voltage protocols, “T-type” Ca2+ currents were stimulated by repetitive depolarisations (0.1Hz) to -20mV (200ms) from a holding potential (Vh) of -

80mV. This evoked Ca2+ currents showing rapid activation and inactivation, the characteristic shape of T-type Ca2+ channel currents. “L-type” Ca2+ channel currents were stimulated by SS voltage protocols to +20mV (200ms, 0.1Hz) from a Vh of -50mV.

When using Ca2+ as the charge carrier this produced currents with rapid activation and both a slowly inactivating and a sustained component. L-type Ca2+ currents are generally characterised by their sustained shape resulting from their very small degree of voltage-dependent inactivation. It should be noted however, that the kinetics and resultant current shape is highly dependent on the L-type Ca2+ channel isoform, splice variant and auxiliary subunits expressed. These factors can dictate varying degrees of Ca2+-dependent inactivation which may account for the slowly inactivating component observed, as reviewed within (Lipscombe et al., 2004).

Interestingly, the amplitude of currents recorded in response to L-type SS protocols were of smaller amplitude than those recorded at the equivalent voltage (+20mV) in I- V protocols, also illustrated in Figure 3.2. This further raises the possibility that summation of L- and T-type Ca2+ currents was occurring. In order to determine whether the currents evoked by the two SS protocols were mediated solely by the channel of interest, we examined the effects of specific T- and L-type Ca2+ channel blockers (CCBs). Currents evoked using the T-type SS voltage protocol were inhibited by the selective T-type CCB NNC55-0396 (3µM, NNC) in a partially reversible manner, whilst the L-type selective CCB nifedipine (2µM) had no significant effect on T-type Ca2+ channel current amplitudes, when applied to a limited number of cells (Figure 3.3). This suggested that only T-type Ca2+ channels were contributing to the Ca2+ influx using this SS protocol. Due to the heterogeneous patterns of T- and L-type Ca2+ current expression in A7r5 cells (Figure 3.2), the observation that some currents evoked using the T-type SS were nifedipine-insensitive does not necessarily translate to the whole A7r5 population

For the L-type SS voltage protocols, Ba2+ (20mM) was examined as an alternative charge carrier. This significantly enhanced the amplitude of L-type Ca2+ currents and almost completely eliminated the slowly inactivating component, resulting in a

sustained current shape more characteristic of L-type Ca2+ currents (Figure 3.4). Reducing the stimulating voltage-pulse duration (100ms from 200ms) whilst using Ba2+ had the added advantage of reducing L-type Ca2+ current rundown (data not shown). Consequently, Ba2+ was used for all SS L-type recordings. Nifedipine (2µM) inhibited currents evoked by the L-type SS protocol in a partially reversible manner. Surprisingly, L-type Ca2+ currents were also inhibited by NNC (3µM), indicating either that NNC was not selective for T-type Ca2+ channels at 3µM or that some of the current recorded using the L-type SS was being facilitated by T-type Ca2+ channels.

Together, these results show that A7r5 cells express functional T- and L-type Ca2+ channels, that both CaV3.1 and CaV3.2 mRNA is expressed with CaV3.1 in greater

abundance and that CaV3.1 channels tend to be localised around the cell membrane,

A